hairy
Hairy, Deadpan and E(SPL) proteins have three evolutionarily conserved domains required for their function: the bHLH, Orange, and WRPW domains. However, the suppression of Scute activity by Hairy does not require the WRPW domain. The Orange domain is an important functional domain that confers specificity among members of the Hairy/E(SPL) family. A Xenopus Hairy homology conserves not only
Hairy's structure but also its biological activity. Transcriptional repression by the Hairy/E(SPL) family of bHLH proteins involves two separable mechanisms: repression of specific transcriptional activators, such as Scute, through the bHLH and Orange domains and repression of other activators via interaction of the C-terminal WRPW motif with corepressors, such as the Groucho protein (Dawson, 1995).
Hairy-related proteins are site-specific DNA-binding proteins defined by the presence of both a repressor-specific bHLH DNA binding domain and a carboxyl-terminal WRPW (Trp-Arg-Pro-Trp) motif. These proteins act as repressors by binding to DNA sites in target gene promoters and not by interfering with activator proteins, indicating that these proteins are active repressors that should therefore have specific repression domains. The WRPW motif is a functional transcriptional repression domain sufficient to confer active repression to Hairy-related proteins or a heterologous DNA-binding protein, Ga14. The WRPW motif is sufficient to recruit Groucho or the TLE mammalian homologs to target gene promoters. Groucho and TLE proteins actively repress transcription when directly bound to a target gene promoter. Thus Groucho family proteins are active transcriptional corepressors for Hairy-related proteins and are recruited by the 4-amino acid protein-protein interaction domain, WRPW (Fisher, 1996).
The repression by Groucho and Hairy-like proteins requires the S/P (Ser-Pro-rich) domain of Groucho and the conserved carboxl-terminal Trp-Arg-Pro-Trp (WRPW) sequence of the Hairy-like transcription factor for direct interactions between Groucho and Hairy-like proteins. In addition to the S/P domain, a WD40 motif is found in the C-terminal half of Groucho. The WD40 motif is a loosely conserved repeat of 40 amino acids, separated by a Trp-Asp dipeptide sequence. The other sequence recognized in the Drosophila protein is a CcN motif, containing Cdc3 and casein kinase sites and a possible nuclear localization signal. A glutamine-rich region, the Q domain, is conserved as well in mammalian Groucho homologs. Both the Q domain and the WD40 domain are highly conserved, suggesting that they perform a highly conserved function. The S/P domain is less well conserved, implying that each Groucho family member binds preferentially to a particular Hairy-like transcription factor. Groucho-related genes in mammals also include one gene that encodes a truncated homolog of Drosophila, containing only the Q domain and part of the S/P sequence. The short protein may act to regulate the activity of the longer protein. The Q domain is used for dimerization between Groucho family members. Groucho proteins are able to dimerize through the Q domain; that dimerization requires a core of 50 amino acids. Surprising, the dimerization does not require the leucine zipper located within the Q domain. It is suggested that the ability of Groucho proteins to dimerize is conserved, and that the presence of truncated Groucho proteins in mammals provides yet an additional level of transcriptional regulation in mammalian systems (Pinto, 1996).
Runt domain family members are defined based on the presence of the 128-amino-acid Runt domain, which is necessary and sufficient for sequence-specific DNA binding. There exists an evolutionarily conserved protein-protein interaction between Runt domain proteins and the corepressor Groucho. However, the interaction is independent of the Runt domain and can be mapped to a 5-amino-acid sequence, VWRPY, present at the C terminus of all Runt domain proteins. Drosophila melanogaster Runt and Groucho interact genetically; the in vivo repression of a subset of Runt-regulated genes is dependent on the interaction with Groucho and is sensitive to Groucho dosage. Runt's repression of one gene, engrailed, is independent of VWRPY and Groucho, thus demonstrating alternative mechanisms for repression by Runt domain proteins (Aronson, 1997).
Unlike other transcriptional regulatory proteins that interact with Groucho, Runt domain proteins are known to activate transcription. The distinction between the Runt domain consensus (VWRPY) and the Hairy-related/HES consensus (WRPW) raises a question: are the C-termini of these families interchangeable? The ability of Runt domain proteins to activate transcription suggests that the interaction with Groucho is regulated: when Runt domain proteins assemble on a promoter that is to be activated, Groucho must either be absent or in a context where it cannot exert its repressive effects. The difference between the Groucho-recruiting C-termini of the Hairy-related/HES family and the Runt domain family may be the difference between a constitutive Groucho interaction and one that is regulated (Aronson, 1997).
A transgenic embryo assay was employed to discover the mode of repression mediated by Hairy. Hairy can act as a dominant repressor capable of functioning over long distances to block multiple enhancers. Hairy is shown to repress a heterologous enhancer, the Rhomboid NEE, when bound 1 kb from the nearest upstream activator. The binding of Hairy to a modified NEE leads to the repression of both the NEE and a distantly linked mesoderm-specific enhancer with a synthetic modular promoter. Two models are proposed for Hairy's long distance repressive function. (1) Hairy could recruit a cofactor that mediates repression at a distance. This factor would inhibit specific upstream activators bound within the proximal promoter. (2) Hairy could interact directly with one or more components of the basal transcriptional complex. Perhaps Hairy binds TFIIE with a high affinity, thereby resulting in a general silencing of the promoter. Additional evidence that Hairy is distinct from previously characterized embryonic repressors (Krüppel and Knirps function over short distances to quench closely linked upstream activators) stems from the analysis of the gypsy insulator DNA. This insulator selectively blocks the Hairy repressor, but not the linked activators Dorsal and Achaete-Scute, within a modified NEE. The linked activators are unaffected by Hairy and continue to direct expression across the gypsy insulator (Barolo, 1997).
How then does Hairy function? Hairy has been shown to interact with the co-repressor protein Groucho through the C-terminal WRPW motif. Gro is not known to bind DNA, but fusions of GRO with heterologous DNA binding domains have revealed that GRO can act as a transcriptional repressor. The Gro protein contains several repeats of a 40-residue motif, termed the WD40 repeat, that is thought to mediate protein-protein interactions. Tup1, a yeast corepressor protein that also contains WD40 repeats, is recruited to DNA by the alpha2 repressor in alpha-type cells for the silencing of alpha-specific genes. Similarly, Hairy may recruit Gro for silencing specific genes in the Drosophila embryo. The yeast mating-type repressors alpha2 and Tup1 have been reported to interact with histones. This observation raises the possibility that Gro mediates transcriptional silencing by influencing chromatin structure (Barolo, 1997)
hairy is a Drosophila pair-rule segmentation gene that functions genetically as a repressor. To isolate protein components of Hairy-mediated repression, a yeast interaction screen was carried out and a Hairy-interacting protein was identified, the Drosophila homolog of the human C-terminal-binding protein (CtBP). Human CtBP is a cellular phosphoprotein that interacts with the C-terminus of the adenovirus E1a oncoprotein and functions as a tumor suppressor. Drosophila To target protein interactions with specific conserved regions of the Hairy protein, a two-hybrid screen was carried out using a LexA-tagged Hairy partial protein bait. This strategy also allowed the circumvention of the reporter system repression that was encountered when using full-length Hairy protein as a bait. A VP16-tagged Drosophila library constructed from 0-4 h embryonic mRNAs was screened with a bait that encodes the Hairy Orange domain through to the C-terminus (h-C, amino acids 93-343. In directed yeast two-hybrid assays, this fragment, h-C28, interacts weakly with full-length Hairy, but more strongly with Hairy partial proteins and with E(spl)m, another member of Hairy-class bHLH proteins. It does not interact with Dpn and interacts poorly, if at all, with E(spl)m3, -m5, -m8, -mbeta -mgamma. h-C28 does not show interaction with proteins from other HLH classes (i.e. Scute, Emc). The region of Hairy required for interaction with h-C28 was mapped using a series of Hairy deletions and partial proteins fused to LexA. h-C28 interacts strongly with a 25 amino sequence immediately upstream of, but not including, the C-terminal WRPW motif. This identifies a previously undefined protein interaction domain within Hairy. dCtBP also interacts with itself (Poortinga, 1998).
Yeast SIR2 (Silent Information Regulator 2) is a nicotinamide adenine dinucleotide (NAD)+-dependent histone deacetylase required for heterochromatic silencing at telomeres, rDNA, and mating-type loci. The Drosophila Sir2 also encodes deacetylase activity and is required for heterochromatic silencing, but unlike ySir2, is not required for silencing at telomeres. Drosophila HREF="../polycomb/sir2-1.htm">Sir2 To identify the earliest stage at which segmentation is affected, the expression of genes at different tiers of the segmentation gene hierarchy were examined. Protein expression patterns of the gap genes Krüppel and knirps are unaffected in progeny from females with reduced maternal Sir2 (Sir205327/+;wimp/+ or Sir2ex10/+;wimp/+ transheterozygous mothers). Sir2 is first required for regulation of segmentation at the level of pair rule gene expression. Pair rule genes can be separated into two classes: primary pair rule genes establish double segment periodicity, whereas secondary pair rule genes respond to this pattern. Pair rule gene products are expressed as a series of seven transverse stripes in wild-type or wimp/+ embryos. Stripes of the secondary pair rule gene fushi tarazu (ftz) are severely derepressed (stripes are broadened) in embryos from mothers with reduced Sir2 expression. Aberrant regulation of Ftz stripe expression in Sir2 mutant embryos is consistent with reduced function of the primary pair rule gene, Hairy, which behaves genetically as a repressor of ftz. Hairy expression was examined in Sir2 mutant embryos: in contrast to Ftz expression, which is significantly altered, Hairy is largely unaffected in these embryos (Rosenberg, 2002).
The Ftz derepression phenotype in Sir2 embryos is reminiscent of the Ftz expression pattern seen in hairy mutants. Sir2 was examined for genetic interaction with hairy; these mutations exhibit a dominant genetic interaction. Progeny from either hairy heterozygous mothers or Sir2 heterozygous mothers mated to wild-type males are viable and exhibit wild-type cuticle phenotypes. In contrast, embryos derived from mothers heterozygous for both Sir2 and hairy (Sir2/+; hairy/+ trans-heterozygous mothers) mated to wild-type males exhibit moderate to severe cuticle abnormalities. Consistent with this segmentation cuticle phenotype, Ftz is derepressed in these embryos, with a reduction in expression of stripes 4, 6, and 7, suggesting that these segmentation defects are largely mediated by interaction of Sir2 with Hairy. Interestingly, Hairy stripes 3 and 4 are also affected in progeny from mothers trans-heterozygous for Sir2 and hairy (Sir2/+; hairy/+ females), suggesting interactions between Sir2 and other developmental regulators. Sir2 was tested for interaction with repression cofactors groucho (gro) and dCtBP, as well as the other primary pair rule genes, even skipped (eve), and runt (run). No dominant synthetic lethal interactions were detected between Sir2 and any of these mutations. Hairy was tested for genetic interaction with the class I HDAC, Rpd3, which has been proposed to be recruited to Hairy via the corepressor Groucho. However, no genetic interaction was detected between Rpd3 and hairy (Rosenberg, 2002).
To test whether Sir2 interacts with Hairy directly, GST pull-down assays were performed using in vitro translated (IVT-) Sir2. Consistent with the genetic results, IVT-Sir2 does not bind to Groucho or dCtBP or to GST alone. However, it does bind specifically to a full-length GST-Hairy fusion protein. To map the region of Hairy required for this interaction, a series of Hairy protein fragments fused in frame to GST were generated. Sir2 binds to all fragments containing the Hairy basic domain, indicating that this domain is sufficient for binding. A series of small basic domain deletions were generated within the context of full-length Hairy protein to identify the smallest region required for Sir2 binding. One of these deletions, DeltaRRAR, disrupts Sir2 binding, while adjacent four amino acid deletions have no effect. The Hairy DeltaRRAR mutation does not affect Hairy homodimerization or binding to other Hairy-interacting proteins, including dCtBP (Rosenberg, 2002).
The basic domain is highly conserved among HES family proteins, including the invariant RRAR residues, so Sir2 was assayed for binding to other bHLH proteins within this family by GST pull-down. IVT-Sir2 binds to GST-Deadpan (Dpn), but, surprisingly, not efficiently to GST-fusions to the E(Spl)m3 and E(Spl)m8 members of the HES family, suggesting that Sir2 may recognize additional features within the basic domain or in distal regions of HES proteins to permit interaction with a specific subset of these similar proteins in a variety of developmental processes (Rosenberg, 2002).
One possible consequence of cofactor binding to the basic domain of HES proteins could be interference with their DNA binding abilities. Since Sir2 is required for Hairy function but binds to the basic domain, a gel electrophoretic mobility shift assay (EMSA) was used to test whether Sir2 and Hairy could be detected in a stable complex on DNA. Both full-length-Hairy and a fragment containing the bHLH domain of Hairy are able to efficiently shift 32P-labeled N-box probe and are competed by cold wild-type competitor. No complex with altered mobility was detected upon addition of Sir2, although these proteins are able to interact in vitro. Addition of Sir2 to Hairy either before or after incubation of Hairy with DNA does not prevent Hairy from binding to target DNA, suggesting that Sir2 does not interfere with Hairy binding to DNA and that there may be other consequences of Sir2 binding to Hairy within this region (Rosenberg, 2002).
In common with several transcription units of the E(spl)-C, including E(spl)m4, Bearded contains two novel heptanucleotide sequence motifs in its 3' untranslated region (UTR), suggesting that all these genes are subject to a previously un-recognized mode of post-transcriptional regulation. These sequence motifs are called the Brd box (AGCTTTA) and the GY box (GTCTTCC). Like known sequence elements that function in post-transcriptional regulation, both of these motifs are found in a single orientation and specifically in the UTRs of the genes that include them. Many mRNAs are translationally inactive until they undergo additional cytoplasmic polyadenylation, a process controlled by cytoplasmic polyadenylation elements (CPEs). Polyadenylation is implicated in Brd box function. Negative regulation by the Brd box motif affects steady-state levels of both RNA and protein. This result indicates that Brd boxes have an additional role in regulating translation, beyond the effect attributable to transcript level differences. Thus, the Brd 3' UTR confers negative regulatory activity in vivo. This activity is spatially and temporally general, in that most or all cells are able to respond to Brd boxes. This suggests that some genes expressed outside of proneural clusters may be regulated by these motifs as well. Three other genes that encode negative regulators of PNS development also contain these sequences in their 3' UTRs. In particular, kuzbanian (kuz) and extramacrochaetae (emc) each include single Brd boxes, while hairy contains a GY box. emc also includes four copies of a GY box-related sequence (GTTTTCC) in its 3' UTR, which may be relevant for its regulation. kuz has functions in SOP selection and lateral inhibition, so its expression certainly includes proneural clusters. However, emc and h are expressed in spatial patterns that are largely complementary to proneural clusters in the leg and wing imaginal discs, and are thus possible examples of genes regulated by the Brd box (and possibly the GY box) in territories outside the clusters. Interestingly, the Emc and H proteins, as members of the HLH family, are structurally related to the E(spl)-C bHLH proteins. In contrast, kuz encodes a metalloprotease/disintegrin protein of the ADAM family (Lai, 1997 and references).
The 3' untranslated regions (3' UTRs) of Bearded, hairy, and many genes of the
E(spl)-C contain a novel class of sequence motif, the GY box (GYB, GUCUUCC);
extra macrochaetae contains the variant sequence GUUUUCC. The 3'
UTRs of three proneural genes include a second type of sequence element,
the proneural box (PB, AAUGGAAGACAAU). The full 13 nt PB is found
once each in ac, l'sc, and ato, along with a second, variant version in
both l'sc and ato. The presence of these motifs in such
distantly related
paralogs as hairy and certain bHLH genes of the E(spl)-C (for the GYB), and
ato and two genes of the AS-C (for the PB), indicates that both classes of
sequence
element are subject to strong selection. Furthermore, both the PB and the
GYB are conserved in the orthologs of ac and E(spl)m4 from the distantly
related
Drosophilids D. virilis and D. hydei, respectively, though
these 3' UTRs are otherwise quite divergent from their D. melanogaster
counterparts. These
findings strongly suggest functional roles for both of these sequence elements (Lai, 1998).
Intriguingly, the central 7 nt of the PB and the GYB are exactly
complementary, and are often located within extensive regions of RNA:RNA
duplex predicted to form
between PB- and GYB-containing 3' UTRs. Indeed, using in vitro
assays, RNA duplex formation has been observed between the ato/Brd and ato/m4
3'
UTR pairs that is PB- and GYB-dependent. It is
noteworthy that the predicted duplex interactions involving the GYB of Brd
are significantly
stronger than those involving the GYBs of the other transcripts. For
example, Brd and ato are perfectly complementary over 18 contiguous
nucleotides.
This difference in the degree of PB:GYB-associated complementarity is
likely to have functional consequences (Lai, 1998).
In C. elegans, small antisense RNAs encoded by lin-4 mediate translational
repression of lin-14 and lin-28 transcripts by binding to complementary
sequences in
their 3' UTRs. In Drosophila,
PB- and GYB-bearing transcripts may likewise participate in a regulatory
mechanism
mediated by RNA:RNA duplexes, but with the feature that both partners are
mRNAs that also direct the synthesis of functionally interacting proteins.
The opportunity
to form such duplexes clearly exists, since transcripts from proneural genes
and their regulators very frequently accumulate in coincident or
overlapping patterns. Moreover, while 7 nt is the minimum length of
complementarity between any PB and any GYB, the longest possible
uninterrupted duplex between a
given GYB-bearing transcript and a given proneural partner is almost always
considerably longer (8-12 nt). It is worth noting that in a lin-4/lin-14
duplex that has
been shown to be sufficient for proper regulation in vivo, the longest
region of uninterrupted complementarity is only 7 nt (Lai, 1998 and references therein).
The formation of the postulated RNA duplexes may serve to regulate
proneural gene function, consistent with the known roles of hairy, emc, and
the bHLH genes of
the E(spl)-C. This might explain occasional C-to-U transitions in the GYB
sequence (in emc and D. hydei m4); these variants retain
complementarity with
the PB due to G:U base-pairing. It is equally plausible that GYB-containing
transcripts are regulated by duplex formation. A third very interesting
possibility is that
RNA:RNA duplexes formed between PB- and GYB-containing transcripts function
to initiate a downstream regulatory activity affecting as-yet-unknown
targets. Ample
precedent exists establishing the trans-regulatory potency of
double-stranded RNA. In any
case, the apparent capacity of transcripts from the proneural genes and
their regulators to form duplexes in their 3' UTRs suggests further
complexity in the already
complex regulatory interactions that control Drosophila neurogenesis (Lai, 1998).
C-Terminal binding protein (CtBP) interacts with a highly conserved amino acid motif (PXDLS) at the C terminus of adenovirus early region 1A (AdE1A) protein. This amino acid sequence has recently been demonstrated in the mammalian protein C-terminal interacting protein (CtIP) and a number of Drosophila repressors including Snail, Knirps and Hairy. The structures of synthetic peptides identical to the CtBP binding sites on these proteins have been investigated using NMR spectroscopy. Peptides identical to the CtBP binding site in CtIP and at the N terminus of Snail form a series of beta-turns similar to those seen in AdE1A. The PXDLS motif towards the C terminus of Snail forms an alpha-helix. However, the motifs in Knirps and Hairy did not adopt well-defined structures in TFE/water mixtures as shown by the absence of medium range NOEs and a high proportion of signal overlap. The affinities of peptides for Drosophila and mammalian CtBP were compared using enzyme-linked immunosorbent assay. CtIP, Snail (N-terminal peptide) and Knirps peptides all bind to mammalian CtBP with high affinity [K(i) of 1.04, 1.34 and 0.52 microM, respectively]. However, different effects were observed with dCtBP, most notably the affinity for the Snail (N-terminal peptide) and Knirps peptides are markedly reduced [K(i) of 332 and 56 microM, respectively] whilst the Hairy peptide binds much more strongly [K(i) for dCtBP of 6.22 compared to 133 microM for hCtBP]. In addition peptides containing identical PXDLS motifs but with different N and C terminal sequences have appreciably different affinities for mammalian CtBP and different structures in solution. It is concluded that the factors governing the interactions of CtBPs with partner proteins are more complex than simple possession of the PXDLS motif. In particular the overall secondary structures and amino acid side chains in the binding sites of partner proteins are of importance as well as possible global structural effects in both members of the complex. These data constitute evidence for a multiplicity of CtBPs and partner proteins (Molloy, 2001).
Localization of cytoplasmic messenger RNA transcripts is widely used to target proteins within cells. For many transcripts, localization depends on cis-acting elements within the transcripts and on microtubule-based motors; however, little is known about other components of the transport machinery or how these components recognize specific RNA cargoes. In Drosophila the
same machinery and RNA signals drive specific accumulation of maternal RNAs in
the early oocyte and apical transcript localization in blastoderm embryos. It has been demonstrated in vivo that Egalitarian (Egl) and Bicaudal D (BicD), maternal proteins required for oocyte determination, are selectively recruited by, and co-transported with, localizing transcripts in blastoderm embryos;
interfering with the activities of Egl and BicD blocks apical localization. It is proposed that Egl and BicD are core components of a selective dynein motor
complex that drives transcript localization in a variety of tissues (Bullock, 2001).
Asymmetric RNA localization is evident during zygotic
development, especially in the unicellular syncytial blastoderm
embryo. At this stage, several transcripts including those of the
pair-rule and wingless (wg) segmentation genes lie exclusively
apically of the layer of several thousand peripheral nuclei. Localization of these transcripts seems to be mediated by signals within their
3' untranslated regions (UTRs), and to be driven on microtubules by the minus-end-directed molecular motor, dynein. The linkers and other factors that provide the cargo
specificity are unknown. Nor is it clear if transcript localization in
blastoderm embryos relates to that in other types of cells (Bullock, 2001).
There is a rapid apical localization of fluorescently
labelled fushi tarazu ( ftz) pair-rule transcripts injected into the basal cytoplasm of the cycle 14 blastoderm embryo. Although these
experiments indicated a requirement for nuclear proteins fluorescein, labelling compromizes the structure of the transcripts, and pair-rule
[even-skipped, hairy (h), ftz, paired and runt] and wg transcripts
labelled with several other fluorochromes localize apically within 5-8 min without the need for exogenous protein. Indeed, injected unlabelled transcripts also
localize apically. The protein-free assay
retains specificity for apical transport, since transcripts that are
normally unlocalized [Krüppel (Kr), huckebein] or enriched in the basal cytoplasm (string) are not transported apically
and instead diffuse away from the site of injection (Bullock, 2001).
Blastoderm localization signals can
drive transcript transport during oogenesis. This view is supported
by more detailed analysis of maternally expressed pair-rule transcripts. The injection assay reveals a minimum region between
positions 1,374 and 1,579 in ftz that is necessary and sufficient for
localization in blastoderm embryos. A similar region of ftz seems to be required for localization
of transcripts into the oocyte. Furthermore, h and
runt transcripts, driven maternally by the Hsp70
promoter, also accumulate specifically in the oocyte and later reside
at its anterior cortex, whereas Kr or truncated h transcripts lacking most of the 3' UTR fail to localize either in
blastoderm embryos or during oogenesis (Bullock, 2001).
Whether Egl and BicD are present in early
embryos was examined. Both proteins are supplied maternally to the embryo.
They are noticeably enriched apical to the nuclei at blastoderm
stages where they colocalize with dynein heavy chain (Dhc) -- a component of the motor associated with apical transcript transport. Nevertheless, a large proportion of both of the
proteins is present in the basal cytoplasm (Bullock, 2001).
Whether endogenous Egl and BicD can associate
with injected localizing transcripts, as might be expected if they are
components of the RNA localization machinery, was tested. Injection of h transcripts leads to marked enrichment of Egl and BicD protein
levels at the sites of RNA localization. Similar results are
found on injection of the other tested maternal and zygotic localizing transcripts ( ftz, bcd, grk, K10, nos, osk and w). Both proteins accumulate basally at the site of injection within 1-2 min. Protein recruitment is not inhibited in
embryos preincubated with colcemid, showing that it is not dependent on intact microtubules. Thus, the proteins are
recruited locally before transport and are transported together
apically with transcripts (Bullock, 2001).
Whether BicD and Egl are required for apical
localization in blastoderm embryos was examined. Strong BicD alleles block oogenesis early, and weaker mutant mothers that lay fertilized
eggs (BicDHA40/BicDR26
and BicDH3/BicDR26) retain
sufficient BicD activity for a normal apical distribution of endogenous pair-rule transcripts. However, the
reduced BicD activity in these embryos no longer supports efficient
transport of injected transcripts: 62% of BicDHA40
/BicDR26
and 73% of BicDH3/BicDR26 embryos show no or weak localization 5-8 min
after injection, compared with 10% of wild-type embryos.
Moreover, an antibody against BicD blocks RNA
transport. Preinjection into the basal cytoplasm of anti-BicD antibody 4C2 strongly inhibits the localization of injected h, ftz, grk and stg-K10TLS transcripts in
70%-75% of embryos. The microtubule
cytoskeleton is not obviously affected by the brief (~20 min) antibody treatment, indicating that the effects on RNA transport
are probably direct. Injection of anti-BicD antibody prevents
apical localization of endogenous pair-rule transcripts, also leading to
anteroposterior smearing of their distribution. Thus, apical
transcript localization seems to be important in restricting the range
of activity of pair-rule genes, and allowing their combinatorial
control of Drosophila segmentation (Bullock, 2001).
Injecting blastoderm embryos with anti-Egl also inhibits apical
localization of both exogenous and endogenous pair-rule transcripts, without overtly disrupting the microtubule network. Moreover, its effect is more potent in embryos from mothers containing only a single copy of the egl gene, indicating that the antibody disrupts RNA
localization by inhibiting the activity of Egl. Egl and BicD are
probably also involved in transporting other cargoes. The arrangement of peripheral nuclei is disrupted after injection of antibodies
to either of the two proteins, consistent with data showing
a requirement for BicD in nuclear migration in eye imaginal
disc cells. Embryos injected with either antibody undergo abnormal morphogenesis, which is also indicative of Egl
and BicD transporting additional cargoes (Bullock, 2001).
These results indicate that Egl and BicD are principal elements of a
complex that transports RNA in blastoderm embryos. Egl and
BicD appear to be present as pools of excess cytoplasmic protein
that associate selectively with localizing transcripts and are transported together apically. Protein recruitment occurs before transport and does not require microtubule integrity; rather, transport
depends on Egl and BicD activity. Egl and BicD probably act directly
to mediate RNA transport associated with establishment and maintenance of the oocyte. Thus, mutant transcripts that are
defective in export from nurse cells into the oocyte fail to recruit
Egl or BicD in blastoderm embryos. grk transcripts are also
recognized by the Egl-BicD-microtubule transport pathway,
which is consistent with the hypothesis that nurse cells are a
source of these transcripts for the early oocyte and that they do
not derive exclusively from the oocyte nucleus (Bullock, 2001).
Egl/BicD is enriched at sites of RNA localization in both blastoderm embryos and oocytes, presumably as the consequence of
protein/RNA co-transport. The complex may have an additional
role in anchoring transcripts at their destination. Alternatively,
maintenance of localized transcripts might not depend on an
independent anchorage step, but result from sustained minus-end-directed transport (Bullock, 2001).
Dhc, Egl and BicD have markedly similar distributions during
oogenesis and in blastoderm embryos, and seem to function
together in specifying oocyte identity. It is proposed that an Egl/BicD complex links specific RNAs to dynein and the microtubules. The same machinery may operate elsewhere in
Drosophila. For example, inscuteable transcripts, which localize
asymmetrically in neuroblasts, also localize apically when
injected into blastoderm embryos. Indeed, germline transcripts localize
apically when expressed in follicle cells. Egl and BicD homologs
have been identified in Caenorhabditis elegans and
mammals, and might comprise part of an evolutionarily
conserved cytoskeletal system for transporting transcripts and
other cargoes (Bullock, 2001).
In several Drosophila cell types, mRNA transport depends on microtubules, the molecular motor dynein and trans-acting factors including Egalitarian and Bicaudal-D. However, the molecular basis of transcript recognition by the localization machinery is poorly understood. The features of hairy pair-rule RNA transcripts that mediate their apical localization have been characterized using in vivo injection of fluorescently labelled mRNAs into syncytial blastoderm embryos. A 121-nucleotide element within the 3'-untranslated region (HLE) is necessary and sufficient to mediate apical transport. The signal comprises two essential stem-loop structures, in which double-stranded stems are crucial for localization. Base-pair identities within the stems are not essential, but can contribute to the efficiency of localization, suggesting that specificity is mediated by higher-order structure. Using time-lapse microscopy, the kinetics of localization has been measured; impaired localization of mutant signals is due to delayed formation of active motor complexes and, unexpectedly, to slower movement. These findings, and those from co-injecting wild-type and mutant RNAs, suggest that the efficiency of molecular motors is modulated by the character of their cargoes (Bullock, 2003).
Efficient recognition of the h transcript depends on two stem-loops, SL1 and SL2a; each is necessary for robust localization, but neither is sufficient alone. The mutagenesis data demonstrate that evolutionarily conserved double-stranded stems of SL1 and SL2a are indispensable for proper transcript transport. However, secondary structure of the stems is not the sole determinant of signal activity because transversions that alter base-pair identities lead to inefficient localization. In addition, although many predicted single-stranded regions are inessential for signal activity, a mutant in which all of these bases are removed or altered localizes only weakly. Thus, higher-order RNA structure is also likely to be important for specific recognition by the localization machinery (Bullock, 2003).
Specificity could reside in the tertiary conformation of the RNA, as has been demonstrated for various well-characterized RNA-protein interactions. Such three-dimensional structures would be difficult to infer solely from mutagenesis data, especially because of the large assortment of potential non-canonical interactions between bases (Bullock, 2003).
Intermolecular RNA interactions could also be involved in h mRNA recognition. Transcript oligomerization appears to be important for localization of bcd transcripts to the anterior of the late oocyte/early embryo, although the signals and trans-acting factors driving this transport process seem to be distinct from those acting in Egl/BicD-mediated early export into the oocyte. Studies of mutant h RNA transcripts have not yet revealed evidence that oligomerization is necessary for transport. Mixtures of up to three different transcripts were injected; rescue of a non-localizing mutant RNA signal by a co-injected localizing transcript has never been detected. Nonetheless, time-lapse studies show transport of injected h RNA in particles that contain numerous transcript molecules, although the imaging is not sensitive enough to detect cargoes of individual molecules. Indeed, particle formation is not sufficient to direct formation of an active transport complex: non-localizing mRNAs are also found in particles (Bullock, 2003).
Although K10 and bcd localization signals share no obvious primary sequence similarities with the HLE, they share structural features, suggesting that they are recognized similarly. The K10 localization signal is only 44 nt long and, unlike the HLE, comprises only a single stem-loop region; nonetheless, it recruits Egl and BicD. bcd transcripts also harbor a stem-loop (the 57-nt stem-loop V) that is required for early transport from nurse cells into the oocyte and for apical localization of injected bcd transcripts in the embryo and their association with Egl and BicD. Like h, the activities of both the bcd and K10 stem-loops rely heavily on double-stranded stems in which exact base-pair identities contribute to, but do not determine, efficient localization; base-pair transversions in all the stems can compromise the efficiency of localization. In common with the h SL1, the bcd stem-loop V is not sufficient for localization, but is fully active when dimerized (Bullock, 2003 and references therein).
The apparent complexity and redundancy of the HLE supports a model for signal recognition in which multiple protein-RNA contacts are needed for the formation of a specific, stable complex. In the HLE, weak binding sites for the machinery may be distributed in SL1 and SL2a. Thus, transcripts with two h SL1 domains are at least as active as those with a wild-type HLE. SL2a may provide quantitatively weaker binding signals; it is unable to support any localization either alone or when multimerized. One possibility is that SL1 alone establishes low affinity interactions with the localization machinery, and binds with high affinity together with SL2. The same mode of recognition could also apply for K10, if, unlike h and bcd, the requisite sites are located within a single stem-loop (Bullock, 2003).
Despite the overall similarities of the structural requirements for localization of bcd, K10 and h, no significant shared base-pair identities were identified within essential regions of the signals. The possibility that different transcripts are recognized by distinct RNA-binding factor(s) and recruited to shared components of the machinery cannot be excluded. However, the same localization signals are active in a variety of cell types. Also, stem-loops from different transcripts, each of which is relatively inactive in isolation, can complement to mediate completely efficient localization when combined in the same transcript. Thus, the view is favored that different transcripts share similar higher-order features, such as tertiary RNA conformations of the stems or RNA oligomers, which are recognized by the same factor(s). Multiple RNA motifs per signal and/or RNA or protein oligomerization would lead to the formation of the multiple protein-RNA contacts that confer specificity (Bullock, 2003).
RNA/BicD/Egl association appears to be a prerequisite for transport. BicD is unlikely to bind RNA directly because it lacks a known RNA-interaction domain, but BicD could hetero- or homo-oligomerize via its heptad repeat domains and thereby increase the numbers of protein-RNA contacts. Egl includes a domain with homology to certain 3'-5' exonucleases and a variety of other nucleic acid-interacting proteins, and thus might recognize RNA directly. However, its ability to recognize specific RNA sequences or structures has yet to be demonstrated (Bullock, 2003).
Establishment of segmental pattern in the Drosophila syncytial
blastoderm embryo depends on pair-rule transcriptional regulators. mRNA
transcripts of pair-rule genes localise to the apical cytoplasm of the
blastoderm via a selective dynein-based transport system and signals within
their 3'-untranslated regions. However, the functional and evolutionary
significance of this process remains unknown. Subcellular
localisation of mRNAs from multiple dipteran species has been analyzed both in situ and by injection into Drosophila embryos. Transcript localisation was assayed in four species that can be cultured in the laboratory. Two of them, Episyrphus (Syrphidae) and Megaselia (Phoridae), are cyclorrhaphan flies (i.e. higher dipterans) but, unlike Drosophila, belong to basal branches of this taxon; the other two, Coboldia (Scatopsidae) and Clogmia (Psychodidae), belong to different branches of lower Diptera. Although localisation
of wingless transcripts is conserved in Diptera, localisation of
even-skipped and hairy pair-rule transcripts is
evolutionarily labile and correlates with taxon-specific changes in
positioning of nuclei. In Drosophila localised pair-rule
transcripts target their proteins in close proximity to the nuclei and
increase the reliability of the segmentation process by augmenting gene
activity. These data suggest that mRNA localisation signals in pair-rule
transcripts affect nuclear protein uptake and thereby adjust gene activity to
a variety of dipteran blastoderm cytoarchitectures (Bullock, 2004).
Apical localisation of pair-rule mRNAs in Drosophila syncytial
blastoderm embryos was first noted 20 years ago, but the developmental and
evolutionary significance of this process has remained unclear. Apical pair-rule mRNA localisation is conserved in cyclorrhaphan species that
diverged over 145 million years ago, indicating that this process has a
significant developmental role under natural conditions. Likewise, the
widespread maintenance of wg transcript localisation in Diptera
supports the importance of this process on a phylogenetic scale, even though,
in Drosophila, wg appears to be less sensitive than pair-rule genes
to a reduction in endogenous transcript localisation (Bullock, 2004).
Unlike wg transcripts, pair-rule mRNAs do not localise in some
branches of lower Diptera, and the phylogenetic occurrence of this process
provides interesting insights into its functional significance. Enrichment of
pair-rule transcripts in the apical cytoplasm correlates with the position of
blastoderm nuclei: efficient apical localisation of pair-rule gene transcripts
is found in species which retain an asymmetric apical position of nuclei
throughout the blastoderm stage (Drosophila, Megaselia); less
efficient localisation is seen when the nuclei move from an apical to a more
central position during blastoderm stages (Episyrphus), and no apical
enrichment of transcripts is seen in species where blastoderm nuclei are
surrounded uniformly by a thin layer of cytoplasm (Coboldia,
Clogmia). Localisation signals are also found in several pair-rule
transcripts of the lower dipteran Anopheles. Like Cyclorrhapha, but
unlike many other lower Diptera and most other insects, this culicid species
has evolved a thickened blastoderm with apically positioned nuclei, probably
to allow rapid development as an adaptation to ephemeral larval habitats: columnar cells that emerge from thickened blastoderms can enter gastrulation directly, whereas cuboidal cells that emerge from thin blastoderms still have to
elongate prior to undergoing the requisite cell shape changes (Bullock, 2004).
In Drosophila, pair-rule proteins are enriched in the
apical cytoplasm prior to import into the nuclei in wild-type blastoderms,
whereas they are detected basally in egl mutant embryos, in which
transcript localisation is inefficient. The apical accumulation of pair-rule
proteins under normal circumstances is consistent with the observation that
apical RNA targeting restricts diffusion of cytoplasmic ß-galactosidase.
Apically targeted protein is most likely confined by the cellularisation
process, in which the plasma membrane invaginates between the nuclei and
encloses the apical compartment first (Bullock, 2004).
It has been speculated that mRNA localisation prevents
pair-rule proteins from moving into inter-stripe regions, where they would
cause dominant patterning defects. However, when pair-rule mRNA localisation
is compromised, either by interfering with the localisation machinery or the
RNA signals, no expansion of RNA or protein stripes or ectopic
phenotypic effects are found. Rather, a reduction of pair-rule activity is seen in their
domains of expression in these experiments, indicating that transcript
localisation augments gene function. Pair-rule mRNA localisation does not
appear to be obligatory for protein activity in Drosophila but makes
the segmentation process more reliable: egl mutants, in which
transcripts localise very inefficiently, have a mild increase in segmentation
defects and are acutely sensitive to the reduction of pair-rule gene dose (Bullock, 2004).
By what mechanism does pair-rule mRNA localisation augment the activity of
their transcription factor products? For h it is demonstrated that
suppression of transcript localisation reduces nuclear levels of its protein.
Pair-rule proteins could be specifically modified in the apical cytoplasm, or
localising transcripts could be translated more efficiently. However, given
the diffuse distribution of pair-rule proteins in the basal cytoplasm when RNA
localisation is disrupted in egl mutants and the correlation between
cytoarchitecture and pair-rule transcript localisation in Diptera, a
third possibility is favored, namely that apical mRNA localisation increases nuclear uptake of their proteins by targeting translation in close proximity to the nuclei. Proteins from
non-localising mRNAs would not be available at high levels in the immediate
vicinity of the nuclei, which would result in a decreased nuclear uptake. Such
a role for apical pair-rule mRNA localisation would be redundant in lower
Diptera with only a thin layer of cytoplasm surrounding the nuclei, which
provides little room for diffusion of pair-rule proteins prior to nuclear
import. A mechanism for perinuclear protein targeting might be particularly
significant for nuclear proteins with short half-lives, such as those encoded
by pair-rule genes. Interestingly, localisation of mRNA in the vicinity of the
nucleus to aid import of nuclear proteins has also been reported in cultured
mammalian cells and may be a widespread mechanism to efficiently exploit a
limited pool of transcripts in cells that are polarised or have a high
cytoplasmic:nuclear ratio (Bullock, 2004).
The relationship between cytoarchitecture and apical pair-rule transcript
localisation does not appear to be absolute because a signal is detected in
eve, but not h, from Haematopota, which has
retained the ancestral, cuboidal blastoderm morphology
and because no localisation signal was detected in Anopheles-eve.
Although the developmental context in which these
signals are used cannot yet be discerned (in situ hybridisation is currently not possible in these
species because of egg shells that are difficult to remove and because of
difficulties in obtaining embryos)
these data raise the possibility that,
within a single species, the differential ability of transcripts to be
recognised by the localisation machinery is used to fine-tune transcriptional
control of target genes in the blastoderm by modulating the nuclear
concentration of pair-rule proteins (Bullock, 2004).
The ability of eve and h pair-rule transcripts to use the
localisation machinery varies in Diptera. A range of localisation
efficiencies is observed in situ that is mirrored in all of 11 cases, upon injection into
Drosophila embryos. Thus, differences in localisation efficiency
appear to reflect changes in the respective localisation signals, rather than
alterations in the specificity of the protein machinery. These findings are
consistent with previous studies with artificial variants of the
Drosophila hairy localisation signal, which suggest that the character of
localisation signals modulates the efficiency of localisation by determining
the kinetics of both the initiation of transport and the transport process
itself. Localisation efficiency appears to be determined by multiple RNA:protein
interactions, the sum of which affects the stability and/or activity of the
RNA:motor complex. Therefore, the efficiency of the localisation process can
be modified gradually during evolution by the addition, loss or modification
of individual recognition sites within mRNAs (Bullock, 2004).
It seems that localisation signals in pair-rule genes have emerged multiple
times within Diptera. For example, although the possibility
that localisation signals in h have been lost in multiple different
lineages of lower Diptera cannot be ruled out, the most parsimonious explanation for the phylogenetic distribution of signals in this transcript is that they evolved independently in response to changes in cytoarchitecture in the lineages
leading to Cyclorrhapha and Culicomorpha. Injection of transcripts from
additional species into Drosophila will determine whether
eve localisation signals emerged independently in the lineages
leading to Haematopota and Cyclorrhapha, or were lost in the lineage
leading to Empis (Bullock, 2004).
Work in mammalian cells has provided insights into how localisation signals
might initially appear. These studies suggest that non-localising mRNAs can also
interact with a motor complex, albeit with a comparatively small probability,
and undergo short movements on microtubules. Localisation signals appear to
augment these interactions and lead to the net translocation of an RNA
population along a polarised cytoskeleton by increasing the frequency and
duration of directed transport. The localisation machinery in Diptera may also have a general, weak affinity for mRNAs because a small proportion of particles of injected non-localising transcripts are transported over short distances in
Drosophila embryos. Asymmetric
accumulation of a population of transcripts may therefore evolve gradually as
a result of selection for increased interaction between a specific transcript
and the localisation machinery (Bullock, 2004).
The histone chaperone Asf1 assists in chromatin assembly and remodeling during replication, transcription activation, and gene silencing. However, it has been unclear to what extent Asf1 could be targeted to specific loci via interactions with sequence-specific DNA-binding proteins. This study shows that Asf1 contributes to the repression of Notch target genes, as depletion of Asf1 in cells by RNAi causes derepression of the E(spl) Notch-inducible genes. Conversely, overexpression of Asf1 in vivo results in decreased expression of target genes and produces phenotypes that are strongly modified (enhanced and suppressed) by mutations affecting the Notch pathway, but not by mutations in other signaling pathways. Asf1 can be coprecipitated with the DNA-binding protein Su(H) and the corepressor Hairless and interacts directly with two components of this complex, Hairless and SKIP. Thus, in addition to playing more general roles in chromatin dynamics, Asf1 is directed via interactions with sequence-specific complexes to mediate silencing of specific target genes (Goodfellow, 2007).
Modulation of the chromatin structure is a key feature in transcriptional regulation. Chromatin remodeling by ATP-dependent enzymes and posttranslational histone modifications are two important mechanisms that affect transcriptional activity, by influencing the accessibility of upstream regions and promoters. A third mechanism involves the breakdown and reassembly of nucleosomes on the DNA, a process that also allows for the incorporation of histone variants, such as H3.3. Histone chaperones, which bind to histone heterodimers, are required both for nucleosome assembly and for their disassembly. They include the H3/H4 chaperone Anti-silencing factor 1 (Asf1), which has roles in replication-dependent and replication-independent chromatin dynamics (Goodfellow, 2007).
In yeast, extensive Asf1-mediated exchange of histones that is independent of replication and of transcription has been detected at gene promoters and is likely to be highly significant in maintaining the balance between induction and silencing of genes. Indeed, there are now several examples of yeast Asf1 contributing to chromatin disassembly at promoters to facilitate binding of the RNA-polymerase complex. Conversely, Asf1 also plays important roles in gene silencing when the reassembly of nucleosomes accompanies transcriptional repression. For example, in the absence of Asf1, there is a delay in promoter closure at the PHO5 gene. However, it remains unclear whether Asf1-mediated nucleosome reassembly occurs via a targeted mechanism, involving sequence-specific DNA-binding proteins, or whether it occurs constitutively by default (Goodfellow, 2007).
A strong correlation between histone loss and gene activation has emerged from genome-wide studies in Drosophila, as it has in yeast, suggesting that transcription in higher eukaryotes is also likely to be regulated by histone loss and replacement at the promoter. However, thus far, the contribution of Asf1 to dynamic gene regulation during cell signaling in multicellular organisms has not been examined. One cell-signaling pathway with very direct effects on transcription is the highly conserved Notch pathway. Activation of the receptor results in the release of a nuclear-targeted intracellular fragment (Nicd), which binds directly to the CSL DNA-binding protein (Suppressor of Hairless, Su(H), in Drosophila) and recruits the coactivator Mastermind, resulting in the activation of target genes. CSL proteins also contribute to the silencing of target genes in the absence of Nicd, through adaptor-mediated recruitment of corepressors such as Groucho (Gro), CtBP, and SMRT. Previous analysis indicates that the activity of Notch target genes correlates with a reduction in histone H3 density, suggesting that nucleosome disassembly and reassembly is likely to be involved in their regulation, and prompting an investigatation of whether Asf1 could play a role (Goodfellow, 2007).
This study shows that Asf1 contributes to the repression of Notch target genes, and that it is recruited to the DNA through interactions with the Su(H)/H complex. Thus, Asf1 is targeted to specific loci by binding to sequence-specific DNA-binding complexes, where it can promote gene silencing during development (Goodfellow, 2007).
To investigate whether Asf1 contributes to the regulation of inducible genes in Drosophila, RNA interference (RNAi) was used to deplete S2-N cells and the levels of transcription were analyzed from the 11 well-characterized Notch target genes clustered in the E(spl) complex. Conditions were established for activating Notch in these cells and it was shown that activation results in Su(H)-dependent stimulation of E(spl) gene transcription (Goodfellow, 2007).
Unlike knockdown of the other chromatin regulators tested, depletion of Asf1 led to a 4-fold increase in E(spl)m7 mRNA levels, but it had no effect on the housekeeping genes rp49 and EF2B. More extensive analysis revealed that mRNA levels for all E(spl) genes were increased after Asf1 depletion in the absence of Notch activation; some showed a greater than 10-fold change in expression, suggesting that these Notch targets are derepressed as they are when the corepressor Hairless is depleted. In contrast, there was little effect of Asf1 depletion on several other repressed genes, including a phagocytosis receptor gene, nimrod. In addition to the derepression observed in resting cells, Asf1 depletion also altered the responsiveness to Notch activation. Many more of the E(spl) genes were susceptible to Notch activation in Asf1-depleted cells; for example, 5 of the 11 genes were expressed at greater than 20-fold higher levels after Asf1 RNAi. There was comparatively little change at the genes, such as E(spl)m3, which normally has the most robust response to Notch and is depleted for histones. Thus, it appears that Asf1 makes important contributions to the silencing of Notch target genes (Goodfellow, 2007).
Previous studies showed that overexpression of Asf1 in the Drosophila eye (ey::Gal4 UAS::asf1/+) causes a 'small-eye' phenotype in which the eye is reduced in size and ommatidia are disorganized. If these small-eye phenotypes are a consequence of Asf1 altering the transcription of Notch targets, they may be modified when combined with mutations in the Notch pathway. To investigate this possibility, flies overexpressing Asf1 were crossed to alleles affecting genes central to Notch or to other signaling pathways, and the eye size was analyzed in the heterozygous progeny (Goodfellow, 2007).
The first dramatic result was that the heterozygous combination of a Notch loss-of-function allele (N55e11) and Asf1 overexpression caused a severe reduction in the eye/head capsule ('pin-head') and resulted in lethality. Thus, the effects of Asf1 overexpression were strongly enhanced by a decrease in Notch function. Significant enhancement of the Asf1 phenotype also occurred with Delta loss-of-function alleles, but not with alleles affecting Hedgehog (smo), EGF-R (Egfr), or Wingless (arm, arrow) pathways or with alleles affecting the SET domain protein Trithorax-related (trr), the histone exchange factor Domino (dom), or the cell adhesion protein Pawn (pwn). Complementary results were obtained by using mutant alleles that increase Notch signaling: both a loss-of-function Hairless (H) allele and a gain-of-function Notch allele (NMcd1) suppressed the small-eye defect caused by Asf1 overexpression. These findings are fully consistent with the results of RNAi-mediated Asf1 depletion, and they suggest that Asf1 is involved in repression of Notch target genes. As asf1 mutant cells failed to proliferate, it was not possible to obtain clones of homozygous mutant cells to test the effects of eliminating Asf1 on Notch target genes in the eye (Goodfellow, 2007).
To investigate whether interactions between Notch and Asf1 occur in other tissues, it was asked whether Asf1 overexpression also perturbed Notch function in the Drosophila wing. Expression of Asf1 in the developing wing pouch (sd::Gal4/+; UAS::asf1/+) resulted in margin loss/wing nicks and mild vein thickening, characteristics of reduced Notch function (Notch/+ heterozygous flies have mild wing nicks due to reduced signaling at the dorsal/ventral (d/v) organizer of the wing). The Asf1 overexpression phenotypes were strongly enhanced when the levels of Notch were reduced; thus, wings had extensive scalloping/margin loss and more extensive vein thickening. Wing phenotypes, similar to the eye phenotypes, produced by Asf1 expression were thus enhanced by reduced Notch (Goodfellow, 2007).
To further assess whether Asf1 affects expression of target genes regulated by Notch (e.g., cut) or by other pathways (e.g., spalt), the effects of overexpressing Asf1 in wing discs was analyzed. In wild-type discs, Notch-dependent expression of Cut is detected in a stripe along the d/v boundary. This was interrupted and reduced in discs in which Asf1 was overexpressed. In contrast, there was no visible effect on Spalt under these conditions. Similar results were obtained when Asf1 was expressed in a more limited domain (by using ptc::Gal4), where a local loss of Cut, but not Spalt, expression was seen. Stronger expression of Asf1 resulted in more pronounced Notch-like phenotypes and loss of Cut expression, which could be rescued by a reduction in Hairless function. Under these conditions, where Asf1 was expressed more strongly, some more generalized effects of Asf1 were sometimes detected, compatible with its proposed role as a histone chaperone during replication. The replication defects became more severe at even higher levels of expression (29°C). Similarly, clones of cells mutant for asf1 failed to proliferate. Thus, as in yeast, Asf1 appears to have roles in replication-dependent as well as replication-independent chromatin dynamics in Drosophila. By moderating the levels of Asf1 expression, it was possible to uncouple these requirements, revealing a contribution to repression of Notch target genes (Goodfellow, 2007).
Complexes implicated in repression at Notch targets are formed by the CSL/Su(H) DNA-binding protein in conjunction with adaptor proteins, such as SKIP and Hairless, which recruit general corepressors, including SMTR or Gro and CtBP. On polytene chromosomes from Drosophila salivary glands, Asf1 is detected at most Su(H)-enriched sites, suggesting that these proteins are present at the same loci. Asf1 is also bound at many other loci, and it is strongly enriched at centromeres and telomeres, reflecting its multiple roles in chromatin dynamics (Goodfellow, 2007).
The colocalization of Su(H) and Asf1 on polytene chromosomes prompted a test of whether Su(H) and/or associated factors could copurify with Asf1 in immunoprecipitation (IP) experiments. For these experiments, extracts prepared from Drosophila embryos, and Su(H) or Asf1 was immunoprecipitated by using moderate salt conditions. Under these conditions, Asf1 was detected in Su(H) IP experiments, and, conversely, Su(H) was precipitated with Asf1, as was the corepressor Gro, but not CtBP. To exclude the possibility that the interaction between Asf1 and the Su(H) complex was mediated by the independent binding of both protein complexes to DNA, IP experiments were performed in the presence of ethidium bromide (EtBr), a DNA-intercalating drug that dissociates proteins from DNA. This treatment did not affect the interaction of Asf1 with Su(H). Thus, these data suggest that Asf1 is present in protein complexes containing the sequence-specific DNA-binding protein Su(H) and the Gro corepressor. A significant suppression of the Asf1-induced small-eye phenotype was observed in flies that were also heterozygous for a strong gro allele (groE48) and an enhancement was seen by Hairless proteins that retained a Gro-binding domain, agreeing with a model linking Gro to Asf1-mediated repression. Therefore whether any of the proteins in the Su(H) repression complex are able to bind to bacterially produced Asf1 (fused to glutathione S-transferase, GST), was examined. Of those tested, both Hairless and the adaptor protein SKIP were bound to GST-Asf1, but not to GST alone or to GST-CAF1p55 (a component of chromatin assembly factor 1). Neither Gro nor Su(H) itself showed direct interactions with Asf1 in this assay (Goodfellow, 2007).
Finally, to test whether Hairless contributes to the recruitment of Asf1 in vivo, chromatin immunoprecipitation (ChIP) was performed with anti-Asf1 antibodies in cells with and without RNAi-mediated depletion of Hairless and association with two E(spl) genes, m3 and m7, was assayed. The E(spl)m7 gene is silenced in the S2 cells and is strongly affected by Asf1 depletion, whereas E(spl)m3 is expressed in S2 cells, is highly induced by Notch activation, and is more mildly affected by Asf1 depletion. Of the two genes, the greatest effects were seen for E(spl)m7; binding of Asf1 to both enhancer and ORF fragments strongly decreased in ChIP after Hairless depletion. A decrease was also seen at the E(spl)m3 ORF region, but not at the E(spl)m3 enhancer. This enhancer is found to have very low histone coverage in these cells, and it was found that it shows only small Asf1 occupancy levels. The decrease in Asf1 from ORFs of both E(spl)m3 and E(spl)m7 after Hairless depletion may indicate that Asf1 spreads from the site of recruitment. Binding of Asf1 to E(spl)m7 and E(spl)m3 regions was confirmed by using affinity-purified anti-Asf1 antibodies raised in a different species. Loss of Hairless does not affect the binding of Asf1 to other loci that do not require Su(H)/H for their regulation, such as eiger or snRNP69D. Similarly, there was no change in the levels of Polycomb protein associated with bxd-PRE after Hairless knockdown. Together, these data support the model that recruitment of Asf1 to Notch targets requires Hairless (Goodfellow, 2007).
The density and precise positioning of nucleosomes are important factors in determining the transcriptional activity of a locus. It is now evident that most nonnucleosomal histones in cells are likely to be complexed with chaperones. It is therefore not surprising that the histone chaperone Asf1 is important for chromatin dynamics and has been shown to have multiple roles in transcription as well as in the disassembly and reassembly of chromatin during replication. These include gene-specific roles in repression, activation, and transcription elongation. For example, Asf1 is required for nucleosome disassembly and transcription activation at the yeast PHO5, PHO8, ADY2, and ADH2 promoters. However, the mechanisms responsible for targeting Asf1 to these loci remain unclear. This study has demonstrated that Asf1 can be specifically recruited to target loci by interactions with sequence-specific DNA-binding transcription factors. Asf1 is present in a complex with Su(H), the central DNA-binding protein in the Notch pathway, and that it interacts directly with two proteins found in CSL complexes, Hairless and SKIP. Importantly, it was found that Asf1 plays a significant role in the repression of Notch target genes. Thus, contrary to effects at many of the inducible loci examined in yeast, these data demonstrate a requirement for Asf1 in silencing rather than in activation of these inducible genes (Goodfellow, 2007).
As the global corepressor Gro is also coprecipitated with Asf1 and is implicated in Asf1-mediated repression through genetic interactions, Gro and Asf1 may cooperate in the repression of Notch target genes. Gro has been postulated to exert long-range repressive effects by nucleating a transcriptionally silent chromatin state, in a similar manner to its yeast relative Tup1. For example, at the STE6 locus, Tup1 recruitment results in increased nucleosomal density and local nucleosome positioning. The recruitment of the histone chaperone Asf1 with Gro to Su(H)/H DNA-binding complexes could facilitate a similar localized increase in histone deposition and participate in the spreading of repressed chromatin. Furthermore, since (H)/H complexes engage in comparatively low-stability interactions with target loci, it is suggested that Asf1 could be critical for translating these transient interactions into stable silencing. However, thus far, the analysis has focused on relatively few targets and tissues; thus, it remains to be determined whether Asf1 is recruited to all targets regulated by Su(H)/H, or whether there are additional factors that influence its recruitment at specific loci. Similarly, it will be important to determine whether other sequence-specific complexes are able to bind directly to Asf1 (Goodfellow, 2007).
In conclusion, these results show that the histone H3/H4 chaperone Asf1 contributes to selective silencing of genes in Drosophila, through interactions with the Su(H)/H DNA-binding protein complexes. In this way, chaperones can act as gene-selective regulators that contribute to the control of gene expression by developmental signaling pathways (Goodfellow, 2007).
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