Hairy and Transcriptional Repression
The early development of the Drosophila embryo is marked by its progressive subdivision into increasingly precise spatial domains. This subdivision is achieved through the actions of a hierarchy of maternal and zygotic segmentation genes, many of which encode transcription factors that both positively and negatively regulate the expression of other transcription factors.
We are particularly interested in the regulation and function of the pair-rule genes, whose correct expression underlies the establishment of metameric pattern in Drosophila. Our work is focused on the pair-rule segmentation gene, hairy (h), that is needed for proper embryonic segmentation. Hairy expression in stripes during blastoderm cellularization serves to establish the reiterated pattern of parasegmental units that represent the basic embryonic body plan. hairy behaves genetically as a negative regulator of a downstream pair-rule gene, fushi tarazu (ftz), during embryonic segmentation. Consistent with Hairy's role as a primary repressor of ftz expression, ftz stripes are expanded in hairy mutant embryos.
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| hairy mutant phenotypes. Hairy is expressed in seven transverse segment-wide stripes in the cellular blastoderm, defining a double segment periodicity. Hairy is also expressed in an antero-dorsal head patch (right). This expression is required to repress Ftz expression, leading to seven transverse Ftz stripes in the opposite parasegments. In hairy mutant embryos, Ftz expression is broadened leading to the loss of alternate segments. The resulting larval cuticle shows a classical pair-rule phenotype with only half the number of segments remaining (left). |
Similar to its role in segmentation, hairy is required for the establishment
of adult bristle pattern during the larval/pupal stages. In this case, hairy
behaves genetically as a negative regulator of achaete bHLH gene (AS-C
T5). hairy is required to suppress ectopic bristle production on various
adult cuticular structures including the wing and the notum. More recently,
hairy has also been shown to play a role in eye development, where it is expressed
ahead of the morphogenetic furrow in the eye imaginal disc and affects furrow
progression when removed in conjunction with another HLH repressor protein,
Extramacrochaetae (Emc). In this case, Hairy does not appear to affect cell
fate as it does during segmentation, but rather appears to affect the rate at
which cells exit from the cell cycle and begin differentiation.
hairy encodes a nuclear protein with a helix-loop-helix domain. hairy belongs to a subclass of repressor bHLH proteins that include the structurally related Drosophila proteins encoded by deadpan (dpn), and seven members of the Enhancer of split complex [E(spl)-C; E(spl)m3, -m5, -m7, -m8, -mß, -mg, -md;], as well as several vertebrate homologs. These proteins are genetically required throughout development as transcriptional repressors of genes necessary for processes such as sex-determination, segmentation, and neurogenesis. Members of the Hairy/E(spl) class share several regions of homology.
They have a conserved HLH domain, required for protein dimerization, that is
preceded by a conserned basic region required for DNA binding with specificity
for N-box sequences (CACNAG). The Hairy/E(spl) proteins are also characterized
by two other conserved domains: the Orange domain that mediates functional specificity
among Hairy/E(spl) family members, and the C-terminal conserved WRPW tetrapeptide
that is necessary and sufficient for the recruitment of Groucho, a WD-repeat
containing protein that is not able to bind DNA on its own, but when brought
to an endogenous or heterologous promoter serves as a strong repressor of transcription.
The Hairy/E(spl) family of bHLH proteins appears to function as dedicated repressors.
Four major models for transcriptional repression mechanisms have been proposed:
1) repressors prevent activators from binding DNA ("competition"); 2) repressors
and activators bind to DNA at independent sites, but the repressors interfere
with interaction between the activators and the general transcriptional machinery
("quenching"); 3) repressors and activators bind to DNA at independent sites,
with the repressors interacting (directly) with the general transcriptional
machinery ("direct repression"); and 4) repressors and activators bind to DNA
at independent sites, with repressors recruiting chromatin-modifying enzymes
("chromatin remodeling").
Existing evidence makes the "competition" model unlikely for Hairy: In particular,
Hairy-binding N boxes are physically separate from the activator-binding E boxes
at target promoters. Identification of the Groucho co-repressor led to the view
that Hairy functions as a promoter-bound repressor: an intact bHLH region is
required for Hairy to bind to specific DNA sites where it then recruits the
Groucho co-repressor protein. For the Groucho-like yeast co-repressor protein
Tup1, experimental evidence supports each of three remaining models: the Tup1/Ssn6-complex
blocks the activation domain of specific DNA binding proteins, interacts with
the general transcriptional machinery, and organizes a repressive chromatin
structure through direct interaction with the N-terminal regions of histones
H3 and H4. Groucho has recently been shown to interact specifically with the
N-terminal region of histone H3 and has been shown genetically to be essential
for H3 transcriptional silencing in yeast, suggesting a repression mechanism
involving chromatin remodeling. In flies, Groucho has been reported to recruit
Rpd3, a class I histone deacetylase (HDAC), suggesting a mechanism involving
chromatin remodeling.
Recruitment of Groucho, however, does not account for all of Hairy's repressor
properties. We find that Hairy can function genetically as a repressor in the
absence of the WRPW motif, and presumably the Groucho co-repressor.
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| Hairy has been proposed to function as a sequence-specific DNA binding protein recruits the Groucho co-repressor protein. Groucho, in turn, has been proposed to recruit Rpd3, a class I histone deacetylase, suggesting a chromatin mechanism***(left). Groucho is required for Hairy-mediated repression: reducing Groucho activity results in de-repression (broadening) of Ftz stripes (left). Hairy can also repress transcription in the absence of Groucho, presumably through a number of chromatin independent mechanisms (right). |
Despite the genetic and molecular characterization to date, it has proved difficult to define the precise molecular mechanisms of Hairy action during segmentation. Mapping of Hairy has shown that there are additional protein domains and interacting factors required for proper Hairy function. It is important to know the scope of interactions that can occur among these proteins and the identity of these proteins to sort out what happens in vivo to achieve transcriptional repression.
Some Hairy co-factors:
dCtBP. dCtBP is required as a co-factor by a number of early developmental repression systems where it functions in a context dependent manner. dCtBP can function as either a co-activator or co-repressor of transcription, with distinct regions of dCtBP being required for activation or repression. While dCtBP is required for Hairy-mediated repression, reduction of maternal dCtBP activity suppresses the hairy phenotype. Several co-repressors including Sin3A and Groucho have been shown to recruit complexes containing histone deacetylase (HDAC), an enzyme proposed to mediate transcriptional repression by remodeling chromatin. dCtBP does not appear to recruit HDACs. However, we find that dCtBP interferes with the formation of mSin3A/HDAC complexes, and consequently Sin3A-mediated transcriptional repression. We hypothesized that when bound to Hairy, dCtBP may modulate the ability of Groucho to recruit HDAC. Consistent with this, dCtBP interferes with Groucho-mediated repression in vivo and in vitro. dCtBP's modulation of Groucho's repression activity may be a crucial form of regulation in the early embryo, a closed system dependent on maternally provided and ubiquitously distributed proteins.
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| Model for CtBP (top) and dCtBP (bottom) action. CtBP binds to the C-terminus of E1A and modulates the binding of factors to conserved region 1 (CR1). DCtBP may function similarly: it binds to the C-terminus of Hairy and may modulate the binding or activity of factors interacting through other domains of Hairy |
dCtBP is encoded by a complex locus encompassing at least three distinct genetic complementation groups: mesA, mesB, and 87De. Alleles from these different complementation groups exhibit distinct as well as overlapping phenotypes. There are 4 major RNA isoforms of dCtBP that differ in their 3' ends - resulting in proteins that differ by 10 to 110 amino acids. Our working model is that the different dCtBP complementation groups remove distinct subsets of dCtBP transcripts. We are currently identifying the molecular lesion(s) associated with each allele in order to correlate these with the phenotypes observed. We have also examined the cellular and subcellular localization of dCtBP using polyclonal antibodies we generated to the common region of this protein. dCtBP is dynamically expressed during the cell cycle and may play a role in regulating nuclear divisions and cellularization. Our current genetic and molecular analysis of the dCtBP locus is crucial to sorting out the roles it plays in these various processes, particularly as multiple early developmental regulatory proteins recruit dCtBP.
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dSir2. Sir2 is a NAD+-dependent histone deacetylase required in yeast for heterochromatic silencing at telomeres, rDNA, and mating type loci. The Drosophila homolog of Sir2 (dSir2) also possesses deacetylase activity and is required for heterochromatic silencing, but unlike ySir2, is not required for silencing at telomeres. While ySIR2 appears to act as a dedicated heterochromatic silencing factor, we find that dSir2 also plays a role in euchromatic repression by interacting with members of the Hairy/Deadpan/E(Spl) family of bHLH repressors that function in processes including segmentation, neurogenesis and sex determination. |
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| dSir2 is required for heterochromatic Position Effect Variegation (PEV). The w+ gene inserted within heterochromatin is silenced (mostly white pigment in eye at left). Reduction of dSir2 rescues this silencing (increased red pigment in eye at right). |
This interaction maps to Hairy's basic domain, a new region for Hairy co-factor
binding. While the basic region is highly similar among members of the Hairy/Deadpan/E(Spl)
family, dSir2 binds to only a subset of the family members, suggesting that
there are additional recognition features within the proteins. dSir2 exhibits a
dominant genetic interaction with hairy, resulting in derepression of
Ftz expression. Our results indicate that Sir2 in higher organisms plays a
role in both euchromatic repression and heterochromatic silencing in a variety
of cellular and developmental processes.
dDrap (NC2a). dDrap is homologous to Dr1-Associated Protein (Drap) or
NC2a, a factor implicated as a negative regulator
of the basal transcription machinery. In yeast and humans, DRAP (NC2a)
has been shown to act as a heterodimer with Dr1 (NC2a)
to globally repress transcription. The transcription of nearly all protein-encoding
genes requires the assembly of the general transcription factors (GTFs) and
RNA polymerase II on class II promoters. The Dr1-DRAP heterodimer (NC2) directly
inhibits the activity of the GTFs in a chromatin-independent manner by interfering
with the interactions between TATA-binding protein (TBP) and TFIIA or TFIIB.
In GST pull down assays, dDRAP and dDr1 interact with one another, dDr1 binds
Drosophila TBP, and dDRAP interacts with Hairy. Additionally, we find
that reducing the dose of either dTBP or dTFIIA maternally leads to enhancement
of the hairy mutant phenotype. Our work suggests that one mechanism of
Hairy repression involves the recruitment of dDRAP, which then acts as a co-repressor
with dDr1 to negatively regulate the basal transcription machinery.
Identifying Hairy targets. We have known for some time that the segmentation
gene, ftz, is Hairy's genetic target, but the mechanism by which Hairy
represses ftz remains elusive. While it has long been assumed that Hairy is
a DNA binding protein that binds to the ftz promoter, such binding has
not been demonstrated. We are using the new chromatin profiling technique developed
by van Steensel and Henikoff to identify additional Hairy targets. E.coli
DNA adenine methyltransferase (Dam) tethered to Hairy leads to specific methylation
of DNA adjacent to Hairy binding sites. This modified DNA can be isolated, labeled
and hybridized to microarray chips to reveal clones containing Hairy binding
sites. We are using this technique in cells and in embryos to identify Hairy,
dCtBP, Groucho, and dSir2 target genes, as well as to examine the relative requirements
of these proteins at different target promoters.
Our present goal is to use developmental, genetic and molecular approaches to examine the properties of Hairy and several of its interacting proteins (dCtBP, dSir2, dNC2, dTopors, and hn13) to help distinguish amongst the possible regulatory mechanisms used by Hairy to mediate transcriptional repression.
