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Division of Endocrinology and Metabolism (B.A.), and Howard Hughes Medical Institute (M.G.R.), Department of Medicine, University of California, San Diego, La Jolla, California 92093-0648
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Abstract |
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 Top Abstract I. IntroductionII. Structure and...III. Development of the...IV. Expression and Function...V. Relevance of POU...References |
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I. Introduction
II. Structure and Transcriptional Function of POU Domain Factors
A. DNA-binding sites for POU domain factors
B. Protein chemistry
C. Protein-protein interactions
D. Mechanisms of transactivation
III. Development of the Hypothalamo-Pituitary Region
IV. Expression and Function of POU Domain Factors in the Neuroendocrine System
A. Pit-1
B. Oct-1
C. Oct-2
D. Overview of expression of Brn-1, Brn-2, Brn-4, and Tst-1 in the neuroendocrine system
E. Brn-2
F. Brn-4
G. Tst-1
H. Oct-3
I. Brn-5 and RPF-1
V. Relevance of POU Domain Factors to Diseases of the Neuroendocrine System
A. Pit-1 mutations as a cause of combined pituitary hormone deficiency in humans
B. Brn-4 mutations in humans
C. Potential implications for other diseases.
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I. Introduction |
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TopAbstractI. IntroductionII. Structure and...III. Development of the...IV. Expression and Function...V. Relevance of POU...References |
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).
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Although some general conclusions can be reached about POU domain factors, more importantly the various members serve a wide variety of functions during development. In fact, it is reasonable to predict that an individual POU factor may play multiple distinct roles in those organs in which it is expressed during development and in the adult. There is evidence for the role of POU domain factors in inhibition and promotion of cell proliferation and determination of cell lineages, as well as in regulation of cell migration, survival, and terminal differentiation. Because of their overlapping expression patterns and similar DNA-binding preferences, POU factors coexpressed in a single organ may be functionally redundant. In addition to a role in early and late development, members of this family play roles in physiological regulation of gene expression in the adult. While POU domain genes are expressed in all organ systems, this review focuses on the role of POU domain factors in the neuroendocrine system with special emphasis on the hypothalamo-pituitary axis.
A great deal of knowledge has accumulated about the biological function of POU domain factors since their initial identification in 1988. In addition to insightful in vitro experiments, progress in this field has been greatly facilitated by the ability to create targeted mutations of POU domain genes in the mouse. These studies, as well as the discovery of human disease mutations involving the POU domain factor Pit-1, have established a crucial role for POU domain factors in neuroendocrine development and function.
The 15 known mammalian POU domain genes have been classified into
six different groups based on homology in the POU domain region,
including the nonconserved linker region (Table 1). The class I POU factor, Pit-1
(GHF-1), is required for the generation and maintenance of three
different cell types in the anterior pituitary gland of humans and
other mammals (1, 2, 27). Expression analyses and in vitro
biochemical/molecular biology experiments have implicated the
octamer-binding proteins Oct-1 and Oct-2, which belong to class II POU
domain factors, in neuroendocrine function and development (28, 29, 30, 31).
The third class II factor, Skn-1a, is selectively expressed in
epidermis and has no role in the neuroendocrine system (32, 33, 34, 35). The
class III POU domain subgroup consists of four closely related factors,
Brn-1, Brn-2, Brn-4, and Tst-1/Oct-6/SCIP, which are all encoded by
intronless genes (36). For members of this subgroup, a role in
neuroendocrine development has been most clearly established for Brn-2,
which is required for the formation of hypothalamic nuclei and the
posterior pituitary gland (37, 38). The other class III POU factors may
also play a crucial role in neuroendocrine function and development.
Brn-3 and two other members of the class IV POU factors have been shown
to be important for various aspects of sensory development, but have no
known roles in the neuroendocrine system and will not be further
discussed in this review (39, 40, 41, 42, 43, 44, 45, 46, 47, 48). The class V POU factor Oct-3 (Oct-4)
is expressed in germ cells, embryonic stem cells, and in early
neurogenesis where its down-regulation may be crucial for proper neural
formation (49, 50, 51, 52, 53). Sprm-1, the second class V factor, is selectively
expressed in spermatocytes and has no role in the neuroendocrine system
(54, 55). The class VI POU factor Brn-5 is widely distributed and
highly expressed in hypothalamic regions (56, 57, 58). The other class VI
member, retina-derived POU-domain factor-1 (RPF-1), is expressed in the
retina, the medial habenula, and in dispersed populations of neurons in
the dorsal hypothalamus (59).
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II. Structure and Transcriptional Function of POU Domain Factors |
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TopAbstractI. IntroductionII. Structure and...III. Development of the...IV. Expression and Function...V. Relevance of POU...References |
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) (62, 63). This principle has been elegantly demonstrated for the
interaction of Oct-1 with an octamer site (ATGCAAAT) where the POU-S
domain and the POU-H domains associate with the ATGC- and AAAT-parts,
respectively (64, 65, 66). The spacing and orientation between the two
halves of a binding element can vary, and the ability of different POU
domain factors to recognize such sites depends in part on the length,
sequence, and flexibility of the linker tethering the POU-S and POU-H
domains (63, 67). In addition, there is evidence that diversity in the
POU DNA binding may stem, in part, from amino acid-base interactions
that are more flexible than those observed for classical homeodomains
(62). Often, high-affinity binding sites for POU factors are flanked by lower affinity binding sites that are capable of promoting cooperative DNA binding of POU factor dimers. These "second" sites are usually highly diverged from high-affinity binding sites and may contain isolated A/T-rich regions or the POU-S recognition sequence ATG (CAT on the opposite strand). In this section, we will first review the DNA binding characteristics of different POU domain factors, and then summarize the main conclusions from the collection of studies on this issue.
1. DNA binding sites for the class I factor Pit-1. Pit-1 was
originally identified based on its ability to bind to multiple sites in
the regulatory regions of its target genes GH and
PRL (1, 2). Similar Pit-1 binding sites have been described
in the regulatory regions of several other pituitary-expressed genes
that are likely targets of Pit-1. These include the Pit-1
gene itself (68, 69, 70), the TSH
gene (71, 72, 73), the rat and
human GHRH receptor gene-regulatory regions (74, 75), the rat
somatostatin receptor 1 gene-regulatory region (76, 77), and the human
TRH receptor gene-regulatory region (78). These sites, which vary
widely around the consensus sequence ATGNATA(A/T)(A/T)(A/T), tend to be
found in multiples in Pit-1 target genes (Table 2). On most sites, Pit-1 appears to bind
as a homodimer with one Pit-1 molecule contacting the high-affinity
octamer-related sequence, ATGNATA, and the second molecule binding
cooperatively to the 3' located lower affinity A/T-rich region
(79, 80, 81). On some sites Pit-1 can bind as a heterodimer with the POU
domain factor Oct-1, and on yet other sites it binds as a monomer (82).
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2. DNA binding sites for the class II factors Oct-1 and Oct-2.
The octamer DNA element, ATGCAAAT, frequently found within
gene-regulatory regions, is conserved and functionally important for
transcriptional regulation of several genes. These include the B
cell-specific immunoglobin genes (83), the ubiquitously expressed small
nuclear (sn)RNA genes (84, 85), the cell cycle-specific histone
H2B genes (86), and a host of other widely and selectively expressed
genes (Table 3). Early studies attempting
to identify the transcription factors binding to octamer elements led
to the isolation of Oct-1 and Oct-2 as the major components of
octamer-binding activity in eukaryotic cells. Review of the literature
indicates that the Oct-1 POU domain has a strong preference for the
core element, ATGCAAAT, but clearly several variants of the core
element are compatible with high-affinity DNA binding (Table 3).
Experiments have also indicated that the bases flanking the core
octamer provide additional binding specificity (60).
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). It is striking that alignment of
these sites results in a consensus sequence remarkably similar to that
identified in the two random site selection studies (Table 3). However,
this approach is unlikely to give a true representation of the type of
sites found in gene-regulatory regions because in their studies,
investigators are much more likely to identify and report on the well
known octamer site rather than more divergent sites.
Oct-1 can also bind to a distinct site, TAATGARAT (R is a purine),
found in the herpes simplex virus immediate early (HSV IE) promoters
(87, 88). The affinity of Oct-1 alone for this site is much lower than
for that of the octamer element (64), but in contrast to the classical
octamer site, this site can promote the formation of a complex of Oct-1
and VP16, which is required for viral transcription (Fig. 2). The critical feature of this site is
the 3'-GARAT motif, which is necessary for the recruitment of VP16,
possibly by a mechanism involving the recognition of specific
conformations of Oct-1 (62, 89) or direct nucleotide recognition (90).
On this site, the POU-S domain is located on the opposite side of the
POU-H domain as compared with Oct-1 bound to a classical octamer site
(62).
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Several divergent sites capable of binding Oct-1 have been identified
in diverse genes, including the GnRH gene-regulatory region
(28, 29) (Table 3). An unusual binding site has been described in the
human interleukin 5 (IL-5) promoter. In this site, inverted POU-S
binding sites seem to flank two POU-H domain binding sites (94, 95).
Yet other sites are similar to the TAATGARAT site in that they only
contain recognizable POU-H domain binding sites (Table 3). The binding
specificity of Oct-1 and Oct-2 has been found to be indistinguishable
in several studies, and other studies indicate that the third class II
POU factor, Skn-1a, has identical binding preferences (35, 96). These
observations suggest that within a POU class, binding preferences tend
to be similar.
3. DNA binding sites for class III factors. Studies have shown
that the class III POU factor Tst-1 binds to and activates octamer
site-dependent promoters (97, 98). Similar to Oct-1, different binding
affinities were observed with perfect octamer sites containing distinct
flanking sequences, indicating that the flanking sequences are also
important determinants of binding affinity for Tst-1 (98). Tst-1 also
binds to five distinct sites in the myelin-specific P0 promoter with a
consensus sequence, GA(A/T)T(T/A)ANA, which appears unrelated to the
octamer site (99). The rat GnRH promoter contains three Tst-1 binding
sites that appear to conform to the consensus derived from the Tst-1
binding sites in the P0 promoter (100) (Table 4). Tst-1 also binds with high affinity
to two sites in the JC virus promoter, but only one of the sites
confers transcriptional activation. While they are AT-rich, neither
site bears any similarity to octamer sites nor the P0 consensus site
(101).
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Brn-4 DNA binding preferences have also been evaluated by a random site
selection method where it was found that the optimal binding site,
CAATATGCTAAT, is related to the smaller octamer site, containing a TAAT
instead of the AAAT (105). Thus, the optimal binding sites selected
with Brn-2 and Brn-4 are different. It is quite possible that these
differences are due to methodological variables rather than bona fide
binding differences between these two class III POU factors. Since it
is likely that all POU domain factors can bind to DNA in different
conformations, it is not surprising that different studies have
identified distinct binding sites for class III POU factors. Nonoctamer
DNA-binding sites for Brn-4 have been found in the striatal D1 dopamine
receptor (106) and proglucagon genes (107). These sites seem to be most
similar to the Tst-1 sites in the P0 and rGnRH promoters (Table 4).
4. DNA binding sites for the class V factor Oct-3. Oct-3 binds
with high affinity to octamer sites (108, 109, 110), but also to so called
palindromic Oct factor recognition elements (PORE), which simulate a
pair of inverted homeodomain sites separated by 5 bp, ATTTG +5 CAAAT,
found in the osteopontin enhancer (Table 5). In addition to Oct-3, other Oct
factors can bind to this site, either as homodimers or heterodimers
(111). Oct-3 is reported to transactivate more robustly from this
element than a classical octamer site (111).
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), selectively recruits Oct-3 and Sox-2
and is incapable of binding complexes containing Oct-1 and Tst-1 (112).
5. DNA binding sites for the class VI factor Brn-5. In
experiments using a random site selection method, Brn-5 was found to
prefer the binding site GCATAA(T/A)TTAT, which is related to the class
IV factor Brn-3 optimal binding site (103). In different experiments
where Brn-5 site selection was performed with a fixed site, GCAT,
followed by eight random nucleotides, an octamer-like sequence was
selected: GCATATGATAAT (56). However, it is clear that Brn-5 binds
poorly to a classical octamer site (56). Alignment of three
high-affinity sites suggested a high-affinity consensus site
GCATN2–3TAAT (56) (Table 6).
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genes are bona fide response
elements. For most other sites identified in such studies, genetic
evidence for their importance is lacking. An alternative approach has
been to use a selection of high-affinity binding sites from a set of
random sites in vitro. While this approach may identify
high-affinity sites, those are not necessarily biologically important
sites. However, from these two approaches several general conclusions
about POU domain binding sites can be reached.
First, members of the same subclass of POU domain factors tend to have
similar DNA-binding preferences, most clearly demonstrated for class II
POU factors. Second, while distinct POU classes may have somewhat
distinct DNA-binding preferences, a single POU factor can recognize
several different DNA-binding sites. Third, POU factors of different
classes can often recognize the same element with similar affinity, and
all POU factors discovered so far seem to be capable of binding to
octamer sites with reasonably high affinity. Fourth, while the core
recognition sequence for monomeric binding is often 8 bp long, bases
outside the core region may be important for binding affinity and
transcriptional activity. Fifth, while POU domain factors can bind as
monomers, some factors, such as Pit-1 and class III factors, can
homodimerize, especially on appropriate symmetrical sites. Sixth, while
transcriptional activation may correlate with binding affinity on
monomeric sites, other features of the site, such as the ability to
allow dimerization and interaction with other factors, may be important
determinants of whether a particular site confers activation (Fig. 2).
Seventh, the DNA-binding site can determine the ability of a bound POU
factor to recruit associated proteins (Fig. 2). Finally, target genes
that are responsive to POU domain factors frequently have a cluster of
POU-binding sites, particularly when the regulatory regions are located
at a distance from the promoter.
B. Protein chemistry
The structure of the POU domain has been extensively studied for
both Oct-1 and Pit-1, using NMR (113, 114, 115, 116) and crystallographic (66, 117) methods. The similarity in structure between these two POU domains
strongly suggests that the basic observations from these studies can be
applied to other POU domains. The POU domain is composed of a 74- to
82-amino acid POU-S domain and a 60-amino acid POU-H domain with a 15-
to 56-amino acid flexible linker tethering the two domains (Fig. 1).
The two subdomains are structurally independent, and individually they
are capable of low-affinity DNA interactions (64). High-affinity DNA
binding, however, requires the intact POU domain, with each subdomain
making major groove DNA contacts. The structure of the POU-S domain is
characterized by four 
-helices surrounding a hydrophobic core
similar to the helix-turn-helix motif of bacteriophage 
and 434
repressor, and 434 Cro DNA-binding domains (113, 115). While somewhat
divergent, the POU-H domain, like classic homeodomains, exhibits a
helix-turn-helix structure with three -helices (118, 119, 120). For both
the POU-S and POU-H domains, the second and third helix form a
helix-turn-helix structure with the third helix docking to DNA in a
major groove where amino acids making direct base contacts tend to be
conserved among POU domain factors (121, 122).
The two subdomains do not contact each other, and the linker is not
visible in the cocrystals, suggesting that it may be unstructured,
perhaps providing flexibility to the POU domain (Fig. 1). This
flexibility would allow the two subdomains to acquire different
orientation and spacing relative to each other, presumably explaining
the versatility in DNA sites that can bind POU domain factors, and the
variability in surfaces available for interactions with coregulatory
proteins (63). This flexibility may depend, in part, on the length of
the linker as evidenced by results showing that the Brn-2 POU domain
can adopt two different orientations on DNA, while this was not
observed with Brn-3, which contains a shorter linker (62, 63). An
effect of the linker on DNA binding specificity was also found in
experiments comparing binding of chimeric molecules from the Oct-1 and
Pit-1 POU domains (123). It has been shown that optimal binding of
Oct-1 to an octamer site requires a minimum linker length between 10
and 14 amino acids (67). Oct-1 molecules with a shorter linker bind DNA
only if the POU-S recognition site is inverted in orientation,
apparently bringing it closer to the POU-H domain. In addition,
mutations of some conserved residues in the Oct-1 linker altered DNA
binding, indicating that there are both sequence and length
requirements for the function of the linker (67).
An example of the subdomain flexibility is provided by the binding of Oct-1 to the octamer element (ATGCAAAT) where the POU-S domain recognizes the 5'-ATGC-part and the POU-H domain recognizes the 3'-AAAT-part. On the TAATGARAT site from the herpes simplex virus (HSV) IE promoter, the Oct-1 POU-H domain recognizes the 5'-TAAT part, and the POU-S domain recognizes the 3'-GARAT part in a flipped orientation as compared with Oct-1 binding to an octamer site (62).
While the primary, secondary, and tertiary structures are similar,
crystallographic studies of Oct-1 and Pit-1 on two different DNA sites
have shown quite distinct structures as to the relationship between the
two POU subdomains (66, 117) (Fig. 1). The Oct-1 POU domain binds to
opposite faces of an octamer DNA-binding site (the histone H2B promoter
octamer). In contrast, the Pit-1 subdomains bind to perpendicular faces
of a palindromic DNA site (derived from the PRL 1P site), with the
POU-S domain exhibiting flipped orientation compared with Oct-1
structure (Fig. 1). In addition, while Oct-1 binds as a monomer to an
octamer site, Pit-1 forms homodimers on the palindromic PRL 1P-related
site via interactions between the C terminus of the DNA-recognition
helix of the homeodomain and helix 1 and the loop between helices
3 and 4 of the POU-S domain. Most likely, these differences in
structure between Oct-1 and Pit-1 are due to the different DNA-binding
sites used for cocrystallization, which is supported by data indicating
that the Pit-1 POU domain binds as a monomer to a classical octamer
site (124).
It has been shown that POU factors can be modified by phosphorylation.
Oct-1 and Pit-1, for example, are phosphorylated on homologous residues
in the POU domain, Ser-385 and Thr-220, respectively, and this
phosphorylation leads to decreased DNA binding to octamer and GH POU
sites (125, 126). On other sites, such as those found in the TSH and
PRL promoters, phosphorylation by protein kinase A or C enhances DNA
binding (127, 128). While cAMP-dependent kinase can phosphorylate these
sites in vitro, the identities of the relevant kinases
operating in vivo are unclear. For Oct-1, this
phosphorylation occurs in a cell cycle-specific manner, thus explaining
the S phase-specific activation of the histone H2B gene by Oct-1 (86, 125). Jun kinase (JNK)/stress- activated protein kinase can bind to the
activation domain of Brn-5 and phosphorylate its POU domain on sites
that are distinct from the aforementioned Oct-1/Pit-1 phosphorylation
sites. This phosphorylation leads to increased binding to DNA and
enhanced transactivation (129). Finally, it has been demonstrated that
Pit-1 can be acetylated by CREB-binding protein (CBP), but the
biological significance of this finding remains to be determined (130).
C. Protein-protein interactions
POU domain factors regulate transcription by interacting with
other proteins. POU interacting proteins can be classified into four
classes: DNA-binding transcriptional activators, coregulators, basal
factors, and replication factors (131). A prominent feature of POU
factors is their ability to form cooperative homodimers, and even
heterodimers with other POU factors, especially when bound to the
appropriate DNA site (80, 82, 132) (Fig. 2). POU factors also have a
striking ability to interact with a variety of divergent structural
domains in heterologous factors, including other DNA-binding proteins
and coregulators that are incapable of independent DNA binding. In this
chapter we will review protein-protein interactions involving those POU
factors expressed in the neuroendocrine system (Table 7). Many of these interactions may not be
directly relevant to regulation of gene expression in the
hypothalamo-pituitary axis, but the principles derived from these
studies are likely to apply to studies of POU domain factors in the
neuroendocrine system (Fig. 2).
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genes (80). This Pit-1
homodimerization has been shown in living cells with fluorescence
resonance energy transfer microscopy (FRET) (133). Functional
experiments indicate that the POU-S domain is important for Pit-1-Pit-1
interactions and that homodimerization is important for efficient
transactivation (80). The ability of some natural Pit-1 mutants to
inhibit Pit-1 action in a dominant negative fashion further supports
the importance of Pit-1 homodimerization in gene activation (see
Pit-1 mutations as a cause of combined pituitary hormone
deficiency in humans). In addition, Pit-1 can form
heterodimers with Oct-1, which results in synergistic transactivation
of the PRL promoter (82). Pit-1 can also interact to varying degree with several heterologous DNA-binding proteins which, when bound to the same promoter, often lead to synergistic transactivation. These interacting factors include the nuclear hormone receptors TR (thyroid hormone receptor), RAR (retinoic acid receptor), RXR (retinoid X receptor), ER (estrogen receptor), VDR (vitamin D receptor), and GR (glucocorticoid receptor). Pit-1, for example, interacts strongly with RXR and more weakly with TR and RAR, which may play a role in synergistic regulation of the GH promoter (134, 135, 136). Pit-1 can also interact with ER (137, 138, 139, 140) and VDR (141) in synergistic activation of the PRL gene. The human PRL promoter is inhibited by glucocorticoids, mediated by a gene regulatory region containing Pit-1 binding sites but no glucocorticoid response element (GRE). Studies show that GR and Pit-1 can interact in solution and that the inhibition does not require GR to bind to DNA (142).
Pitx1, a bicoid-like homeodomain factor, interacts with the N terminus of Pit-1 (143) and synergistically transactivates the PRL promoter with Pit-1 (144, 145). A direct interaction of Pit-1 with the related Pitx2 removes an inhibitory effect of a 39-amino acid C-terminal sequence of Pitx2, thus promoting DNA binding and transactivation (146). Pit-1 also interacts in solution with the LIM homeodomain protein P-Lim (Lhx3) and acts synergistically with P-Lim to regulate the PRL enhancer/promoter (147).
Synergistic interactions between Ets-1 and Pit-1 may be important in
regulation of the PRL promoter. The synergistic gene activation
requires direct Pit-1-Ets-1 protein-protein interaction and is
not observed with Ets-2 (148). This interaction, which has been shown
in living cells with FRET microscopy (133), maps to the POU domain
(149). Pit-1, an isoform of Pit-1 containing 26 additional amino
acids in the N terminus, is incapable of transcriptional synergism with
Ets-1, apparently because of additional interaction surface that maps
to the N-terminal sequence specific to this isoform (149).
Interactions between Pit-1 and the transcription factor GATA-2 are more
complex and have distinct features depending on whether both factors
bind to DNA (150, 151) (Fig. 2A). Pit-1 and GATA-2 can interact in
solution (150, 151), and the interaction interface maps to the POU-H
domain of Pit-1, specifically the N-terminal basic residues and the
surface of the second helix (P26, Q29) (151). In GATA2, the C-terminal
DNA-binding zinc finger and an adjacent cluster of basic residues seem
to be important. Pit-1 can inhibit binding of GATA-2 to cognate DNA
sites on promoters that do not have Pit-1 binding sites, such as the
gonadotrope-specific SF-1 promoter (151) (Fig. 2A). This seems to be
important for generation of the gonadotrope phenotype. In contrast,
Pit-1 leads to synergistic activation with GATA-2 on promoters
containing adjacent Pit-1 and GATA-2 sites, such as the
thyrotrope-specific TSH promoter (150, 151) (Fig. 2A). As described
later, these interactions may be important for the formation of both
thyrotrope and gonadotrope cell types.
b. Interactions with coregulator proteins.
In addition, Pit-1
has been demonstrated to interact with coregulators. The POU domain of
Pit-1 interacts with CREB binding protein (CBP) through two
cysteine-histidine rich domains (C/H1 and C/H3) (152, 153) (Fig. 3). Pit-1 can also, via its POU-H domain,
interact with the corepressor N-CoR (nuclear receptor and corepressor)
(153). As will be described later, these interactions may be important
for transactivation and the role of Pit-1 in signal transduction.
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Both Oct-1 and Oct-2 have been shown to interact with DNA-binding factors from other classes, including nuclear hormone receptors, such as RXR (138). The interaction interfaces seem to involve the Oct-1 POU-H domain, but not the POU-S domain, and the DNA-binding domain and hinge region of RXR. Functional studies suggest that Oct-1 may negatively regulate nuclear signaling pathways by altering the DNA-binding ability of nuclear receptor-RXR heterodimers. In contrast, on artificial promoters containing vitamin D response elements and octamer sites, Oct-1 and the vitamin D receptor exhibit cooperative binding and transcriptional synergism mediated by the DNA-binding domain of the vitamin D receptor (155).
In solution, the POU domains of Oct-1 and Oct-2 can bind to the DNA-binding domain of GR, including an interface that contains amino acids C500 and L501 (156). Again the POU-H domain of Oct-1 seems to be responsible for the interaction (156). Microinjection experiments in zebrafish embryos suggest that such DNA-independent effects of GR involving interactions with the Oct-1 and Oct-2 POU-H domains are biologically important (157). The GR-Oct-1 interaction can both modulate the transcriptional activity of Oct-1 on certain promoters, like the GnRH promoter as detailed later (158), and the activity of GR on its target genes (138). On the mouse mammary tumor virus (MMTV) promoter, it has been shown that Oct-1 and GR bind in a cooperative fashion and that this leads to a synergistic transcriptional regulation on naked DNA templates in vitro (159). Oct-2 can also bind cooperatively with GR, and this also results in synergistic transcriptional regulation (156, 160). In vivo results indicate that Oct-1 binding to the MMTV promoter is strictly glucocorticoid dependent, consistent with a role for GR in remodeling of chromatin to promote access of other factors to the MMTV promoter, such as nuclear factor 1 (NF-1) and Oct-1 (161, 162).
On templates containing GREs and octamer sites, Oct-2A and GR can synergize transcriptionally (156, 163). However, when GR sites are not present, GR strongly inhibits transactivation by Oct-2A, while the reciprocal is not true. This effect does not seem to depend on interference with Oct-2A DNA binding, but more likely is due to competition for rate-limiting coactivator molecules (163). A minimal promoter driven by Oct-1, binding to an octamer element, can also be inhibited by GR in a hormone-dependent manner. This interaction seems to be mediated by a strong solution interaction between GR and Oct-1, resulting in inhibition of Oct-1 DNA-binding (164). This interaction, which seems to involve the POU-H domain of Oct-1, can be inhibited by GR binding to a GRE (156). While the mechanism is unclear, functional analyses indicate that androgen-mediated repression of the dehydroepiandrosterone sulfotransferase gene promoter requires Oct-1 binding to the promoter. Since there seems to be no direct AR binding to this promoter, AR-Oct-1 interactions may be important (165).
Oct-1 can interact with several other DNA-binding factors, including
Sp1 (166, 167), AP-1 (168, 169, 170), CCAAT/enhancer binding protein-
(C/EBP) (171), NF-1 (172, 173), and MEF2 (174). Oct-1/2 interactions
with heterologous DNA-binding proteins can either result in
transcriptional synergism, as exemplified by the AP-1/Oct-1 interaction
(168, 169, 170), or interference as exemplified by C/EBP inhibition of
Oct-1 function (171).
Interaction of the high mobility group protein HMG2 with the POU domains of Oct-1 and Oct-2 leads to cooperative binding (175). The related HMG I(Y), a known octamer-binding protein, functions as a coactivator with Oct-2A but not Oct-1 for HLA-DRA transcription. Apparently HMG I(Y) facilitates the binding of both Oct-1 and Oct-2 to octamer sites (176) by a mechanism that involves interactions between HMG I(Y) and the POU domains of Oct-2 and Oct-1. However, synergistic transactivation of the HLA-DT gene is only found with Oct-2 and seems to depend on sequences within the C terminus of Oct-2 (177).
b. Interactions with coregulator proteins.
The B cell-specific
coactivator OCA-B (also referred to as OBF-1 and Bob-1), a 256-amino
acid, proline-rich protein with no close homologs in the database,
interacts specifically with the POU domains of Oct-1 and Oct-2 and is
required for B cell-specific activation of immunoglobin genes
(178, 179, 180, 181, 182, 183). By interacting with Oct-1 and Oct-2, OCA-B can be recruited
to a subset of octamer sites where it serves a coactivator function for
transcription (184) (Fig. 2B). In part, a strong transactivation domain
in the C terminus of OCA-B mediates this effect, while the N terminus
contains the domain interacting with POU domains (185). This
interaction is specific for Oct-1 and Oct-2 (Fig. 2B) and not observed
for other POU domain factors such as Oct-3, Oct-6, and Pit-1. The
specificity of the interaction of OCA-B with both the POU-S and the
POU-H domains is determined by residues in the first helix of the POU-S
domain (L6 and E7) and the end of the third POU-H domain helix (K155
and I159) (124, 184). Other residues in the POU domain, including L53
and N59 in the POU-S domain, have been shown to be important for the
Oct-OCA-B interactions. However, these residues are conserved in POU
domain factors that do not interact with OCA-B and are thus unlikely to
determine the specificity of the interaction.
Immunoglobin promoters are strongly activated by OCA-B, whereas the
histone H2B promoter is poorly activated, suggesting that there is some
promoter-specific restriction of the ability of OCA-B to transactivate.
Further specificity is observed on the immunoglobin promoters because
OCA-B only seems to activate on sites in the proximal promoter but not
on distally located enhancer sites (124). In addition, the
Oct-1/Oct-2-OCA-B complex can only occur on octamer sites that contain
an A in the fifth position of the octamer site, such as found in the
classical octamer site (Fig. 2B). Position +3, +4, and +6 also appear
to be important (124, 184). Apparently, OCA-B makes direct contacts
with the DNA backbone in the middle of the octamer site, via its N
terminus, in addition to POU domain contacts (186, 187). It has been
proposed that OCA-B clamps the two POU subdomains together and at the
same time bridges the DNA within the octamer motif at several
positions. According to this model, OCA-B may help organize the
conformation and stabilize the DNA binding of the Oct POU domain on DNA
(124, 184). There is also evidence that binding of OCA-B to the Oct-1
POU domain/octamer complex induces a partial folding of OCA-B (186).
Another extensively studied coregulator is VP16. In human cells
infected with the herpes simplex virus (HSV), viral gene expression is
initiated with the viral protein VP16, which regulates the HSV
immediate-early (IE) genes. VP16 does not bind DNA directly, but is
instead recruited to IE promoters by its association with Oct-1 (87, 188, 189, 190, 191, 192) (Fig. 2B). Another cellular protein, host cell factor (HCF),
is required for nuclear import of VP16 (193) and stabilization of the
VP16-Oct-1 interaction (194, 195, 196, 197). After infection, VP16 first forms a
complex with HCF, which then promotes its interaction with Oct-1 and
binding to the TAATGARAT motif in the HSV IE promoters (Fig. 2B). The
interaction of the Oct-1 POU domain and VP16 is selective and not
observed with the highly homologous Oct-2 POU domain (198) (Fig. 2B).
Selective residues, particularly a glutamic acid residue (E22) located
on the surface of the second helix of the POU-H domain, are critical
for the recognition by VP16 (199, 200). In the Oct-2 POU-H domain, the
analogous residue is an alanine, thus precluding interaction with VP16.
In addition, the interaction between Oct-1 and VP16 requires the 3'-end
GARAT of the TAATGARAT motif to be intact for ternary complex formation
(89, 90) (Fig. 2B). VP16 alters gene regulation by Oct-1 in two
distinct ways. First, it provides a transactivation domain that is
highly active on mRNA promoters where the Oct-1 transactivation domain
is inactive, and second, it stabilizes the Oct-1-binding to
VP16-responsive elements (184).
c. Interactions with basal transcription factors.
SNAPc (snRNA
activating protein complex; also referred to as PTF) is a basal
transcription factor, composed of several well defined peptides, that
binds to promoter elements called the proximal sequence element (PSEs)
in both RNA Pol II and Pol III snRNA promoters (84, 201, 202). Oct-1
binds to the enhancer or distal sequence elements (DSEs) in snRNA
promoters, which leads to cooperative binding with SNAPc via a
protein-protein interaction involving the POU-S domain of Oct-1,
especially residue E7, and the C-terminal region of SNAP190, a major
component of the SNAPc (203). The result is an enhanced transcriptional
activation by Oct-1-mediated recruitment of SNAPc to the PSE of snRNA
genes (204). The carboxy terminus of SNAP190 acts as an inhibitor of
SNAPc DNA binding, but the interaction with the Oct-1 POU domain
relieves the inhibition and promotes cooperative binding; SNAPc binds
with 8- to 10-fold higher affinity when Oct-1 is bound to the DSE (205, 206).
The POU domains of Oct-1 and Oct-2 have been shown to interact with the TATA binding protein TBP (207). Since this interaction was in solution and not on DNA, its significance is somewhat unclear. However, this finding is consistent with the observation that Oct-1 and Oct-2 can facilitate preinitiation complex formation (208). In addition, the binding of Oct-1 and Oct-2 to the lipoprotein lipase promoter octamer site was shown to be stimulated by transcription factor II B (TFIIB), apparently via protein-protein interactions (209).
A yeast two-hybrid screen with the Oct-1 POU domain identified MAT1 as a POU domain-binding protein capable of binding to Oct-1, Oct-2, and Oct-3 POU domains. MAT1 is required for the assembly of cyclin-dependent kinase (CDK)-activating kinase (CAK), which is functionally associated with the general transcription factor IIH (TFIIH). The interaction between POU domains and MAT1 can target CAK to octamer binding factors and promote their phosphorylation. This interaction may play a role in the recruitment of TFIIH to the preinitiation complex (210).
d. Interactions with replication factors.
Oct-1 is involved in
adenovirus replication where it binds to the highly conserved core
origin and interacts via its POU domain with one of the three viral
proteins required for replication, the precursor terminal protein (pTP)
(122, 211, 212). The interaction seems to involve several regions of
the pTP protein (213). The POU-H domain binds to the pTP-DNA polymerase
complex (213), but more weakly than the intact POU domain. No binding
is observed with the POU-S domain alone (214). Presumably, this
interaction facilitates the recruitment of the pTP-DNA polymerase
complex to the origin of replication. The stimulation of adenovirus DNA
replication seems to depend on a property of the POU domain that is
common to many POU domains because other POU domain factors, including
Pit-1 and Tst-1, can also stimulate DNA replication (215).
3. Class III POU interactions.
a. Interactions with DNA-binding proteins.
Based on
far-Western and mammalian two-hybrid experiments, all class III POU
domain factors can interact with each other via the POU homeodomain
(216). Consistent with this observation, Brn-2 exhibits highly
cooperative homodimerization on a consensus nonoctamer binding site
(103).
Sox/HMG proteins are transcription factors that contain Sry boxes that mediate binding to the minor groove of DNA. Such factors may frequently interact with POU domain factors that bind to the major groove of DNA. Tst-1 (Oct-6) can interact with HMG I(Y) in solution, which facilitates Tst-1-binding to an AT-rich site in the JC viral regulatory region (217). Tst-1 can also interact with the related HMG2 (218). Both Tst-1 and Brn-1 can cooperate with different Sox proteins in transcriptional activation. Specifically, Brn-1 can synergize with Sox-11 when both proteins are bound to adjacent elements; the synergism depends on the transactivation domains in both proteins. Such POU-Sox interactions may be important for a combinatorial code required for cell-specific transcriptional activation (219, 220).
b. Interactions with coregulator proteins.
Tst-1 can interact
with the large T antigen from the human papovavirus JC virus, and this
interaction seems to lead to transcriptional activation on the early
and late promoters from the JC virus (101, 221, 222). The
amino-terminal 82 residues of the large T antigen and the POU domain of
Tst-1 mediate this interaction, but for a functional effect on
transcription, the N-terminal transactivation of Tst-1 is also
required. This functional effect is specific for Tst-1, because while
Brn-1 is also capable of interacting with large T antigen, this
interaction is not transcriptionally productive, apparently because of
differences in the transactivation domains of Brn-1 and Tst-1 (223).
4. Oct-3 interactions.
a. Interactions with DNA-binding proteins.
Oct-3 can interact
with the HMG-box protein Sox2 (224), and these two factors have been
implicated in synergistic regulation of the fibroblast growth factor 4
(FGF-4) gene (49) and the embryonic stem cell coactivator gene UTF-1
(112). The synergism between Oct-3 and Sox2 on the FGF-4 promoter seems
to be due to cooperative binding, which can be disrupted by increasing
the distance between the two binding sites (109). In contrast, Sox2
functionally antagonizes Oct-3 on the ostepontin gene-regulatory region
where its binding site is adjacent to that of Oct-3 (111). Oct-3 and
Sox2 interact directly in solution, with the interaction interface
being the POU domain and the Sox2 HMG domain (109).
b. Interactions with co-regulator proteins.
In embryonic stem
cells Oct-3 transactivation is independent of the distance of the
octamer site from the promoter, but in differentiated cells, Oct-3
transactivation from a distance seems to require stem cell-specific
coactivators that bridge the remotely bound Oct-3 to the basal
transcriptional machinery (225). While these factors remain to be
identified, it is known that transactivation by Oct-3 is strongly
stimulated by E1A in 293 and HeLa cells and depends on both the intact
POU domain and the transactivation domain of Oct-3. This effect is
specific because it is not found with Oct-1 and minimally with Oct-2.
Direct binding of Oct-3 to E1A was also demonstrated (225). The Oct-3
POU domain can associate with two distinct domains on the E1A protein,
CR1 and CR2, and it can also associate with the HPV E7 protein (226).
These data suggest that viral oncoproteins have characteristics that
mimic a stem cell-specific activity (226).
5. Conclusions.
From studies on the protein-protein
interaction characteristics of POU domain factors several general
conclusions can be drawn. First, most factors that interact with POU
domain proteins have been found to interact with the POU domain rather
than other parts of the proteins. This finding may be due, in part, to
technical difficulties in identifying factors that interact with the
transactivation domains outside the POU domain. However, these results
indicate that the POU domain is not only a DNA-binding domain but also
an important interface for regulatory proteins. Second, POU domain
factors seem fairly promiscuous as to their ability to interact and
synergize with diverse DNA-binding proteins bound to the same promoter.
Most POU domain factors have been shown to interact in such a way with
nuclear hormone receptors and HMG proteins. Third, while the POU domain
is highly conserved, most POU domain interactions with coregulators are
remarkably selective; apparently proteins are able to discern small
differences in POU domain sequence (Fig. 2B). Thus, the E1A protein
interacts strongly with Oct-3 but not Oct-1 or Oct-2. VP16 interacts
strongly with Oct-1 but not Oct-2, Oct-3, or Tst-1 (Fig. 2B). OCA-B
interacts selectively with Oct-1 and Oct-2, but not with Oct-3 or Tst-1
(Fig. 2B). This selectivity for protein-protein interactions by POU
domains is in stark contrast to the lack of selectivity in DNA-binding
preferences. Fourth, the association of a coactivator with a POU domain
factor may be stabilized by a simultaneous direct interaction of the
coactivator with DNA, such as that found with Oct-1/2 and OCA-B (Fig. 2B). Fifth, some POU factor-coactivator complexes can only form on
selective DNA sites, perhaps in part due to direct DNA contacts of
coactivators or due to DNA-induced conformational changes of the POU
domain as has been described with OCA-B/Oct-1 and VP16/Oct-1
interactions (Fig. 2B). For example, the Oct-1/2-OCA-B interaction
requires an "A" in position 5 of an octamer site
(ATGCAAAT) (184), and the Oct-1-VP16 interaction requires
the GARAT part of a TAATGARAT site for ternary complex
formation (90). Sixth, additional functional specificity is provided by
sequences outside the POU domain, such that two distinct POU domain
factors may be capable of physical interaction with a coregulator, but
only one of the interactions will result in transcriptional activation,
apparently because of specific sequences in the transactivation domain.
Finally, while some protein-protein interactions involve only one of
the subdomains of the POU domain, e.g., the interactions of
VP16 with the POU-H domain and SNAPc with the POU-S domain, others such
as OCA-B interact simultaneously with the POU-S and the POU-H domains.
D. Mechanisms of transactivation
1. Transactivation domains.
While the N- and C-terminal
domains in POU domain factors are thought to act as transactivation
domains, little is known about their structures. These domains, which
frequently contain polymeric amino acid stretches, are poorly conserved
between different POU domain factors, consistent with the idea that
they confer specificity to the action of different factors, often
binding to the same DNA element. The N terminus is serine/threonine
rich in Pit-1, alanine/glycine rich in Tst-1, proline rich in Oct-3,
and glutamine rich in Brn-2. The N- and C-terminal transactivation
domains of Oct-2 have been most extensively characterized (227, 228, 229, 230).
The N terminus contains an 18-amino acid glutamine-rich motif, and the
C terminus contains a proline-rich domain (231). The function of these
motifs is enhanced when they are combined or reiterated independently
(229, 232), suggesting that they have a modular structure. The Oct-1
transactivation domain appears to be fairly specific for snRNA
promoters and has less effect on mRNA transcription, in contrast to the
Oct-2 transactivation domains that are active on mRNA promoters (184, 228).
Studies on Oct-3 suggest that transactivation domains may work in a cell-specific manner (233). The N-terminal sequence of Oct-3 fused to the GAL4 DNA-binding domain can transactivate in all cell types tested, whereas the C-terminal sequence shows cell-type specificity in the same assay. This cell type specificity of the C-terminal domain seems to be imposed by the Oct-3 POU domain because it functioned in all cell types tested when the C terminus was fused to Pit-1 POU domain. It is further possible that the transactivation specificity is regulated by phosphorylation as the variation in transactivating ability of the C-terminal domain correlates with differences in the phosphorylation status of Oct-3.
Studies on Pit-1 have defined additional specificity conferred by the N-terminal transactivation domain (234). Thus, synergistic activation by Pit-1 and the estrogen receptor on a PRL reporter construct requires an N-terminal 25-amino acid domain of Pit-1 that is not required for analogous synergism on the GH promoter. This synergy, which depends on two of three tyrosine residues, spaced by six amino acids, is preferentially used on sites where Pit-1 binds as a monomer instead of a dimer. These data suggest that the DNA-binding site, dictating whether Pit-1 binds in dimeric or monomeric form, can alter the segment of the N terminus used as a synergy domain.
The N terminus of Brn-2 contains a glutamine-rich region that is mainly encoded by CAG residues (36). Similar nucleotide triplets that can be expanded are found in disease genes causing fragile-X syndrome and myotonic dystrophy (235). It is unknown whether the same mutational mechanism might involve the Brn-2 gene. A polyglutamine tract-binding protein, PQBP-1, capable of binding to the polyglutamine tract of Brn-2 and other such tracts of triplet repeat disease, has been identified (236). While the function of this novel protein is unknown, it is known to be localized in the nucleus and capable of interfering with transactivation by Brn-2.
The N-terminal sequences of POU factors can also mediate repression, including regions from the N terminus of Oct-2 (237, 238, 239, 240, 241, 242). Oct-3 binds to the promoter of Rex-1, which encodes an acidic zinc finger protein expressed at high levels in embryonic stem cells (108). Depending on the cellular environment, Oct-3 can either activate or repress the promoter, with distinct domains within the N terminus being responsible for repression and activation.
2. Transactivation and chromatin.
Gene expression is
influenced by chromatin structure (243). Histones are regulated by
acetylation of lysines in their N-terminal tails, allowing changes in
histone conformation generally associated with increased access of
DNA-binding proteins to promoters. In contrast, histone deacetylation
is coupled to transcriptionally silent chromosomal domains. Many
transcriptional coactivators possess intrinsic histone acetylation
activity that is targeted to specific genes through interactions with
DNA-binding proteins. Repressors, on the other hand, can recruit
deacetylating enzymes to promoters, either directly or via the
corepressor mSin3 (244, 245).
N-CoR, originally identified as a nuclear receptor corepressor (246),
has been reported to bind to the homeodomain of Pit-1 and actively
suppresses transactivation by Pit-1. This suppression is dependent on
mSin3, SAP30, and histone deacetylase (153, 247). N-CoR may be limiting
under certain conditions, and its binding to other transcription
factors, such as nuclear hormone receptors, may indirectly activate
transcription by Pit-1. The Pit-1 POU domain also associates with the
coactivator complex consisting of CBP/p300 and P/CAF, both of which
possess histone acetylase activity (152, 153) (Fig. 3). Thus, the
transcriptional activity of Pit-1 may be mediated by the competing
binding of complexes responsible for either acetylation or
deacetylation, resulting in activation or repression (Fig. 3).
3. Conclusion.
Ultimately, it is likely that POU domain
factors mediate their transcriptional activation by a combinatorial
mechanism involving the N- and C-terminally located transactivation
domains and coactivators/corepressors that interact with the POU domain
(Fig. 3). Since the involved coregulators participate in gene
activation by many different transactivators, it is likely that the
unique transactivation domains of POU domain factors provide
specificity to the activation (228).
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III. Development of the Hypothalamo- Pituitary Region |
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) (139, 248, 249, 250): 1) the anterior lobe
with five major hormone-producing cell types that respond to
neuropeptides released from hypothalamic nuclei carried to the anterior
pituitary gland via the portal circulatory system; 2) the posterior
lobe containing specialized astroglia referred to as pituicytes, and
magnocellular neuronal axons projecting from hypothalamic nuclei,
releasing vasopressin and oxytocin (OT); and 3) the intermediate lobe
containing melanotropes and endorphin- secreting cells (Fig. 4).
|
Most of the hypothalamus appears to arise from the neuroepithelium ventral to the hypothalamic sulcus while the preoptic region arises rostral to the optic sulcus (256). During development of the hypothalamus, between embryonic days 11 and 18 (e11 and e18) in the rat, periventricular neurosecretory cells become organized into several nuclei. These include the paraventricular (PVH), supraoptic (SO), arcuate, and periventricular nuclei (PVN). Development of the hypothalamus appears to progress in an outside-in gradient with the late-arising neurosecretory cell types occupying the most medial or periventricular zone of the hypothalamus. For descriptive purposes, four rostrocaudal levels of the hypothalamus can be described: preoptic, anterior, tuberal, and mammillary.
Within the hypothalamus, two distinct neurosecretory systems are
organized (Fig. 5) (256). One system is
composed of magnocellular neurons that project axons directly into the
posterior lobe of the pituitary gland (Fig. 5). The magnocellular
neuronal system includes neurons of the PVH and SON that synthesize OT
and vasopressin (VP) and release these peptides into the posterior lobe
of the pituitary gland. The second system is composed of parvocellular
neurons that synthesize neuropeptides released into the pituitary
portal circulation for regulation of the anterior pituitary gland (Fig. 5). In addition to the magnocellular neurons, the PVH also harbors
parvocellular neurons that synthesize, among other neuropeptides, CRH
and TRH for regulating the ACTH/adrenal axis and the TSH/thyroid axis,
respectively. In the anterior periventricular and arcuate nuclei are
parvocellular neurons that provide dopaminergic control of lactotrophs,
and somatostatin/GRH control of somatotrophs. GnRH neurons originate in
the olfactory placode and migrate during embryonic development to the
hypothalamus (257). GnRH is synthesized in scattered neurons throughout
the hypothalamus, especially in preoptic hypothalamic neurons. It is
involved in the initiation of puberty and regulation of FSH and LH
synthesis in the anterior pituitary gland and thereby regulates
reproduction.
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). When this oral ectoderm makes contact
with the overlying neuroectoderm, around e9 in the mouse, a cascade of
signaling events is initiated that ultimately lead to the generation of
the anterior and intermediate lobes of the pituitary gland containing
six distinct hormone-producing cells by e17.5 (139, 258). These cell
types are corticotropes secreting ACTH, melanotropes secreting MSH,
gonadotropes secreting LH and FSH, thyrotropes secreting TSH,
somatotropes secreting GH, and lactotropes secreting PRL.
|
). Thyrotropes and gonadotropes are most
ventral and somatotropes and lactotropes reside in a dorsal position
(139, 258) (Fig. 6). During this stage of pituitary organogenesis, at
e15, corticotropes are distributed throughout the anterior pituitary
gland (258, 259, 260). The spatial restricted organization of thyrotropes,
gonadotropes, somatotropes, and lactotropes may be regulated by
gradients of signaling molecules, including sonic hedgehog, bone
morphogenic proteins (BMPs), FGFs, and Wnts, which in turn create
overlapping spatial gradients of transcription factors (Fig. 6). Thus,
the transcription factors Nkx3.1, Six3, and Prop1 are expressed
predominantly dorsally; Isl1, Brn-4, P-Frk, SF-1, and GATA-2 are
expressed predominantly ventrally; and Pit-1 is expressed in an
intermediary location, the site where lactotropes and somatotropes
initially appear (248, 249). BMP4 signaling from the ventral diencephalon seems to be important for the early formation of the Rathke’s pouch and seems to be linked to the induction of the transcription factor Isl1 (261). Later, FGF8 released from the ventral diencephalon and BMP2 released from mesenchymal cells ventral to the pouch and within the pouch seem to be important for patterning and cell proliferation (261, 262, 263). Embryological studies in the rat suggest that the programs required for specification of all hormone-producing cell types is completed as Rathke’s pouch closes (around e12 in the mouse).
Concurrent with the appearance of the anterior and intermediate lobes
of the pituitary gland, formed from the ventral and dorsal aspects of
Rathke’s pouch, respectively, the posterior gland develops from neural
ectoderm (Fig. 6).
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IV. Expression and Function of POU Domain Factors in the Neuroendocrine System |
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Several alternatively spliced isoforms of Pit-1 have been described
(81, 265, 266, 267, 268). These include Pit-1, an isoform containing a
26-amino acid insertion, which is conserved among mammals, in position
48 of the N-terminal transactivation domain (265, 266, 269). While
Pit-1 is expressed at levels that are 7- to 8-fold lower than that
of Pit-1, it has been shown in transient transfection assays that the
Pit-1 isoform has distinct properties from Pit-1 (266, 270, 271).
Another isoform is Pit-1T, which contains a 14-amino acid insertion at
the same location and may play a specific role in TSH stimulation
(268, 272).
As previously described, Pit-1 can also be regulated at a posttranscriptional level. For instance, phosphorylation is reported to lead to decreased or increased DNA binding depending on the DNA-binding site (126, 128). Activin leads to increased Pit-1 phosphorylation that not only decreases DNA binding, but also decreases the stability of Pit-1 (273).
2. Regulation of terminal differentiation. Ontogenic analyses
showed that the initial expression of Pit-1 in the caudomedial region
of the pituitary gland at e14.5 in the mouse correlated both temporally
and spatially with the subsequent activation of the GH,
PRL, and TSH genes (139, 274) (Fig. 6).
Throughout development and in the adult, Pit-1 expression continues in
somatotropes, lactotropes, and thyrotropes (Fig. 6). Consistent with a
role for Pit-1 in these cell types was the observation that the
developmental-specific gene regulatory regions of the GH,
PRL, and TSH genes contain AT-rich elements to
which Pit-1 binds and which mediate Pit-1-stimulated activation in
transient transfection assays (71, 275, 276). Subsequent analyses of
the Pit-1-deficient Snell and Jackson dwarf mice suggested
that these in vitro findings are of biological importance
because, with the exception of TSH expression in Pit-1-independent
rostral tip thyrotropes (72), activation of the GH,
PRL, and TSH genes fails in
Pit-1-mutated mice (27) (Fig. 4).
In addition to regulating target genes by binding to regulatory regions in close proximity to promoter regions, there are data implicating Pit-1 in regulating locus control regions (LCRs). The human pituitary GH (GH-N) gene contains Pit-1, Zn-15, and Sp1 binding sites in the first 140 bp upstream of the transcription start site. This region, however, is not sufficient for efficient expression in transgenic mice (277) and an LCR appears to be required for efficient and consistent expression of the hGH-N gene. LCRs, which mediate an efficient and position-independent expression of associated genes, are thought to perform their function by establishing open chromosomal domains as well as by acting as enhancers. Two pituitary-specific DNAse-hypersensitive sites were found in a 1.6-kb region located approximately 15 kb upstream of the transcription initiation site (277) and within this region enhancer activity was localized to a 404-bp region (278). This region contains three A/T-rich Pit-1-binding sites that seem to be important for the activity of this LCR in transgenic mice, suggesting that Pit-1 is involved in the enhancer activity of the GH LCR, consistent with a chromatin-mediated developmental role for Pit-1 (279, 280). Interestingly, the A/T-rich Pit-1-binding sites bear some similarity to nuclear matrix anchorage sites (281), but it has been demonstrated that the POU-specific domain of Pit-1 contains information for targeting to the nuclear matrix (282).
3. Regulation of cell proliferation. In addition to regulating
expression of the terminal differentiation marker genes GH,
PRL, and TSH, Pit-1 stimulates cell
proliferation of pituitary cells, as evidenced in the Snell and Jackson
dwarf mice, which are characterized by marked failure in the expansion
of somatotropes, lactotropes, and thyrotropes (27). In addition,
microinjection of Pit-1 antisense sequences resulted in decreased cell
proliferation of GC somotatroph cells (283). Recent findings have
suggested three mechanisms involved in Pit-1-induced expansion of
pituitary cells. First, experiments with adenovirus suggest that Pit-1
can directly regulate DNA replication in vitro (215).
Second, Pit-1 may directly regulate IE genes involved in cell-cycle
initiation, such as c-fos (284). It has been shown that
Pit-1 expression in PC12 cells, which do not normally express Pit-1,
leads to increased c-fos expression. In addition, decrease
of Pit-1 protein levels, by means of an antisense strategy, leads to
decreased cAMP-induced c-fos promoter activity in pituitary
cell lines. Finally, Pit-1 is required for expression of the GHRH
receptor, which acts to stimulate proliferation of somatotrophs (74, 75, 285, 286). However, the ontogeny of GHRH receptor mRNA expression,
which is highest at 19.5 in the rat with subsequent decrease until
postnatal day 12, does not correlate with that of Pit-1, indicating
that other factors, in addition to Pit-1, are important for GHRH
receptor expression (287). Pit-1 binding sites have also been described
in the TRH receptor gene regulatory region (78), suggesting a possible
mechanism for Pit-1 regulation of proliferation of thyrotropes.
4. Regulation of Pit-1 gene expression. The Pit-1 gene is located on chromosomes 3p11 and 16 in humans and mice, respectively. In the mouse, the Pit-1 gene is initially activated on e13.5–14 under control of a specific early enhancer that remains to be precisely characterized, but appears to be located between –5.1 and –10.2 kb upstream of the start site (288). From e16.5, Pit-1 expression requires a different autoregulated distal enhancer, located 10 kb upstream of the Pit-1 start site (70, 288). The distal enhancer contains three functional Pit-1 binding sites, a vitamin D receptor-binding site, and an retinoic acid response element (RARE) that mediates a Pit-1-dependent RAR induction (70). In the Snell dwarf mouse, Pit-1 gene expression at e14.5 is initiated normally but is extinguished in the perinatal period, apparently due to failure of autoregulation (27). The proximal promoter region of the Pit-1 gene also contains Pit-1 binding sites (68, 69). Mutations in the transcription factor gene Prop-1 in the Ames dwarf mouse lead to a failure in the initial activation of Pit-1 gene expression (289); this effect, however, may be indirect. The transcription factor GATA-2 may be responsible for restricting Pit-1 gene expression from ventral cell types by repressing the Pit-1 early enhancer (151).
5. Restriction and selective facilitation of Pit-1 action.
Pit-1 is expressed in three distinct cell types in the anterior
pituitary gland, and in each of these cell types it seems to be
required for the expression of the characteristic hormone-encoding gene
(Fig. 4). Therefore, there must be mechanisms in place that limit the
function of Pit-1 to prevent it, for example, from activating the
GH gene in thyrotropes. There are some data suggesting that
sequences in the target genes mediate active repression in heterologous
pituitary cells (290, 291). The abundance of evidence, however,
suggests that a complex combinatorial code based on synergistic
interactions with other transcription factors is responsible for
cell-specific target gene activation by Pit-1.
The GH promoter is synergistically regulated by Pit-1 and TR/RXR or
RAR/RXR (136, 292), apparently based on protein-protein interaction
where Pit-1 can interact strongly with RXR but also to a lesser extent
with TR and RAR (135). The synergism between Pit-1 and TR depends, in
part, on Pit-1 residues that when mutated have no effect on Pit-1
acting alone. Thus, while the synergism amplifies the intrinsic
activity of Pit-1, there are also functions that are specific to
synergistic activation (134). Other transcription factors that bind to
the GH promoter are a zinc finger transcription factor, Zn-15, which is
required for activation of the GH gene (293) and C/EBP
(294), both binding to and activating the rat GH promoter in a
synergistic fashion with Pit-1.
The PRL gene promoter is regulated by estrogens only when it is bound both by Pit-1 and the ER, and this synergistic regulation depends on the intact AF-2 domain of ER (137, 139, 141, 295, 296). There is evidence for direct interaction between ER and Pit-1 that is estrogen dependent (140). The Pit-1/ER synergism is modulated by nuclear receptor coactivators with RIP140 inhibiting and SRC-1 and GRIP1 stimulating (295). In addition, there is strong synergism between Pit-1 and Pitx1 (143, 144) and Pitx2 (146) on the PRL promoter.
Members of the ETS family of transcription factors can bind to a composite Ets-1/Pit-1 binding site in the rat PRL promoter (148, 297, 298, 299). Synergistic gene activation between Pit-1 and Ets-1 may be important for basal activity and in mediating signals from growth factors and the Ras/mitogen-activated protein kinase (MAPK) pathway to the PRL gene (297, 298, 299). The synergistic gene activation correlates with direct Pit-1-Ets-1 protein-protein interaction; this interaction is specific and not observed with Ets-2 (148). In addition, ETS factors do not bind to or activate the GH promoter and are therefore good candidates for factors contributing to the combinatorial control that is specific for PRL gene expression (148).
ETS-2 repressor factor (ERF) contains an ETS DNA binding domain and a C-terminal repressor domain (300). This factor can bind to the PRL promoter and inhibit its activity. In cell lines, overexpression of ERF blocks induction of PRL transcription by protein kinase A and acts in an additive manner with dopamine to suppress PRL promoter activity, suggesting that dopamine and ERF may function by complementary mechanisms to suppress PRL promoter activity. In addition, it can block the cooperative activation of PRL gene expression by Pit-1 and ETs-1. ERF seems to act by inhibiting Pit-1 binding to the PRL regulatory elements and could be responsible for cell-specific repression of the PRL gene.
Studies on the human TSH promoter suggest that there are functional
interactions between Pit-1 and AP-1, with both factors binding to DNA,
resulting in synergistic transcriptional regulation (301). The activity
of the transcription factor GATA-2, which appears to be required for
the formation of both thyrotrope and gonadotrope cell types, can
be modulated by Pit-1 (150, 151). The presence of Pit-1 represses
the gonadotrope phenotype via direct interactions with GATA-2,
inhibiting its binding to cognate DNA sites important for generation of
the gonadotrope phenotype (151) (Fig. 2A). Pit-1 can thus inhibit
promoters that contain GATA-2 binding sites and no Pit-1 binding sites,
such as the gonadotrope-specific SF-1 promoter. In contrast, Pit-1 can
also synergize with GATA-2 to promote the thyrotrope phenotype on
promoters containing adjacent Pit-1 and GATA-2 sites, such as the
thyrotrope-specific TSH promoter (150, 151) (Fig. 2A).
6. The role of Pit-1 in hormonal regulation of target genes.
In addition to a role in regulating the appearance of cell types and
expression of target genes during development, there is evidence that
Pit-1 functions in the homeostatic hormonal regulation of gene
expression in the anterior pituitary gland (302). Experimental results
suggest that Pit-1 participates in pathways for acute regulation of the
GH, PRL, and TSH genes.
GH gene regulation is stimulated by GHRH released into the
tuberoinfundibular system and acting on the GHRH receptor located on
somatotropes (303). This G protein-coupled transmembrane receptor in
turns acts by stimulating the production of cAMP and activating protein
kinase A. While there are cAMP response elements (CREs) within the
human GH promoter, experiments suggest that the protein kinase A signal
is mediated by phosphorylation of CBP associating with and acting as a
cofactor for Pit-1 (Fig. 3).
Transcription of the PRL gene is regulated by dopamine,
which acts by reducing intracellular cAMP concentration and lowering
protein kinase A activity (304). This response is unusual in that it
does not appear to require CREB and CREs and instead may be mediated by
the most proximal Pit-1-binding site of the rat PRL promoter.
Mutagenesis of the three phosphoacceptor sites in the POU domain of
Pit-1 showed that these are not required for hormonal regulation of PRL
(127, 305), suggesting that other proteins binding to this site or
Pit-1-associated cofactors may be the substrate. Recently it was shown
that Pit-1 and CBP synergistically regulate the PRL promoter,
suggesting that association of CBP with Pit-1 may be responsible for
cAMP regulation of the PRL gene (152) (Fig. 3). It has also been shown
that the cAMP response is enhanced by Oct-1 and the Pit-1 isoform
(306), suggesting that Oct-1 can act as a protein kinase A signaling
cofactor. TRH, which is known to regulate PRL gene transcription, can
stimulate PRL reporter plasmids in GH3 cells, apparently also via Pit-1
interacting with its binding sites in the PRL promoter (307, 308).
Glucocorticoids mediate negative regulation of the human PRL promoter. This inhibition, which requires both Pit-1 and GR, is conferred by a gene-regulatory region containing Pit-1 binding sites but no GRE (142). The inhibition does not require GR to bind to DNA, and coimmunoprecipitation studies show that GR and Pit-1 interact.
B. Oct-1
Oct-1 is ubiquitously expressed (309) and encodes a 100-kDa
protein that has been implicated in the cell cycle-regulated expression
of histone H2B (86) and in the ubiquitous expression of small nuclear
RNA genes (84, 85). Based on these data, and the fact that Oct-1 can
stimulate DNA replication (212, 310, 311), it has been suggested that
Oct-1 may play an important role in regulating cell proliferation.
However, there is also a large amount of data suggesting that Oct-1 may
play roles in regulating expression of cellular genes not associated
with cellular proliferation. No knockout study of Oct-1 has been
published.
1. Oct-1 in GnRH gene regulation. Studies in the brain have suggested that Oct-1 is not highly expressed in neurons, especially nondividing mature neurons (312), and is mostly found in glial cells (313). However, there is evidence suggesting that Oct-1 may be directly involved in neuroendocrine gene regulation. Based on data from work with the hypothalamic cell line GT1–7, a model for GnRH-expressing neurons, the rat GnRH gene contains two regulatory regions: an evolutionary conserved proximal promoter region located between—170 and –1, and a 300-bp distal enhancer located approximately 1.7 kb upstream of the start site (29, 314). The proximal promoter of the rat GnRH gene, which is critical for regulated expression of the GnRH gene by protein kinase C (29, 314) and progesterone (315), is bound by several nuclear factors in the GT1–7 cell line (29). In addition, this region contains at least two octamer-binding elements that are critical for transcription directed by the GnRH promoter in GT1–7 cells (29). Two octamer sites are also found in the rat GnRH neuron-specific enhancer located 1.7 kb upstream of the promoter, and the activity of the enhancer is critically dependent on Oct-1 binding to these sites (28). These data suggest that Oct-1 plays critical roles in regulation of GnRH transcription, by interacting with both enhancer and proximal promoter sequences, perhaps through homotypic interactions between Oct-1 bound to the enhancer and the promoter (28, 29).
In the GT1–7 cell line, GnRH gene expression is repressed by the NO/cGMP signal transduction pathway (316). This repression, which is mediated by the 300-bp distal enhancer, is obliterated when either an Oct-1 or a C/EBP binding site is mutated, suggesting that these factors play a role in the NO/cGMP pathway (317). Consistent with this observation are experiments showing that NO analogs increase Oct-1 binding to octamer sites in GT1–7 cells (317). However, this effect of NO on Oct-1 binding may be cell-specific because in cultured vascular smooth muscle cells, NO inhibits Oct-1 DNA binding (318). Such cell-specific effects of NO might be due to differential activation of signal transduction pathways in different cell types.
As previously described, experiments with the mouse GnRH promoter in the GT1–7 cell line suggest that GR can confer negative regulation on this promoter by associating with Oct-1 bound to one of the octamer elements (158, 319, 320). Apparently, this particular Oct-1 binding site imposes conformational changes that allow docking of GR to Oct-1 and repression of transcription (158).
2. Oct-1 in vasoactive intestinal peptide (VIP) gene regulation. Oct-1 has also been implicated in regulation of VIP, which is widely distributed throughout the central and peripheral nervous system, including the hypothalamus and anterior pituitary gland (321, 322). VIP can function as a neuromodulator, growth regulator, and neuroendocrine releasing factor. Experiments using subclones from the SK-N-SH neuroblastoma cell line to study the VIP promoter have identified an upstream tissue-specific element (TSE), located between –4.6 and –4.0 from the start site in addition to a proximal promoter element containing a CRE. The 425-bp TSE contains two octamer sites, similar to those found in the GnRH neuronal-specific enhancer, both of which are important for expression (30). Both elements bound Oct-1 with the upstream element also binding Oct-2 in neuroblastoma cell extracts.
3. Oct-1 and TSH gene silencing. Oct-1 has been implicated
in silencing of the hTSH gene in heterologous cells
(291). Kim et al. localized an AT-rich region between –128
and –480 bp upstream of the start site. This element was bound by
Oct-1 in multiple locations, and silencing was mediated by the
alanine-rich C-terminal domain of Oct-1. Oct-1, again acting through
multiple binding sites, has also been implicated in silencing of the
3-hydroxysteroid/dihydrodiol dehydrogenase gene (323). In addition,
a silencer in the B cell-specific B29 (Ig) gene is an AT-rich region
shown to bind Oct-1 and Oct-2 (324). Together, these studies and the
fact that such AT-rich regions may serve as nuclear matrix attachment
sites suggest that Oct-1 may have a general role in gene silencing.
Consistent with this idea are observations that a significant amount of
Oct-1 is found in the nuclear matrix fraction of nuclei (325, 326).
C. Oct-2
Oct-2 encodes a 60-kDa protein, preferentially
expressed in B lymphocytes, especially in the most mature cell types,
as well as in the developing and adult nervous system (313, 327, 328).
In contrast to the widespread embryonic nervous system expression,
Oct-2 is confined to specific areas in the adult, which include the
suprachiasmatic and medial mammillary nuclei, hippocampus, olfactory
tract, and the olfactory bulb (313). Oct-2 proteins are present in both
neuronal and oligodendroglial cells, but are more abundant in glial
cells (313). At least eight alternatively spliced mRNAs have been
detected (329), and there is evidence that the different splice
variants, which are differentially expressed to a certain extent, may
have different functions (327, 330). In addition to the most
predominant form, Oct-2A, two of the splice variants, Oct-2.5 and
mini-Oct, are expressed at high levels in neural tissue (327). Oct-2
has also been detected in intestine, testes, and kidney (313).
1. Oct-2 gene-deleted mice. Oct-2 gene-deleted mice develop normally, including B cell development, but show an abnormal function of mature B lymphocytes with defective immunoglobin secretion in response to polyclonal antigens (331, 332). These mice die within hours of birth, and while no gross structural abnormalities within the nervous system have been described (333), it has been suggested that the cause of death may be abnormalities in the nervous system (332).
2. Oct-2 in GnRH regulation. Other experiments have implicated
Oct-2 in the regulation of GnRH gene expression and the onset of
puberty (31). While transsynaptic regulation of GnRH neurons is clearly
important for the onset of puberty and regulation of reproduction,
there is also evidence for GnRH neural regulation by astroglial cells
within the hypothalamus. Lesions of the anterior hypothalamus lead to
precocious puberty apparently caused by increased expression of TGF
by astrocytes formed in response to the lesion. TGF is involved in
stimulatory control of GnRH secretion and mimics some of the events in
normal puberty. It has been demonstrated that in both lesion-induced
puberty and normal puberty there is increased expression of Oct-2a and
Oct-2c in the hypothalamus. Both Oct-2 forms can transactivate the
TGF gene promoter, and inhibition of Oct-2 synthesis with antisense
technology reduces TGF expression and the onset of puberty. These
results suggest that the Oct-2 gene is an important component of a
glia-to-neuron signal pathway leading to the onset of female puberty in
mammals (31). Since Oct-2 (-/-) mice die around the time
of birth, a more rigorous testing of this hypothesis will require the
use of a tissue-specific Oct-2 knockout.
D. Overview of expression of Brn-1, Brn-2, Brn-4, and Tst-1 in the
neuroendocrine system
Several studies have described the expression patterns of class
III POU factors (97, 256, 328, 334, 335, 336). At e10 to e11,
Brn-1, Brn-2, and Brn-4 are expressed
widely in the nervous system, including in the primordium of the
endocrine hypothalamus adjacent to the third ventricle (328). By e14,
four separate rostrocaudally arranged areas (corresponding to the
preoptic, anterior, ventromedial, and mammillary nuclei) of the
hypothalamus are labeled with Brn-2 and Brn-1 (256). At that time, both
Brn-2 and Brn-4, but not Brn-1, are colocalized in the region of the
developing PVH and SON and Brn-4 expression extends ventrally to
potential precursors of the anterior hypothalamus. Brn-1-expressing
cells are located immediately dorsolaterally in the presumptive zona
incerta and in a dorsoventral stripe lateral to Brn-4-expressing cells.
In contrast to the nearly identical widespread expression pattern of
Brn-1 and Brn-2 in the developing nervous system, Tst-1 has restricted
expression in the developing embryo, but shows dense hybridization
signal in the mammillary region at e13. Tst-1 exhibits a much more
widespread expression pattern in the adult (256) but is excluded from
the hypothalamus.
Both Brn-2 and Brn-4 are expressed at high levels in the PVH and SON of
the mature hypothalamus (37, 38, 328, 334, 335). These nuclei contain
the parvocellular neurons that synthesize high levels of CRH and the
magnocellular neurons that synthesize arginine vasopressin (AVP) and OT
(Fig. 5). Double labeling studies show that Brn-2 is coexpressed with
each of these neuropeptides, but not in a significant number of
parvocellular neurons that produce TRH (38). Double labeling
experiments show that Brn-4 is not expressed in OT containing neurons
of the PVN and SON, but is present in the magnocellular neurons of the
PVN and SON that contain dynorphin and vasopressin (335). Brn-2 levels
in the PVH in response to ether stress have been studied, and it was
found that Brn-2 levels were unchanged (337).
The early neuronal expression of Brn-2, Brn-1, and Brn-4 has suggested a role for these factors in early neurogenesis (50, 328). Multipotential stem cells in the central nervous system are characterized by the expression of the intermediate filament protein nestin and the brain fatty acid binding protein (B-FABP). Regulatory regions of both genes, mapped in transgenic mice, contain POU binding sites close to hormone response elements (HRE), both of which are essential for expression of these stem cell markers (338). In the rat nestin gene, the POU site is recognized by the class III POU proteins Brn-1, Brn-2, Brn-4, and Tst-1 and is critical for general central nervous system (CNS) expression, whereas the HRE is required for full expression in the anterior CNS. In the B-FABP gene a composite POU/Pbx site is essential for neuroepithelial expression. This site can bind Pbx-1, Brn-1, and Brn-2 in CNS extracts, suggesting the possibility that expression of class III POU domain factors may define the stem cell state. Exit from the stem cell state is indeed characterized by changes in expression with many class III genes being turned off and Brn-5 turned on (50, 339). So far, gene deletion experiments of Brn-2, Brn-4, and Tst-1 have shown no early defects consistent with such a model, indicating that either class III factors do not play important roles in early neural development or that redundancy between different members has prevented an observable phenotype in mice deleted for single class III genes.
As described below, with the exception of Brn-1, knockout experiments for all individual members of the class III POU factors have been reported.
E. Brn-2
Deletion of the Brn-2 gene in mice has provided the
best evidence for the involvement of class III POU domain factors in
neuroendocrine development and function (37, 38). Brn-2
(-/-) mice are born normally but exhibit a decrease in size and
weight from postnatal day (p) 3. Most die before p6 and exhibit a
marked decrease in brown adipose tissue, consistent with severe
malnutrition. Analyses showed dramatic hypothalamic/posterior pituitary
gland abnormalities, but no obvious defects outside the hypothalamus
(Fig. 5). Among the most striking findings was a complete lack of CRH
expression in parvocellular neurons of the PVH. This effect was
extremely site specific because CRH expression was normal in other
regions of the brain. In addition, parvocellular neurons of the PVH
that produce TRH, but do not express Brn-2, were not
affected. Further, OT and AVP expression was absent from the
magnocellular neurons of the PVH and SON, but AVP expression in the
non-Brn-2expressing cells of the suprachiasmatic
nucleus was maintained normally, again indicating that the defects were
cell specific (38). Similarly, anterior periventricular neurons that
produce somatostatin and arcuate neurons that produce GHRH, which do
not normally express Brn-2, were not affected. The lack of
abnormalities in cells of the hypothalamus that do not express
Brn-2 suggests that the phenotype of the Brn-2
(-/-) mouse is due to cell-autonomous effects. By e19.5, a striking
decrease in cellularity of the PVH and SON was also observed.
The developing posterior pituitary gland in Brn-2 (-/-) mice appears to be normal until e16.5 when axonal projections from magnocellular neurons fail to make contact (37, 38). By e19 Brn-2 (-/-) mice show complete loss of pituicytes and a fold of the intermediate lobe instead fills the space normally occupied by the posterior lobe of the pituitary gland. Therefore, Brn-2 appears to be essential for magnocellular neurons to invade the posterior pituitary gland structure. These axons are then required for the survival of the pituicytes of the posterior pituitary gland. Surprisingly, despite the lack of CRH and AVP, ACTH expression in the pituitary gland of Brn-2 (-/-) mice is normal, as are the adrenal glands. This finding may indicate that alternative mechanisms are involved in regulating the ACTH/cortisol axis in the mouse.
Thus, the Brn-2 gene is required for the terminal
differentiation and survival of cell types that compose the
magnocellular system and one of the parvocellular cell types that
comprise the central control of the pituitary-adrenal axis (Fig. 5).
Detailed ontogenic analyses suggest that precursors for neurons of the
PVH and SON die around e12.5 during migration (37). While the
Brn-2 (-/-) mice show a severe phenotype in the PVH and
SO, no obvious abnormalities are observed in the vast majority of
neurons where Brn-2 is expressed. It has been proposed that overlapping
expression with other class III POU factors, such as Brn-1, may provide
redundancy to Brn-2 function in some neurons. However, in the PVH and
SO, Brn-4 is clearly incapable of compensating for the loss of Brn-2. A
proof for a model proposing both overlapping and specific
neuroendocrine functions for each of the class III POU factors will
require the analyses of double and triple mouse mutants.
While no morphological changes were observed in Brn-2 heterozygous (+/-) mice, these mice only expressed half the level of Brn-2 compared with wild-type mice. Intriguingly, this decrease in Brn-2 levels correlated with 50% reduction in the levels of AVP and OT in Brn-2 (+/-) mice (37). These results suggest that Brn-2 levels are limiting for the expression of AVP and OT and that in addition to a developmental function in the specification of AVP and OT-producing neurons, Brn-2 has a direct role in regulating the expression of these genes in the adult. Consistent with this notion, several elements capable of binding class III POU factors have been identified in the region –1,535 to –1,270 in the rat OT gene (340). In addition, the 5'-region of the CRF gene contains Brn-2-binding sites (63), which seem to be responsible for the activation of the CRF promoter (38). Further support for this model comes from experiments with the neuroblastoma cell line BE(2)-M17, where retinoic acid induces expression of CRF. Expression of Brn-2 antisense RNA abrogates this response, suggesting that Brn-2 acts in a pathway mediating retinoic acid-induced CRF expression in terminally differentiated neurons (341).
The basic helix-loop-helix (bHLH)-PAS transcription factor SIM1 is expressed in three hypothalamic nuclei during development: the PVN, the SO, and the anterior periventricular nucleus. In SIM1 (-/-) mice, the hypothalamus is hypocellular and neurons expressing OT, vasopressin, TRH, ACTH, and somatostatin (SS) are absent from these nuclei (342). Thus, the phenotype of SIM1 gene-deleted mice is overlapping but more severe than that of Brn-2 gene-deleted mice. Evidence suggests that SIM1 functions to maintain Brn-2 expression, and part of the observed phenotype may be due to loss of Brn-2 expression (342). The relationship between SIM1 and Brn-2 in the hypothalamus is therefore somewhat analogous to the relationship between Prop1 and Pit-1 in the anterior lobe of the pituitary gland.
F. Brn-4
Deletion of the Brn-4 gene in mice resulted in multiple
developmental defects in the inner ear, causing deafness, but no
detectable defects in the development or function of the central
nervous system (343, 344). The inner ear phenotype is consistent with
the expression of Brn-4 in the otic vesicle and derived structures. The
lack of phenotype in most neurons expressing Brn-4 suggests
the possibility that related POU domain factors may function on
Brn-4-responsive genes in the absence of functional Brn-4.
Brn-4, which is highly expressed in proglucagon-expressing cells of the pancreas, binds to the G1 promoter element of the proglucagon promoter, and in transient transfection experiments, Brn-4 appears to be a major regulator of proglucagon gene expression (107). It is unclear whether neuronal expression of proglucagon is regulated by the same mechanisms. The biological relevance of these in vitro studies is unknown because analysis of pancreatic structure or function in Brn-4 (-/-) mice has yet to be presented. Brn-4 has also been implicated in the regulation of striatal D1A dopamine receptor gene transcription (106).
G. Tst-1
In addition to the previously described neuronal expression in the
hypothalamus, Tst-1 is prominently expressed in
myelin-forming glia of the central and peripheral nervous system during
a period of rapid cell division (97, 98, 99, 328, 345, 346). In cultured
Schwann cells where cAMP induces the expression of myelin-specific
genes, Tst-1 gene expression is also induced, preceding the
induction of myelin-specific markers (345). In addition,
Tst-1 is expressed in embryonic stem cells and in the
developing nervous system (97). Expression in the brain is prominent in
certain areas of the telencephalon, mesencephalon, brain stem, cortex
anlagen, and in the developing colliculi (97, 328).
1. Tst-1 gene-deleted mice. Deletion of the Tst-1 gene in mice produces a severe defect in peripheral myelination by arresting Schwann cell maturation before axonal wrapping (347, 348). Most neuronal development appears to progress normally except for the phrenic nucleus, which is disrupted, and neurons of the nucleus of the lateral olfactory tract, which mismigrate. Most Tst-1 (-/-) mice die soon after birth but the occasional survivor is severely runted, consistent with a possible undefined defect in neuroendocrine function.
2. Tst-1 in GnRH gene regulation. Tst-1 transcripts are found in the neuronal cell line GT1–7, which produces GnRH, and in a subset of GnRH neurons in the hypothalamus of prepubertal female rats. In vitro studies using transfected cell lines have shown that Tst-1 can repress the GnRH promoter, apparently by binding to three sites in the promoter (100). Since neurons capable of synthesizing GnRH seem to fluctuate between GnRH positivity and negativity depending on the reproductive cycle, it has been proposed that Tst-1 can determine the ratio of phenotypically active and inactive GnRH neurons during postnatal life (100).
H. Oct-3
The Oct-3 gene is expressed in male and female
primordial germ cells, unfertilized oocytes (but not spermatocytes),
and embryonic stem cells of the preimplantation embryo (51, 53, 349).
The Oct-3 protein is also found in totipotent and pluripotent cells of
the inner cell mass of the pregastrulation embryo, but not in the
trophoectoderm. Expression of Oct-3 is required for the formation of
totipotent cells of the embryo since mutation of the Oct-3
gene in mice causes all cells of the embryo to acquire a trophoblastic
cell fate (49).
Oct-3 is expressed in the early embryo with expression in the headfold ectoderm extinguished around e8.5, when neural induction occurs (349), suggesting that its disappearance might be important for early neural induction (50). The embryonic carcinoma P19 cell model for neural induction is consistent with this idea. Treatment with RA, which produces a neuronal like phenotype, leads to down-regulation of Oct-3 while forced expression of Oct-3 in certain differentiated P19 cells specifically represses expression of Brn-2 and nestin. Together, these data suggest the possibility that POU domain factors may control the acquisition and loss of stem cell characteristics in the CNS (50). Oct-3 is not expressed in the hypothalamus or pituitary gland and is not likely to play later roles in the neuroendocrine system.
I. Brn-5 and RPF-1
Brn-5 exhibits widespread expression in neurons and tissues
outside the nervous system (56, 57, 58, 350, 351). Within the hypothalamus,
expression of Brn-5 is reported in the mammillary nucleus and ventral
area (339). Brn-5 is also expressed in the anterior pituitary gland
(56). During development, predominant expression is found in
postmitotic neurons and not in proliferating neuronal precursors,
suggesting that Brn-5 may have a role in regulating the postmitotic
fate of these cells (339). In addition, ectopic expression of Brn-5 in
dividing NG108–15 cells, a neuroblastoma/glioma hybrid cell line,
reduces proliferating cell nuclear antigen (PCNA) mRNA levels
and inhibits DNA synthesis, suggesting the possibility that Brn-5 may
suppress continued cell proliferation (352). No specific function has
been proposed for Brn-5 in the neuroendocrine system and a knockout
study has not been reported.
The second class VI POU factor, RPF-1, is expressed only within the CNS, where its expression is restricted to the medical habenulla, to a dispersed population of neurons in the dorsal hypothalamus, and to subsets of ganglion and amacrine cells in the retina (59). No specific function has been proposed for RPF-1 in the hypothalamus, and a knockout study has not been reported.
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V. Relevance of POU Domain Factors to Diseases of the Neuroendocrine System |
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TopAbstractI. IntroductionII. Structure and...III. Development of the...IV. Expression and Function...V. Relevance of POU...References |
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). Clinically,
patients usually have serum GH and PRL levels that are below detection
in standard assays, indicating loss of all secretory function in
somatotropes and lactotropes. Initially, some patients have detectable
TSH levels, but ultimately all patients develop secondary
hypothyroidism (Fig. 4). In other cases, a severe central
hypothyroidism has been diagnosed at birth (357). NMR studies have
shown variable normal or small anterior pituitary glands.
|
). Those
inherited in an autosomal recessive pattern are often, but not always,
due to disruption of DNA binding of Pit-1. Examples include Arg172Stop,
Glu250Stop, Ala158Pro, Arg143Gln, Phe135Cys, and Pro239Ser. These
mutations are similar to the Pit-1 mutation in the Snell
dwarf mouse where a tryptophan at residue 261 is replaced with
cysteine. A tryptophan at this position is critical for DNA binding and
conserved in all POU-H domains. Other mutations, which occur both
familially and in sporadic cases, are inherited as dominant traits and
therefore clinically evident in heterozygous individuals. These
mutations, like Arg271Trp, Pro14Leu, and Pro24Leu, tend to retain
DNA-binding ability but somehow dominantly interfere with the function
of wild-type Pit-1 transcribed from the other allele.
The Ala158Pro Pit-1 mutation, affecting the second helix of
the POU-S domain (Fig. 7), was described
in two homozygous Dutch families with severe GH and PRL deficiency but
mild hypothyroidism (358). An NMR study showed normal size anterior
pituitary glands in the two affected patients (358). The Ala158Pro
protein binds normally to cognate DNA sites but is transcriptionally
inactive on the GH, PRL, and Pit-1 promoters. Apparently, this region
of the Pit-1 molecule is crucial for transactivation, either directly
or through recruitment of coactivators.
|
A homozygous Pit-1 mutation, Pro239Ser, is located at the
beginning of the second -helix of the POU-H domain, in a position
that is strictly conserved among all POU proteins. The Pro239Ser
protein has been reported to bind to DNA, but fails to activate the GH
promoter. This mutation has been reported in seven individuals from
three unrelated Middle Eastern families (360).
An isolated case has been described in which a patient harbored
compound heterozygous mutations (361). One mutation, Arg172Stop (Fig. 7), results in a protein lacking part of the POU-S and all of the POU-H
domains, predicting a complete loss of function. The other mutation,
Glu174Gly, affects the DNA-recognition helix of the POU-S domain. This
protein is reported to have drastically decreased affinity for DNA
(361). The Arg172Stop mutation has also been described in a homozygous
form (362).
Other human Pit-1 mutations have been described, but not
extensively characterized. These include a homozygous mutation,
Arg143Gln (Fig. 7), found in a child of consanguineous parents. This
mutation is located within the first helix of the POU-S domain and
presumably disrupts a basic region important for DNA binding (363).
Glu250Stop (Fig. 7) has been described in a single homozygous patient
and would delete the DNA-recognition helix of the POU-H domain of
Pit-1, thus interfering with DNA binding (364).
A heterozygous Arg271Trp mutation (Fig. 7), found both in sporadic and
familial cases, seems to be a common cause of Pit-1 dysfunction, with
at least 11 cases reported in unrelated families of diverse ethnic
backgrounds (357, 363, 365, 366, 367, 368, 369). The Arg271Trp mutant protein, which
is altered in the basic region of the DNA-recognition helix of the
homeodomain, binds DNA with normal or increased affinity but fails to
stimulate transcription from Pit-1-responsive promoters and acts as a
dominant repressor of transcription (366). One patient was described to
have abnormal facial features with prominent forehead, marked midfacial
hypoplasia with depressed nasal bridge, deep-set eyes, and a short nose
with anteverted nostrils (365). These changes, however, are probably
not specific for Pit-1 mutations because similar facial
features, sometimes found in GH deficiency of other etiology, become
less prominent with GH therapy. Interestingly, within a family some
individuals harboring the Arg271Trp mutation may be clinically normal,
possibly due to preferential expression from the normal
Pit-1 allele (367).
Other less well characterized dominant mutations include Pro14Leu and Pro24Leu, which are located within the transactivation domain of Pit-1 and found in heterozygous individuals (363, 370, 371).
An unusual heterozygous mutation, Lys216Glu, was described in a single patient. This mutation has no effect on DNA binding nor does it directly interfere with GH or PRL gene expression. Instead it blocks RA induction of Pit-1 gene expression. Cohen et al. (372) suggest that this mutation interferes with RA-induced activation of the Pit-1 gene during a crucial period of development (372). This mutation affects the C-terminal part of the linker between the POU-S and POU-H domains and therefore may disrupt important protein-protein interactions between Pit-1 and other transcription factors (372).
The Prop1 (Prophet of Pit-1) gene acts upstream of Pit-1 in the pituitary developmental pathway as evidenced by the failure of effective Pit-1 gene activation in the Prop-1 defective Ames mouse (289, 373). However, mutations in the human Prop1 gene lead to a CPHD in humans that is different in several respects from CPHD caused by Pit-1 mutations (353, 355). First, the clinical presentation is more variable and while all patients ultimately develop GH, PRL, TSH, and gonadotropin deficiency, the age of diagnosis varies significantly. Apparently, Prop1 mutated individuals can have remnant secretion of GH and PRL. Second, gonadotropin deficiency, which is not found in Pit-1- mutated individuals, is common in Prop1-mutated patients. Third, one family with a Prop1 deletion has been found to have ACTH deficiency (374). Fourth, while most Pit-1 and Prop1 mutated individuals will have small anterior pituitary glands on NMR examination, occasionally Prop1 patients develop parasellar tissue masses that regress over time.
Thus, whereas the phenotypes resulting from mutations in the human Pit-1 gene seem to mirror quite well the phenotype of the Pit-1-mutated Snell dwarf mouse, mutations in the human Prop1 gene can lead to a phenotype distinct from the Prop1-mutated Ames dwarf mouse. This observation has been interpreted that, in contrast to human disease mutations, the Prop1 mutation in the Ames dwarf is a hypomorph, leading to a partial loss of function. Regardless of the explanation, these data suggest that in humans, the Prop1 gene plays a role in the differentiation of at least gonadotropes, in addition to acting upstream of Pit-1 in generating the Pit-1-dependent cell lineages.
B. Brn-4 mutations in humans
Brn-4 mutations have been described in humans where
they cause an X-linked nonsyndromic mixed deafness disorder referred to
as DFN3 (343, 375). As in the Brn-4 (-/-) mouse, no
metabolic changes have been reported in these individuals, but specific
metabolic tests might be needed to detect subtle differences.
C. Potential implications for other diseases
1. Pituitary adenomas. Several studies on the expression of
Pit-1 in pituitary tumors have been published, showing Pit-1 commonly
expressed in GH-, PRL-, and TSH-secreting tumors (376, 377, 378, 379, 380, 381, 382, 383, 384). No
major abnormalities in the amount or form of Pit-1 transcripts have
been observed in these tumors, and Pit-1 mRNA levels seem to be similar
in somatotrope and lactotrope adenomas (382, 385). In addition, Pit-1
is expressed in many clinically nonfunctioning pituitary adenomas, but
in most of those, mRNAs for GH, PRL, or TSH were also found (381).
Pit-1 transcripts have also been found in ACTH-secreting tumors, again
with expression seeming to correlate with GH-, PRL-, or
TSH-expressing cells found in these tumors (381). Since Pit-1 has
been implicated in proliferation of certain pituitary cell types during
development, it is reasonable to propose that Pit-1 is at least a
component in the pathways leading to the formation of these tumors.
Tuberoinfundibular dopamine, acting through dopamine D2 receptors, tonically inhibits PRL synthesis and secretion from lactotropes. As previously described, the dopamine signal may ultimately be transmitted through Pit-1 action on the regulatory region of the PRL gene (386, 387). Female mice deleted for the dopamine D2 receptor develop lactotroph hyperplasia and prolactinomas. Male mice deleted for the dopamine D2 receptor develop prolactinomas without any accompanying hyperplasia. In each case, these adenomas showed both Pit-1 and ER expression, suggesting that Pit-1 may participate in the pathway ultimately leading to experimental prolactinomas (388). In humans there seems to be a correlation between D2 receptor mRNA and Pit-1 mRNA levels in prolactinomas (382, 389).
Activating mutations in the G protein (S) subunit, the
gsp oncogene, may be responsible for as many as 40% of
somatotropinomas (389). In transient transfection assays, a
constitutively active (S) subunit stimulates expression from a
reporter construct under the control of the proximal 200 bp of the
Pit-1 promoter. Full induction seems to depend on Pit-1 and CREB, which
both bind to this region (390). Thus the overactivity of protein kinase
A in this system may increase Pit-1 levels. In addition, the
constitutive activation of this pathway may directly stimulate target
genes such as the GH and PRL promoters (391). As previously described,
this action seems to be mediated through Pit-1 binding sites in the GH
gene, perhaps by direct interaction of CBP with Pit-1 (153, 303).
While there is evidence implicating Pit-1 as a target of signaling pathways that lead to pituitary adenoma formation in humans and mice (392), no specific abnormalities in Pit-1 expression or function implying a more causative role have been associated with the genesis of human pituitary tumors. One could hypothesize that putative activating mutations of Pit-1 may contribute to hormone-secreting tumor formation. However, even if Pit-1 abnormalities do not cause tumor formation, understanding Pit-1 function may help in designing strategies to control the secretion and proliferation of pituitary tumors of the somatomammotrope lineage.
2. Developmental defects. Septo-optic dysplasias, characterized by optic nerve hypoplasias, pituitary gland hypoplasias, and other midline abnormalities of the CNS, such as absence of the corpus callosum and septum pellucidum, are most likely caused by mutations in genes that act earlier than POU domain factors in the development of the hypothalamic/pituitary axes. Indeed, at least some cases are caused by a mutation in the paired like homeobox gene, Rpx/Hesx1, which acts early in pituitary development (393, 394).
3. Hypothalamic disease. It is clear that the hypothalamus is important for several symptoms and diseases that fall outside classic neuroendocrine disorders. These include psychiatric diseases such as depression (395), disturbances in the biological clock (396), and obesity and other eating disorders (397). So far no such abnormalities have been demonstrated in mice deleted for POU factors highly expressed in the hypothalamus.
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Acknowledgments |
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Footnotes |
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1 Supported by NIH Grants AR-044882 and AR-02080 and the Irving
Weinstein Foundation. 
2 Investigator with the Howard Hughes Medical Institute.
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