Polycomb
See the embryonic expression pattern of Pc at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
Polycomb transcripts are most abundant in unfertilized eggs and early cleavage embryos, when PC mRNA is homogeneously distributed. At blastoderm stages PC transcripts are still uniformly distributed but there is less staining at anterior and posterior poles. PC transcripts are found in almost all cells through gastrulation [Images] and organogenesis though at a much lower level than in earlier syncytial stages. In late stages PC transcripts are predominantly expressed in the central nervous system (Paro, 1992).
The subcellular three-dimensional distribution of three polycomb-group (PcG) proteins (Polycomb, Polyhomeotic and Posterior sex combs) in fixed whole-mount Drosophila embryos was analyzed by multicolor confocal fluorescence microscopy. All three proteins are localized in complex patterns of 100 or more loci throughout most of the interphase nuclear volume. The rather narrow distribution of the protein intensities in the vast majority of loci argues against a PcG-mediated sequestration of repressed target genes by aggregation into subnuclear domains. In contrast to the case for PEV repression, there is a lack of correlation between the occurrence of PcG proteins and high concentrations of DNA, demonstrating that the silenced genes are not targeted to heterochromatic regions within the nucleus. There is a clear distinction between sites of transcription in the nucleus and sites of PcG binding, supporting the assumption that most PcG binding loci are sites of repressive complexes. Although the PcG proteins maintain tissue-specific repression for up to 14 cell generations, the proteins studied here visibly dissociate from the chromatin during mitosis, and disperse into the cytoplasm in a differential manner. Quantitation of the fluorescence intensities in the whole mount embryos demonstrate that the dissociated proteins are present in the cytoplasm. Less than 2% of Ph remains attached to late metaphase and anaphase chromosomes. Each of the three proteins that were studied has a different rate and extent of dissociation at prophase and reassociation at telophase. These observations have important implications for models of the mechanism and maintenance of PcG- mediated gene repression. The findings reported in this paper do not exclude the possibillity that the minor fraction of the PcG proteins, which remains bound to mitotic chromosomes, may be associated with specific nucleation sites within the repressed genes. Repression could then be initiated in telophase from these sites via cooperative binding of previously dispersed PcG protein complexes, insuring that promoters are blocked before reassembly of functional transcription complexes. A similar marking mechanism has been proposed for active genes by residual transcription factors on mitotic chromosomes. In the BX-C, possible candidates for repression nucleation sites are regulatory elements, which are preferentially associated with PC (Buchenau, 1998).
Polycomb group proteins (PcG) repress homeotic genes in cells where these
genes must remain inactive during Drosophila and vertebrate
development. This repression depends on cis-acting silencer sequences, called
Polycomb group response elements (PREs). Pleiohomeotic (Pho), the only known
sequence-specific DNA-binding PcG protein, binds to PREs, but pho
mutants show only mild phenotypes compared with other PcG mutants. pho-like, a gene encoding a protein with high similarity
to Pho, has been characterized. Pho-like binds to Pho-binding sites in vitro and pho-like; pho double mutants show more severe misexpression of homeotic genes than
do the single mutants. These results suggest that Pho and Pho-like act
redundantly to repress homeotic genes. The distribution of five
PcG proteins on polytene chromosomes was examined in pho-like, pho double
mutants. Pc, Psc, Scm, E(z) and Ph remain bound to polytene chromosomes at
most sites in the absence of Pho and Pho-like. At a few chromosomal locations,
however, some of the PcG proteins are no longer present in the absence of Pho
and Pho-like, suggesting that Pho-like and Pho may anchor PcG protein
complexes to only a subset of PREs. Alternatively, Pho-like and Pho may not
participate in the anchoring of PcG complexes, but may be necessary for
transcriptional repression mediated through PREs. In contrast to Pho and
Pho-like, removal of Trithorax-like/GAGA factor or Zeste, two other
DNA-binding proteins implicated in PRE function, do not cause misexpression
of homeotic genes or reporter genes in imaginal discs (Brown, 2003).
The distribution of the PcG proteins Pc, Psc,
Polyhomeotic (Ph), Sex combs on midleg (Scm) and Enhancer of zeste [E(z)] on
polytene chromosomes was examined. Pc, Ph and Psc are all core components of the PcG
protein complex called PRC1. Scm has also been reported to co-purify with PRC1. Scm and
Ph may also be present in protein complexes other than PRC1. E(z) is
a component of the Esc-E(z) complex, which is distinct from PRC1. The analysis focused on PcG protein binding sites on the X chromosome and on the right
arm of chromosome 3, which includes the bithorax and Antennapedia gene
complexes (BXC and ANTC) (Brown, 2003).
Pho, Pc, Psc, Ph and Scm all bind the same three sites
in wild-type chromosomes. As expected, in phol; pho double mutants, no Pho protein is detected. Binding of Pc, Psc and Scm is lost at polytene subdivision 2D in phol; pho double mutants; binding of these proteins to all other sites on the X
chromosome is unaffected. Binding of Ph is completely unaffected in
phol; pho double mutants. In particular, the Ph signal at 2D is
present, suggesting that Ph can bind at this site even if other PcG proteins
are removed. Pc binding to 2D is not lost in either
pho or phol single mutants, suggesting that the presence of either of these two proteins is sufficient for Pc to bind to this site (Brown, 2003).
Taken together, the immunolocalization data suggest that binding of PcG
proteins to most sites is unaltered in the absence of Pho and Phol protein,
but that two proteins are redundantly required for PcG protein binding
at a few specific sites. Intriguingly, it appears that all PcG proteins tested
in this study are still associated with the BXC and ANTC loci. Nevertheless, the BXC genes Ubx and Abd-B are derepressed in
phol; pho double mutant wing discs. Several different
explanations for this paradox are proposed. (1) Derepression of homeotic genes and
binding of PcG proteins were not assayed in the same tissues. It was not possible
to detect derepression of Ubx in salivary gland cells of phol,
pho double mutants. (2) Pho and Phol may only be
required for anchoring PcG proteins at some PREs in the BXC. Different
DNA-binding proteins may provide this function at other PREs. This is
supported by the finding that binding of PcG proteins is lost at some sites in
phol; pho double mutants. Moreover, several different
PREs have been identified in the Ubx gene. The
resolution of antibody signals on polytene chromosomes is not refined enough
to resolve distinct PREs in a single gene and, hence, loss of only a fraction
of PcG protein complexes may not be detectable. Finally, Phol and Pho may not
be necessary for the anchoring of PcG protein complexes to the DNA, but may
confer the actual transcriptional repression mediated by PREs in imaginal
discs, while the PcG protein complexes might function in the propagation and
memory of the repression. Thus, PcG protein complexes might serve to recruit
Phol and Pho or their co-repressors to the DNA (Brown, 2003).
These results show a strong requirement for the DNA-binding proteins Pho and
Pho-like in homeotic gene silencing in imaginal discs. In fact, the strong
misexpression of homeotic genes observed in phol; pho double mutant
imaginal cells is comparable with that seen in imaginal disc clones mutant for
Pc, Scm, Sce or Pcl. The
loss of PcG protein binding at only a small number of sites in phol,
pho polytene chromosomes is consistent with the idea that Phol and Pho
are required to recruit PcG protein complexes at only a subset of PREs.
Alternatively, Phol and Pho may be required for transcriptional repression
mediated by PREs, but not for anchoring of PcG protein complexes (Brown, 2003).
Fluorescence recovery after photobleaching (FRAP) microscopy was used to
determine the kinetic properties of Polycomb group (PcG) proteins Polycomb and Polyhomeotic in whole living
Drosophila organisms (embryos) and tissues (wing imaginal discs and salivary
glands). Translational diffusion constants of PcG proteins,
dissociation constants and residence times for complexes were determined in vivo at different
developmental stages. In polytene nuclei, the rate constants suggest
heterogeneity of the complexes. Computer simulations with new models for
spatially distributed protein complexes were performed in systems showing both
diffusion and binding equilibria, and the results compared with the experimental
data. Forward and reverse rate constants for complex
formation were determined. Complexes exchange within a period of 1-10 minutes, more than an
order of magnitude faster than the cell cycle time, ruling out models of
repression in which access of transcription activators to the chromatin is
limited and demonstrating that long-term repression primarily reflects
mass-action chemical equilibria (Ficz, 2005).
Most FRAP studies of nuclear proteins have involved components in transcription
complexes or transcriptional activators that exchange in less than 2 minutes.
The only repressor protein that has previously been
investigated is heterochromatin protein 1 (HP1), a protein targeted to
heterochromatin in higher eukaryotes.
Although HP1 is loaded directly onto the chromatin during replication, it
was found by FRAP to bind only transiently to chromatin with a maximum residence
time of 60 seconds. Thus, both HP1 and PcG repression complexes appear to
function by dynamic competition with other chromatin-binding proteins rather
than by formation of a static, higher-order chromatin structure with immobilized
bound repressors. FRAP measurements on polytene chromosomes revealed
differences in the dissociation rate constants between individual bands -- this implies that a flexible repression system of complexes with various compositions that influence the binding affinity of other members and whose turnover is in the
order of a few minutes. It is concluded that: (1) activation and repression can be dynamically controlled by simple chemical equilibria; (2) reduction in PcG levels will facilitate epigenetic change and may explain why non-cycling cells can be reprogrammed more easily than cycling cells, and (3) PcG complexes are
exchangeable protein assemblies that maintain repression over many cell cycles
by simple chemical equilibria (Fitz, 2005).
Polycomb transcriptional silencing machinery is implicated in the maintenance of precursor fates, but how this repression is reversed to allow cell differentiation is unknown. Testis-specific TAF (TBP-associated factor) homologs required for terminal differentiation of male germ cells may activate target gene expression in part by counteracting repression by Polycomb. Chromatin immunoprecipitation revealed that testis TAFs bind to target promoters, reduce Polycomb binding, and promote local accumulation of H3K4me3, a mark of Trithorax action. Testis TAFs also promoted relocalization of Polycomb Repression Complex 1 components to the nucleolus in spermatocytes, implicating subnuclear architecture in the regulation of terminal differentiation (Chen, 2005).
Male germ cells differentiate from adult stem cell precursors, first proliferating as spermatogonia, then converting to spermatocytes, which initiate a dramatic, cell type–specific transcription program. In Drosophila, five testis-specific TAF homologs (tTAFs) encoded by the can, sa, mia, nht, and rye genes are required for meiotic cell cycle progression and normal levels of expression in spermatocytes of target genes involved in postmeiotic spermatid differentiation. Requirement for the tTAFs is gene selective: Many genes are transcribed normally in tTAF mutant spermatocytes. Tissue-specific TAFs have also been implicated in gametogenesis and differentiation of specific cell types in mammals. In addition to action with TBP (TATA box–binding protein) in TFIID, certain TAFs associate with HAT (histone acetyltransferase) or Polycomb group (PcG) transcriptional regulatory complexes. To elucidate how tissue-specific TAFs can regulate gene-selective transcription programs during development, the mechanism of action of the Drosophila tTAFs was investigated in vivo (Chen, 2005).
The tTAF proteins were concentrated in a particular subcompartment of the nucleolus in primary spermatocytes. Expression of a functional green fluorescence protein (GFP)–tagged genomic sa rescuing transgene revealed that expression of Sa-GFP turns on specifically in male germ cells soon after initiation of spermatocyte differentiation and persists throughout the remainder of the primary spermatocyte stage, disappearing as cells entered the first meiotic division. Some Sa-GFP was detected associated with condensing chromatin. However, most Sa-GFP localized to the nucleolus, in a pattern complementary with Fibrillarin, which marks a fibrillar nucleolar subcompartment. Staining with antibodies against endogenous Sa, Can, Nht, or Mia proteins showed similar temporal expression and nucleolar localization in primary spermatocytes, consistent with collaborative function of the tTAFs. In contrast, the generally expressed sa homolog TAF8 and its binding partner TAF10b are excluded from the nucleolus (Chen, 2005).
Several components of the Polycomb Repression Complex 1 (PRC1) transcriptional regulator appear in the nucleolus in spermatocytes, coincident with tTAF expression and dependent on tTAF function. Polycomb (Pc) protein expresses from a Pc-GFP genomic transgene localized on chromatin, but in addition becomes concentrated in the nucleolus in primary spermatocytes. Both Pc-GFP and staining of endogenous protein with antibody against Pc (anti-Pc) revealed localization to the same nucleolar subcompartment as the one containing tTAFs. Recruitment of Pc to the nucleolus exactly coincides with onset of expression of the tTAFs after early G2 phase in spermatocytes. Relocalization of Pc depends on wild-type tTAF activity: Pc localizes to chromatin but is not concentrated in the nucleolus in tTAF mutant spermatocytes. Two other components of the PRC1 core complex, Polyhomeotic (Ph) and Drosophila Ring protein (dRing), also become concentrated in the nucleolus in primary spermatocytes dependent on tTAF function. Failure of PRC1 components to localize to the nucleolus in tTAF mutants is not caused by nucleolar loss because Fibrillarin staining appears normal in the mutants. H3K27me3 laid down by action of the PRC2 complex acts as a docking site for the Pc chromodomain to recruit PRC1 and block transcription initiation. H3K27me3 localizes on chromatin in spermatocytes, along with Pc. However, no H3K27me3 was detected in the nucleolus in spermatocytes, suggesting that PRC1 components may be recruited to the nucleolus by a different mechanism independent of chromatin (Chen, 2005).
The tTAFs are required for activation of robust transcription of several spermatid differentiation genes, whereas the PcG proteins are known to mediate transcriptional repression. Chromatin immunoprecipitation (ChIP) suggested that the tTAFs might allow robust transcription of spermatid differentiation genes in part by counteracting repression by Pc, perhaps causing dissociation of PRC1 from cis-acting control sequences at target genes (Chen, 2005).
ChIP from wild-type testes using anti-Sa revealed enrichment of tTAF binding at three different known target genes (fzo, Mst87F, and dj), compared with binding at intergenic regions 10 to 20 kb away or at a tTAF-independent gene expressed in the same cell type (cyclin A or sa itself), suggesting that the tTAFs are in occupancy at target genes. Real-time polymerase chain reaction (PCR) analysis revealed ~10-fold enrichment of Sa at a target (mst87F) compared with a non-target gene (sa) (Chen, 2005).
ChIP analysis also revealed that Pc protein binds to tTAF-dependent target genes in tTAF mutant testes, and that wild-type function of the tTAFs reduce Pc binding. ChIP with anti-Pc from can mutant testes preferentially precipitates the three tTAF target promoters, compared with intergenic regions or promoters from two different nontarget controls. Quantification by real-time PCR showed more than 50-fold enrichment of Pc at the target gene mst87F compared with the tTAF-independent control sa. In contrast, relative occupancy of Pc at the tTAF targets was not significantly different from that at the non-targets in wild-type testes (Chen, 2005).
The tTAFs may act near the promoter of target genes (fzo) to allow expression by directly or indirectly reducing nearby binding of PRC1. ChIP using primer pairs across the promoter region of fzo revealed that the tTAF enrich most strongly for sequences just upstream of the transcription start site. In contrast, Pc-containing protein complexes (in tTAF mutant testes) enrich for a broader distribution, including sequences near and downstream of the transcription start site, consistent with localization of Pc at Ultrabithorax (Ubx) locus in wing discs and on the hsp26 promoter in vivo (Chen, 2005).
Binding of the tTAFs at target promoters may allow expression through recruitment or activation of the Trithorax group (TrxG) transcriptional activation complex, which often acts in opposition to repression by PcG proteins. Trx, like its mammalian homolog MLL, creates an H3K4me3 epigenetic mark. ChIP from wild-type testes revealed H3K4me3 at or near the promoter regions of the three tTAF targets tested, as well as at nontargets. Analysis using primer pairs across the tTAF target fzo region revealed that H3K4me3 associated most strongly with sequences spanning the promoter. In contrast, ChIP with anti-H3K4me3 from can mutant testes did not enrich for the tTAF target promoters. Quantitative PCR revealed 36-fold enrichment of the promoter region of the tTAF-dependent mst87F gene by ChIP for H3K4me3 in wild-type compared with can mutant testes (Chen, 2005).
Consistent with the presence of H3K4me3 at target promoters in wild-type testes, trx function appears to be required for continued expression of two different kinds of tTAF-dependent targets. Boule triggers the G2/M transition in meiosis I by allowing translation of twine and requires tTAFs for protein accumulation, setting up a cross-regulatory mechanism so that meiotic cell cycle progression awaits expression of terminal differentiation genes. When temperature-sensitive trx1 flies grown at permissive temperature were shifted to nonpermissive temperature as adults, the Boule protein level in mutant testes substantially decreased over time at nonpermissive temperature compared with the level in wild-type flies shifted in parallel or trx1 flies held at permissive temperature. Likewise, analysis of mRNA levels by semiquantitative PCR revealed a ~40% decrease in transcript level for the tTAF target gene fzo, but not for the tTAF-independent gene cyclin A, in testes from trx1 mutant flies shifted to non-permissive temperature compared with the level in testes from similarly treated wild-type flies (Chen, 2005).
In summary, occupancy of tTAFs and Pc at target promoters appears to be mutually exclusive in wild-type and tTAF mutant spermatocytes, suggesting that the tTAFs may turn on target gene expression by counteracting repression by Polycomb, either directly or indirectly reducing Pc binding and allowing local action of Trx. Loss of function of Pc in marked clones of homozygous mutant cells does not restore terminal differentiation in a tTAF mutant background, suggesting that in addition to counteracting repression by Pc, tTAFs may also be required at the promoter region independent of Pc, possibly to recruit Trx or other cofactors for transcription activation. Transcriptional derepression by sequestration of PcG proteins has been observed during HIV-1 infection, when the viral Nef protein recruits the PRC2 component Eed to the plasma membrane. Likewise, the tTAFs may sequester Pc to the nucleolus. The tTAFs Nht, Can, and Mia are homologs of the generally expressed TAF4, TAF5, and TAF6, which are found as stoichiometric components of the PRC1 complex purified from fly embryos, raising the possibility that the tTAFs might associate with a population of Pc-, Ph-, and dRing-containing complexes in the nucleolus. If so, interactions in the nucleolus are likely to differ from interactions at the promoters of target genes, because the ChIP results indicate immunoprecipitation of tTAFs without Pc (Chen, 2005).
The PcG and TrxG proteins act to maintain cell fates set during embryogenesis throughout development. Emerging evidence indicates that PcG and TrxG complexes also play critical roles in decisions between proliferating precursor cell fates and terminal differentiation, for example, in the blood cell lineages. In particular, the mammalian PcG protein Bmi-1 promotes proliferation and blocks differentiation of normal and leukemic stem cells, and is required for establishment or maintenance of adult hematopoietic stem cells in mouse. Transcriptional silencing by PcG action may allow self-renewal and continued proliferation of precursor cells by blocking expression of terminal differentiation genes. This repression must be reversed to allow production of terminally differentiated cells, whereas failure may allow overproliferation of precursors and eventually cancer. Although central for both normal development and understanding the genesis of cancer, little is known about the mechanisms that reverse such epigenetic silencing to allow expression of the terminal differentiation program. These findings in the male germ line provide an example of how cell type– and stage–specific transcriptional regulatory machinery, turned on as part of the developmental program, might allow onset of terminal differentiation by counteracting repression by the PcG and highlight the importance of subnuclear localization in regulation of transcriptional regulation (Chen, 2005).
The brahma gene is required for the activation of multiple homeotic genes in Drosophila.
Loss-of-function brm mutations suppress mutations in Polycomb, a repressor of homeotic genes,
and cause developmental defects similar to those arising from insufficient expression of the
homeotic genes of the Antennapedia and Bithorax complexes (Tamkun, 1992).
Polyhomeotic immunoprecipitates in a multimeric complex
that includes Polycomb. Duplications of ph suppress homeotic transformations of Pc
and Polycomb-like, supporting a mass-action model for Pc-G function. ph alleles have been crossed to all
members of the Polycomb group. Synergistic effects are found suggesting that these gene
products might interact directly with ph. Embryonic phenotypes of ph mutant embryos that are
lethal when heterozygous or homozygous for other mutations suggest that ph may perform different functions in conjunction with differing subsets of Pc group
genes (Cheng, 1994).
moira (mor) is a member of the trithorax group of homeotic gene regulators in Drosophila. moira is required for the function of multiple homeotic
genes of the Antennapedia and bithorax complexes (HOM genes) in most imaginal
tissues. Heterozygous mor mutations suppress the following Polycomb-induced phenotypes: The requirement for moira function is at the level of transcription. The ability of moira mutations to supppress Antp homeotic phenotypes is dependent on the promoter.
moira is also required for transcription of the engrailed segmentation gene in the
imaginal wing disc. Because homozygous mor clones have phenotypes similar to those seen in clones of cells that have lost en function, en transcription was examined in clones of cells in the posterior wing. In the absence of transcriptional activation by mor, the pattern of en is altered. Greatly reduced en expression is found in wing clones. The abnormalities caused by the loss of moira function in germ
cells suggest that at least one other target gene requires moira for normal oogenesis (Brizuela, 1997).
Recently, the gene encoding a large protein related to the trithorax group protein brahma has been cloned and maps to the same three polytene chromosome bands as moira (Goldman-Levi, 1996). This new gene 89B helicase, is missing in a cytologically-invisible moira deletion. Since this small deletion also affects at least one other vital gene in this region, attempts are being made to determine whether moira encodes the 89B helicase, and if so, whether the Mor protein is part of a large protein complex similar to the SWI/SNF complex that may interact with specific cis-regulatory regions of its target genes (Brizuela, 1997).
Arresting cell division using the string mutation or blocking DNA
replication with aphidicolin failed to prevent ectopic expression of the homeotic gene Ultrabithorax
in Polycomb mutants. Thus, even in the absence of DNA replication, Pc is required to maintain spatially
restricted patterns of homeotic gene expression (Gould, 1990).
The observation of a shared pathway in the function of a chromatin insulator and trithorax group (trxG) and Polycomb group (PcG) gene activation and silencing is suggestive of a common mechanism at work. If this is the case, mutations in trxG and PcG genes, known to be involved in activation and silencing, might also affect the ability of the insulators to interfere with enhancer-promoter interactions. To test this possibility, the effect of trxG and PcG mutations on the abdominal coloration of flies carrying the yellow2 mutation (affecting coloration) was measured using insertion of an insulator-containing gypsy retrotransposon. Males hemizygous for the y2 allele show brown abdominal pigmentation in the fifth and sixth abdominal segments, instead of the black pigmentation observed in wild-type males, due to the effect of the insulator on the upstream body cuticle enhancer. This insulator effect on the body enhancer is altered by hypomorphic mutations in mod(mdg4), which gives rise to a variegated phenotype resulting from different expression levels of the yellow gene in adjacent groups of cells. In some cuticle cells, the effect of the insulator is reversed, resulting in normal expression of the yellow gene; in other cells, the effect of the insulator on enhancer-promoter communication appears to be enhanced, further repressing yellow gene expression. To examine the effect of trxG mutations on insulator function, the partially nonfunctional insulator, renderend such by hypomorphic alleles of mod(mdg4), was tested. An examination was carried out of the consequence of mutations in trxG genes, such as trithorax, on the frequency and severity of a mod(mdg4) phenotype engendered by Mod(mdg4) action at the gypsy insulator (Gerasimova, 1998).
Both the penetrance and severity of a variegated phenotype due to insulator function are enhanced by mutations in trxG genes. trx mutation results in a decrease in the number of dark spots
with respect to that observed in hypomorphilc mod(mdg4) males, with only a few spots visible in a light brown-colored background. A stronger effect can be seen when trx is combined with brahma or ash1. Mutations in polycomb cause the opposite result, reversing the effect of the insulator on enhancer-promoter interactions and resulting in a wild-type expression of the yellow gene in the body cuticle. These results indicate that mutations in trxG genes cause an enhancement of the variegated phenotype induced by mod(mdg4) mutations in the yellow gene, suggesting that decreased levels of these proteins enhance the inhibitory effect of the insulator on enhancer-promoter interactions. In contrast, mutations in Pc impair the ability of the insulator to inhibit enhancer-promoter interactions, restoring normal expression of the gene. The effects of trxG and PcG mutations on insulator function at the yellow gene are not a result of homeotic transformations in abdominal segments that cause changes in the pigmentation of the cuticle, since these effects are not observed in flies carrying a wild-type copy of the yellow gene. In addition, the same effect can be observed with other gypsy-induced mutations such as scute-1 and cut-6. Flies of the genotype ct6; brm+ trx+ mod(mdg4)T16/brm2 trxB11 mod(mdg4)+ display a much stronger cut phenotype than ct6; mod(mdg4)T16/mod(mdg4)+ individuals, suggesting that the effect of TrxG and PcG proteins on gypsy insulator function is general and does not depend on the nature of the affected gene. A similar result was obtained with the sc1 mutation. The effects of trxG and PcG mutations on insulator function suggest that the proteins encoded by these genes might be structural components of the gypsy insulator or they might regulate its function (Gerasimova, 1998).
Drosophila Mi-2 protein binds to a domain in the gap protein
Hunchback which is specifically required for the repression of HOX genes. dMi-2 protein was tested to see if it participates in PcG repression. As in the case of dMi-2, maternally deposited PcG product often
rescues homozygous mutant PcG embryos to a considerable extent. Extensive derepression of HOX genes can be observed if
such homozygous embryos are also mutant for another PcG gene. Thus embryos homozygous for the PcG gene
Posterior sex combs (Psc) and dMi-2 were examined and it was found that Ubx and Abd-B are derepressed more extensively in this double mutant than
in Psc homozygotes alone. A similar result was found if dMi-2 is combined with other PcG mutations; these
double mutants consistently lead to much enhanced homeotic transformations compared with the single PcG mutants. Thus, there
is a synergy between dMi-2 and PcG genes. dMi-2 behaves like the PcG mutations Enhancer of Polycomb and Suppressor 2 of zeste,
neither of which on their own cause a homeotic phenotype but do so in combination with other PcG mutations. This suggests that
dMi-2 functions in PcG repression (Kehle, 1998).
Imaginal discs were examined for derepression of HOX genes as well as the phenotypes of their adult derivatives. Clonal analysis
suggests that dMi-2 is required for the survival of somatic cells. Do dMi-2 mutations exhibit
gene-dosage interactions with PcG mutations? While larvae heterozygous for Polycomb (Pc) mutations show slight derepression of
Ubx, larvae transheterozygous for both Pc and dMi-2 mutations show more extensive derepression.
Furthermore, derepression of the HOX gene Sex combs reduced (Scr) in the second and third leg discs of Pc heterozygotes results in
the formation of a first leg structure, the sex comb, on the second and third legs. The extent of this homeotic transformation reflects
the number of cells that misexpress Scr protein. This homeotic transformation is far stronger in dMi-2/Pc
transheterozygotes than in adults heterozygous for Pc alone, which is consistent with more extensive derepression of Scr in
the double mutant. These results are further evidence that dMi-2 acts together with PcG proteins to repress HOX genes (Kehle, 1998).
It has been proposed that Hb directly or indirectly recruits PcG proteins to DNA to establish PcG silencing of homeotic genes. The present data suggest that dMi-2 might function as a link between Hb and PcG repressors. Although dMi-2 contains two
motifs with similarity to DNA-binding domains (the myb and HMG domains), dMi-2 does not seem to bind to DNA on its own.
Therefore, Hb may recruit dMi-2 to DNA. Xenopus Mi-2 was recently purified as a subunit of a histone deacetylase complex with
nucleosome remodeling activity. In yeast and in vertebrates, several transcription factors repress transcription by recruiting histone
deacetylases. It is possible that in Drosophila, nucleosome remodeling and deacetylase activities of a dMi-2 complex, recruited to
homeotic genes by Hb, may result in local chromatin changes that allow binding of PcG proteins to the nucleosomal template.
Alternatively, the proposed Hb-dMi-2 complex might directly bind a PcG protein and recruit it to DNA. Finally, the involvement of dMi-2
in PcG silencing suggests that this process may involve deacetylation of histones (Kehle, 1998 and references).
Animals doubly mutant for two different PcG genes often show phenotypes more extreme than either single mutant alone. It has been suggested that this phenotypic enhancement results from PcG protein complexes that are more severely impaired by simultaneous reduction or alteration of multiple components. Having defined the molecular lesions and relative strengths of many Sex combs on midleg (Scm)
mutations, it was of interest to test the Scm alleles for interactions with other PcG mutations. In particular, a comparison was made of genetic interactions exhibited by mutations affecting different parts of SCM protein (Bornemann, 1998).
Animals were generated that were doubly heterozygous for each of the Scm alleles
and for a lethal allele of Polycomb (Pc3). Transheterozygous Pc3/Scm adults display more severe homeotic phenotypes than Pc3/+ adults, and this enhancement is seen with all Scm alleles tested. These phenotypes include transformations of wing to haltere, antenna to leg, second and third leg to first leg, and fourth abdominal segment to fifth. However, three Scm alleles, Su(z)302, R5-13, and ET50, produce much stronger interactions with Pc3 than do other Scm alleles. These three are the only Scm alleles to cause partial lethality in combination with Pc3. The transheterozygous progeny classes for Su(z)302, R5-13, and ET50 are reduced to about one-third that expected for full viability. In contrast, the Scm null alleles H1 and M56 are fully viable with Pc3. The surviving Su(z)302, R5-13, and ET50 transheterozygous progeny also exhibit more severe homeotic phenotypes than do other Pc3/Scm combinations. A marked male sex bias was observed among these survivors. In the most severe case, only about 5% of the surviving Pc3/ScmSu(z)302 progeny were female. Similarly, partial lethality and a male sex bias were seen with the reciprocal crosses consisting of Su(z)302, R5-13, or ET50 females mated to Pc3 males. These interactions likely result from the Scm lesions rather than mutations at other loci because these three Scm alleles produce similar phenotypic effects and were isolated independently on different genetic backgrounds. Each of the three alleles maps to the first mbt repeat in SCM protein. Thus, Su(z)302, R5-13, and ET50 are hypomorphic mutations based upon their behavior as homozygotes, yet they interact with Pc3 more strongly than do null Scm alleles (Bornemann, 1998).
Two types of transgene silencing were found for the Alcohol dehydrogenase (Adh) transcription unit. Transcriptional gene silencing (TGS) is Polycomb dependent and occurs when Adh is driven by the white eye color gene promoter. Full-length Adh transgenes are silenced posttranscriptionally at high copy number or by a pulsed increase over a threshold. The posttranscriptional gene silencing (PTGS) exhibits molecular hallmarks typical of RNA interference (RNAi), including the production of 21-25 bp length sense and antisense RNAs homologous to the silenced RNA. Mutations in piwi, which belongs to a gene family with members required for RNAi, block PTGS and one aspect of TGS, indicating a connection between the two types of silencing (Pal-Bhadra, 2002).
Despite the fact that posttranscriptional silencing appears to be a matter of RNA metabolism, some indications of chromatin modification of the homologous endogenous gene under certain circumstances have emerged. (1) Transgene copies of viral genes present in the nucleus only become methylated upon infection of the plant by the homologous virus, which has a double-stranded RNA genome and which does not enter the plant nucleus. (2) In plants and rodent cells, the introduction of DNA constructs into cells can trigger the RNA degradation reaction. (3) Mutations in Arabidopsis selected for reduced DNA methylation, ddm1, a SWI2/SNF chromatin component, and met1, the major DNA methyltransferase, will relieve gene silencing, including a stochastic reversal of posttranscriptional silencing. (4) Transgene arrays in the C. elegans germline are desilenced and appear less condensed in mutant backgrounds for mut-7 and rde-2, which are both required for RNAi. (5) The ectopic transcription of promoter sequences or their introduction to the plant cell in a virus will trigger transcriptional silencing of another reporter construct in the same cell with a homologous promoter, which also becomes hypermethylated. Evidence is presented for a link between posttranscriptional and transcriptional modes of gene silencing in Drosophila (Pal-Bhadra, 2002 and references therein).
The silencing of the promoter-reporter construct white-Alcohol dehydrogenase (w-Adh) and full-length Adh transgenes occurs in Drosophila . The w-Adh effect is modulated by mutations in the Polycomb group (Pc-G) of chromatin-repressive proteins, and the silenced transgenes are associated with the Pc-G complex, whereas single highly expressed copies are not. The endogenous Adh gene is drawn into the silencing pool and is capable of further extending the effect to an Adh-w construct, which has no portion in common with w-Adh but which does share homology with the endogenous Adh 5' sequences. The silenced Adh-w construct also accumulates the Pc-G complex. Deletion of the endogenous Adh gene eliminates the silencing interaction between the two nonhomologous, reciprocally constructed transgenes, w-Adh and Adh-w (Pal-Bhadra, 2002 and references therein).
The full-length Adh transgene silencing operates posttranscriptionally as determined by nuclear run-on transcription assays that directly assess the distribution of transcriptionally-engaged Pol II. In contrast, the w-Adh and Adh-w silencing is transcriptional. The posttranscriptional silencing of Adh correlates with the appearance of 21-25 bp sense and antisense RNAs as occurs with virus and PTGS silencing in plants and RNAi in flies (Pal-Bhadra, 2002).
Mutations have been recovered in C. elegans that are defective for RNAi. Homologs to these genes exist throughout the plant and animal kingdoms. One of these mutations, rde1, has several related gene family members in flies. A homozygous viable member of this group was tested for its impact on transgene silencing. The piwi mutation drastically reduces the magnitude of posttranscriptional silencing. Surprisingly, piwi also had a strong impact on the transcriptional silencing of Adh-w by w-Adh. This result indicates that under certain circumstances the two types of silencing can be mechanistically related (Pal-Bhadra, 2002).
When one to five copies of full-length Adh transgenes are introduced in the genome, the steady-state RNAs accumulate in direct correlation with copy number. However, at higher dosage (six to ten), Adh RNA levels depart dramatically from a linear relationship with transgene copy number. The transgene analyzed contains all the Adh sequences required for normal function. Five single insert strains showing minimal positional effects were selected. By combining these insertions via genetic crosses, a series of Adh stocks was generated that carry one to ten copies. Each stock is also homozygous for the endogenous transformation recipient allele, Adhfn6. This allele is defective at the 3' splicing acceptor of the first intron, resulting in a longer transcript. The endogenous allele produces only 5%-10% of the steady-state level of a normal Adh gene (Pal-Bhadra, 2002).
To determine whether the endogenous gene was silenced in concert with the transgenes, an RNase protection assay was used that distinguishes the two RNAs. The normal full-length Adh RNA protects two fragments, 142 and 160 bp, while the Adhfn6 RNA protects a 355 bp fragment due to defective splicing. To correct for loading differences, a ß-tubulin probe was included, which protects a 70 bp fragment. The level of each protected fragment (142/70 or 160/70 ratio) was reduced at higher dosage. The amount of endogenous Adhfn6 transcripts did not show any significant difference in one to five copies, suggesting an equal expression. However, these transcripts followed a similar trend as those from the transgenes at higher dosage (Pal-Bhadra, 2002).
To test for any positional influence on Adh transgene silencing, separate sets of one to nine copy stocks were examined. A similar level of Adh expression at any one dosage suggests that the silencing is not significantly affected by the insertion sites. RNA in situ analyses in embryos the Adhfn6 strain, as well as with one, five, and seven Adh transgene copies, indicates a linear increase of Adh expression to five copies. Silencing of the seven copy stock was already evident during blastoderm, the stage where Adh RNA is accumulated initially (Pal-Bhadra, 2002).
To determine whether the Adh transgene silencing is transcriptional or posttranscriptional, nuclear run-on assays were performed. ß-tubulin was included as an internal control in order to determine the relative amount of Adh transcription, while lacZ acted as a negative control (Pal-Bhadra, 2002).
Transcription levels were estimated in adult flies carrying zero to ten copies of full-length Adh transgenes. The results reveal that the endogenous Adhfn6copies, present in each stock, are transcribed similarly to two normal copies. In the one to ten copy series, the amount of transcription increases proportionally to the transgene copy number relative to ß-tubulin (Pal-Bhadra, 2002).
A similar experiment was performed using flies that contain selected genotypes from zero to six copies of the w-Adh hybrid constuct. In the absence of transgenes, the Adhfn6 allele is transcribed at the normal level. When one copy of the w-Adh transgene is present, the transcript level is increased as expected. In contrast, two copies of w-Adh exhibit a plateau of transcript accumulation. In the presence of more w-Adh copies (four to six), total Adh transcription is reduced progressively. The transcribed RNA in four to six copy stocks is below the level produced by the Adhfn6 alleles alone. The amount of Adh transcription in males is always greater than in females with equal dosage (Pal-Bhadra, 2002).
Adh transcriptional level (as detected using run-on assays), produced by multiple full-length Adh or w-Adh transgenes, was compared to steady-state mRNA levels produced by the same genotypes, as determined in Northern analyses. In the Adh series, the amount of mRNA is proportional to dosage from one to five, while in six to ten copies, the steady-state levels depart from linearity. This difference from the run-on assays indicates that silencing of full-length Adh transgenes is posttranscriptional. On the contrary, with the w-Adh dosage series, a similar curve was found when the transcription level and steady-state RNA were compared. A parallel pattern of reduction of both nascent and mature RNA suggests that w-Adh silencing, which includes an effect upon the endogenous Adh gene, is transcriptional. The reason for the different modes of silencing of the two types of transgenes is not known (Pal-Bhadra, 2002).
The silencing of w-Adh and the endogenous Adh locus can also be extended to an Adh-white (Adh-w) transgene that is the reciprocal construct of w-Adh. Run-on transcription analysis of the Adh-w transgene in a w deficiency background with varying numbers of w-Adh indicates that this type of silencing also occurs on the transcriptional level. A comparison of the run-on data with a previous Northern analysis of Adh-w with increasing w-Adh copies shows a similar relationship. The Adh-w + 1 w-Adh and Adh-w + 4 w-Adh genotypes have nearly identical levels of reduction in gene expression in the two assays. There is a residual amount of transcription in the Adh-w + 2 w-Adh genotype but no detectable RNA in the Northern analysis, which might suggest that a combination of PTGS and TGS is operating. However, these data points lie near the limit of detection for the two techniques and likely differ by chance due to exposure time because the same genotype assayed subsequently by both methods shows a similar low level of expression. Thus, at the developmental stage examined (adults), the silencing is predominantly, if not exclusively, transcriptional (Pal-Bhadra, 2002).
Biochemical experiments on RNA interference (RNAi) in Drosophila have shown that double-stranded RNAs in the form of 21-25 nt fragments are generated and these fragments are used in the sequence-specific degradation of mRNA. Therefore, the in vivo existence of such RNAs was tested in the case of Adh silencing. The 21-25 nt antisense Adh RNAs were strongly accumulated in the stocks that contain six to ten copies of the Adh transgene. At the lower doses, the same RNA was not detected or was present at very low amounts in the four and five copy stocks. Using an antisense probe for the detection of sense fragments, a similar-sized RNA was found in the silencing doses (Pal-Bhadra, 2002).
Whether transcriptional silencing induced by w-Adh transgenes was associated with small RNAs was tested by analyzing stocks containing zero to six copies of w-Adh. Hybridization with sense or antisense Adh probes revealed that low molecular weight RNA was not found in abundance at any w-Adh dosage (Pal-Bhadra, 2002).
To investigate the nature of PTGS, it was reasoned that an induced hsp70-Adh construct with four Adh transgenes would bring the Adh expression over the silencing threshold. The hsp70-Adh construct contains a heat shock promoter and the Adh reporter gene. Adh RNA from adult flies was measured relative to ß-tubulin controls in three different circumstances: no heat, heat shock (37°C for 45 min), and 20 hr after heat shock. In flies with five full-length Adh copies, the amount of RNA in heat shock and 20 hr after heat shock did not change relative to that of the untreated flies (Pal-Bhadra, 2002).
The heat treatment of adult flies carrying two copies of the hsp70-Adh gene increased their RNAs as expected. In contrast, the heat treatment of a stock that carries four copies of the Adh transgene and in addition an hsp70-Adh gene causes a rapid loss of Adh mRNA. A similar response was found in embryos. The heat shock induced increase in Adh RNA appears to surpass the threshold limit that triggers silencing. A time course sampling of RNA after heat shock showed that Adh mRNA levels were partially recovered 20 hr subsequent to heat treatment. Interestingly, the transient silencing in this case is in contrast to the systemic spread and prolonged silencing that occurs in C. elegans and plants following a localized induction of posttranscriptional silencing (Pal-Bhadra, 2002).
Whether pulsed threshold induced RNA degradation is correlated with the small species of Adh RNA was tested. The small RNAs were found at a high level in flies with the combination genotype after heat shock. Treatment of the control stock composed only of two hsp70-Adh copies does not produce the small RNAs. This result suggests a threshold level must be exceeded to initiate the synthesis of the small RNAs, which in turn participate in the destruction of the homologous mRNA (Pal-Bhadra, 2002).
Because Polycomb and Polycomb-like (Pcl) mutations have a significant effect on w-Adh transcriptional silencing and Pc-G proteins are bound at the sites of the transgenes under silencing conditions, binding of Pc-G proteins at the Adh transgene insertion sites was tested cytologically. Those sites that do not overlap (1CD and 53B) normal positions of Pc labeling were examined for evidence of binding in the single insert stocks and for the same insert in the ten copy larvae. The Pc proteins were not observed at these sites in either case. Therefore, Pc protein recruitment is not a consequence of posttranscriptional silencing of Adh (Pal-Bhadra, 2002).
A group of genes has been characterized that affects PTGS or RNAi in fungi, plants, and animals. Some of the mutations show sequence similarity to members of the piwi/sting/eIF2C/argonaute gene family conserved from plants to vertebrates. Members of this family, including piwi, have been characterized in Drosophila . To determine whether this mutation has any effect on PTGS and TGS of Adh, the role of this mutation on posttranscriptional silencing was tested. Two or four copies of fully functional Adh genes (two normal endogenous copies ± two transgenes) plus a hsp70-Adh transgene in combination with either heterozygotes or homozygotes of piwi were examined. In the stocks analyzed, a normal Adh allele, rather than Adhfn6, resides on both the balancer chromosome present in heterozygotes and on the chromosome carrying the piwi mutations. These endogenous Adh copies contribute greater amounts of mRNA to the total pool than Adhfn6 (Pal-Bhadra, 2002).
The heat shock and nonheat shock RNAs of the respective stocks were compared in quantitative Northern blots. The stocks that are heterozygous for the piwi 1 or piwi 2 alleles with two copies of the normal Adh gene plus hsp70-Adh exhibit an increase of RNA after heat incubation. In contrast, a sharp reduction of the Adh RNA was found in the five copy (four Adh + hsp70-Adh) stock following heat shock. In piwi homozygotes, the presence of two endogenous genes + hsp70-Adh has a similar pattern of expression as in heterozygotes following heat shock. However, the Adh mRNA level in the four Adh + hsp70-Adh stocks that are homozygous for piwi alleles is restored to nearly normal after heat shock. An almost equal level of Adh expression in nonheat shock and heat shock flies suggests that the piwi mutation disrupts the threshold-based posttranscriptional Adh silencing. The two separate piwi alleles examined as well as their heteroallelic combination, which was used to minimize any influence of linked modifiers, produced the same results (Pal-Bhadra, 2002).
Examination of 21-25 nt RNAs in the piwi-segregating classes shows that the small RNAs are present in the heat shocked class with four Adh genes plus a hsp70-Adh construct when the flies are heterozygous for piwi. In contrast, these RNAs are diminished in the piwi homozygotes that carry the same constellation of Adh genes and that were subjected to heat shock. This result suggests that piwi acts before or during the formation of these small RNAs (Pal-Bhadra, 2002).
As a first measure of the effect on transcriptional silencing, the interaction between the reciprocal w-Adh and Adh-w transgenes was examined because the expression of the latter can be observed phenotypically. One copy of w-Adh reduces and two copies nearly eliminate the expression of Adh-w. The w-Adh transgenes among themselves participate in silencing and include endogenous Adh. The w-Adh/Adh-w silencing interaction is eliminated when the endogenous Adh is deleted from the genome, suggesting that it mediates the reaction via mutually homologous sequences. The expression of the Adh-w transgene can be assayed on the RNA level by probing for white RNA because the transgene is present in a stock with the normal white gene deleted. In the same RNA populations, the silencing of w-Adh and its effect on endogenous Adh can be assayed by probing for total Adh messenger RNA (Pal-Bhadra, 2002).
piwi mutations were introduced into a background with an Adh-w and a w-Adh transgene. The eye color of Adh-w flies, which is reduced in the interaction with one w-Adh copy, is restored to a normal level in piwi1 or piwi2 homozygotes. The heteroallelic combination piwi1/piwi2 shows a similar level of restoration. These combinations of piwi alleles did not have any effect on the eye color of Adh-w in the absence of w-Adh, indicating that their effect is due to a relief of silencing (Pal-Bhadra, 2002).
w mRNA was measured in genotypes with Adh-w and zero to two copies of w-Adh segregating for the piwi alleles. The accumulation of white transcripts in the Adh-w/Y flies without a w-Adh transgene was not significantly different in the presence of the mutations. As expected, the w transcripts from Adh-w were reduced from the normal level in the presence of one copy of w-Adh. However, the presence of the homozygous mutations restored the levels significantly but not completely to the normal amount. Moreover, the w transcripts from Adh-w were significantly restored in mutant homozygotes carrying two w-Adh copies (Pal-Bhadra, 2002).
The same RNA blots were hybridized using an Adh probe to estimate the effect on w-Adh/endogenous Adh silencing. The data revealed that the chromosomes carrying either allele of piwi slightly increased the Adh mRNA in the Adh-w flies without w-Adh copies compared to the balancer chromosomes. The effect of the mutant-bearing chromosome may reflect variation at linked modifiers or at Adh itself. In the Adh-w/Y;w-Adh/+ and Adh-w/Y;w-Adh/w-Adh flies, the total Adh expression is significantly reduced as expected when piwi is heterozygous. This Adh expression is minimally increased in piwi homozygotes, although the increase in these classes is likely due to the variation on chromosome 2, rather than a release from silencing. The magnitude of this slight increase is similar with Adh-w alone and in the presence of one or two w-Adh copies, suggesting that the mutations do not interfere with silencing at this step. These data suggest that piwi has no effect on w-Adh/Adh silencing in contrast to the effect on Adh-w (Pal-Bhadra, 2002).
Run-on analysis was conducted on heterozygous and homozygous genotypes to determine the influence of piwi on the transcriptional silencing of Adh-w. The heteroallelic mutant combination has no impact on Adh-w expression in the absence of w-Adh transgenes. In the heterozygous genotypes, introduction of one or two w-Adh copies causes a progressive reduction of Adh-w expression. The magnitude of reduction in expression in the transcriptional assay is quite similar to the Northern analysis, again indicating that the silencing of Adh-w at the adult stage does not appear to have a posttranscriptional component. In the heteroallelic piwi mutant flies, Adh-w transcription is only slightly diminished by the addition of one or two copies of w-Adh. These results indicate that piwi mutations interfere with the transcriptional silencing of Adh-w (Pal-Bhadra, 2002).
Thus, a single transcription unit, namely Alcohol dehydrogenase, can experience two types of transgene silencing: transcriptional and posttranscriptional. The w-Adh/Adh/Adh-w silencing is transcriptional as might have been anticipated from the involvement of the Polycomb chromatin complex. In contrast, the full-length Adh transgene silencing is posttranscriptional. The molecular features of this silencing follow those established from biochemical studies of RNAi. In other words, the specific loss of Adh messenger RNA is accompanied by the appearance of small sense and antisense 21-25 nt length RNAs (referred to as small interfering RNAs, siRNAs). The appearance of these RNAs and the degradation of Adh messenger RNA require a certain threshold over which the silencing is triggered. When these in vivo data are considered together with in vitro analyses of RNAi, it is reasonable to suggest that at a certain concentration of Adh messenger RNA, a double-stranded RNA moiety is transiently formed, presumably by a form of RNA-dependent, RNA polymerase activity. These molecules would then be cleaved to form the siRNAs by an RNase type III nuclease and subsequently incorporated into a larger RNase complex (RISC) to specifically target the homologous mRNA for enzymatic destruction. It is noted, however, that until further study is performed, it remains a formal possibility that the siRNAs form by an alternative pathway that does not involve double-stranded RNA (Pal-Bhadra, 2002).
The piwi gene is a member of a family including the RNAi defective 1 (rde1) gene of C. elegans, which was isolated for its failure to support RNAi. Family members contain a PAZ domain (Cerutti, 2000) that is characteristic of several gene products involved with RNAi. This study demonstrates that piwi mutation blocks the posttranscriptional silencing of Adh, including the production of siRNAs. In contrast, rde1 does not inhibit siRNA formation during RNAi. Another member of this gene family, the aubergine locus, interferes with the germline silencing of the Stellate genes present on the X chromosome (Aravin, 2001). This silencing must occur for male fertility and is accomplished by repeated genes on the Y chromosome that generate siRNAs in conjunction with Stellate. The product of another family member, the argonaute2 gene, is associated with the RISC complex. Thus, several members of this gene family have been implicated in PTGS at various steps (Pal-Bhadra, 2002).
It was of interest to determine whether genes involved with posttranscriptional silencing might also have an impact on transcriptional silencing. Several lines of evidence from plant research have suggested a connection between silencing via RNA in the cytoplasm and changes in the nucleus, although no previous data in animal species have indicated such a relationship. For example, cDNAs of plant viruses (or other sequences incorporated into the virus) transformed into the nucleus are undermethylated until infection by the corresponding virus. The presence of the virus in the cytoplasm undergoing silencing causes a hypermethylation of the sequences in the nucleus. These DNA modifications are coincident in length with the homologous portion carried in the virus. While the mechanism of this RNA-directed DNA methylation is unknown, it is presumed to involve an RNA-DNA interaction. In addition, ectopic transcription of promoter sequences will cause transcriptional silencing of transgenes with a homologous promoter driving another reporter gene (Pal-Bhadra, 2002).
The piwi mutations inhibit the transcriptional silencing of Adh-w. Two aspects of the Adh-w silencing can be assayed by probing for either white or Adh messenger RNA. The silencing of w-Adh and its effect on endogenous Adh can be monitored by examining the amount of Adh RNA. With increasing dosage of w-Adh, the total Adh RNA declines. This trend is not affected by the mutations. The silencing of Adh-w itself can be determined phenotypically and by measuring the amount of w RNA. Typically, the expression of Adh-w declines with increasing dosage of w-Adh, but this response is strongly diminished in homozygotes of piwi (Pal-Bhadra, 2002).
While the evidence presented here suggests a relationship between posttranscriptional and transcriptional silencing, the basis for this connection is unknown. The step in the silencing of Adh-w that is affected by piwi requires the presence in the nucleus of the 5' regulatory sequences of Adh. These sequences are not known to be transcribed under normal circumstances (Pal-Bhadra, 2002).
There are several possibilities to draw a connection between an RNAi-like mechanism and this transcriptional silencing. First, there may be undetected, transient transcription of the Adh regulatory sequences, which in turn form homologous siRNAs. These may act in the nucleus to trigger a chromatin change at the complementary regulatory regions in an analogous fashion to experimental transcription of promoters in tobacco. The piwi mutations might block such a mechanism by inhibiting the formation of the siRNAs homologous to the regulatory sequences. Considering this scenario, in the case of the w-Adh/Adh-w interaction, one must postulate that increased dosage of w-Adh would increase the amount of ectopic transcription of the endogenous Adh regulatory sequences and that the Polycomb complex becomes targeted to Adh-w in conjunction with a homologous interaction between the siRNAs and the regulatory region of Adh-w (Pal-Bhadra, 2002).
If small RNAs trigger transcriptional silencing, it is of interest why w-Adh/Adh silencing is unaffected by piwi. One potential explanation is that the RNA involvement is needed only for the establishment of transcriptional silencing but not its maintenance through to the adult stage. The initiation of w-Adh/Adh silencing in early embryogenesis precedes that of Adh-w. It is possible that maternal contributions of piwi product in early embryos are sufficient for initiation of w-Adh/Adh silencing but are depleted to effective levels by the time of the establishment of Adh-w silencing (Pal-Bhadra, 2002).
Another explanation suggests that the piwi gene product may play dual roles in an RNAi-like mechanism and transcriptional silencing. One possibility is that it could affect some aspect of gene-to-gene association that might trigger transcriptional silencing. Pairing of transgenes or other intranuclear interactions appears to have an impact on transcriptional gene silencing. Indeed, the w-Adh transgene is silenced much more effectively when paired between homologs than when two dispersed copies are present in the nucleus. If the piwi product participates in some aspect of sequence recognition, nucleic acid associations or protein-nucleic acid interactions, its elimination might block certain steps of both posttranscriptional and transcriptional silencing. It remains a possibility that gene-to-gene associations might trigger silencing as well as dsRNA-to-gene interactions with the two acting independently but by a related mechanism. Finally, the connection between the two types of silencing could be indirect, with piwi blocking a function secondarily removed from the transcriptional silencing but that is required nevertheless (Pal-Bhadra, 2002).
Certainly, members of this gene family have diverse roles in the cell. A previously identified function of the nucleoplasmic product of piwi indicates its requirement for germline stem cell renewal. The aubergine product is required for dorsoventral patterning of the early embryo, which is mediated by an enhancement of the translation of the oskar mRNA. The aubergine product is also implicated with the expression of the Stellate (Ste) repeats and Suppressor of Stellate [Su(Ste)] genes on the X and Y chromosomes. The argonaute1 gene is required early in Drosophila embryogenesis for proper development. In mammals, a member of this gene family, eIF2C, functions in translation initiation via tRNA association with messenger RNA, while in Arabidopsis the argonaute mutation affects the functions of the meristem. In C. elegans, Drosophila, and human cells, related genes play a role in the maturation of small regulatory RNAs involved in the temporal control of development. The data presented here suggest that they can also play a direct or indirect role in transcriptional regulation. Clearly, many aspects of development and cellular metabolism use these gene products, raising the possibility that the transposon and virus defense functions may have been co-opted early in evolution from genes involved with other regulatory processes (Pal-Bhadra, 2002).
The homeotic genes controlling segment identity in Drosophila are repressed by the Polycomb group of genes (PcG) and are activated by genes of the trithorax group (trxG). An F1 screen for dominant enhancers of Polycomb yielded a point mutation in the heat shock cognate gene, hsc4, along with mutations corresponding to several known PcG loci. The new mutation is a more potent enhancer of Polycomb phenotypes than is an apparent null allele of hsc4, although even the null allele occasionally displays homeotic phenotypes associated with the PcG. Previous biochemical results had suggested that HSC4 might interact with Brahma, a trxG member. Further analyses now show that there is no physical or genetic interaction between HSC4 and the Brahma complex. HSC4 might be needed for the proper folding of a component of the Polycomb repression complex, or it may be a functional member of that complex (Mollaaghababa, 2001).
The hsc4 gene encodes the major heat shock
cognate protein, which is expressed at all times of development, but is
particularly abundant in ovaries and embryos. A variety of
phenotypes have been associated with hsc4 mutations. Removal
of both the zygotic and maternal contributions of hsc4
causes embryonic lethality accompanied by variable segmental deletions. Loss of zygotic activity alone leads to larval lethality.
These dying larvae develop melanotic tumors and are defective in
the development and projection of the larval optic nerve, referred to
as Bolwig's nerve. Heterozygotes for all alleles show malformations of the fourth abdominal tergite in a small fraction of adults. A dominant negative allele of hsc4, hsc4195, modulates Notch signaling during development. The same allele enhances the dominant adult phenotypes of mutations in the ecdysone receptor. This study describes the interaction with mutations in the PcG, that constitutes another distinctive class of phenotypes. A seemingly contradictory enhancement of trithorax mutation, itself an antagonist of Polycomb, is reported. One mechanism that might connect all of these phenotypes is the one suggested by the protein sequence of HSC4, namely, the protein folding process. Protein targets of the HSC4 chaperone function might be involved in many diverse developmental pathways (Mollaaghababa, 2001).
The interaction of the hsc454.1
allele with Polycomb is distinctive. The HSC4 protein is not
particularly limiting, because heterozygotes for a null allele appear to be nearly wild type. Heterozygotes for
hsc454.1 do show a phenotype,
suggesting that the hsc454.1 protein
product might act as a poison. Perhaps the hsc454.1 protein product sequesters a target protein or misfolds it into a nonfunctional product. If Polycomb were the target, this could inactivate 50% of the Polycomb protein, assuming half of the Polycomb nascent chains interact with the mutant
form of HSC4. A 50% reduction in functional Polycomb would explain the
weak phenotypes of hsc454.1/+, and the
lethality of hsc4 alleles with ph410, because another ph-proximal null, ph409,
is lethal in a Pc-/+ background (Mollaaghababa, 2001).
An alternative explanation for the
hsc454.1 phenotype is that HSC4 is
more directly involved in the process of Polycomb group repression. HSC4 has been identified among the components of the Polycomb complex purified from Drosophila embryos. Because HSC4 is so abundant, it could be merely a contaminant, but the purification includes immunoprecipitation of an epitope-tagged complex, and it was demonstrated that some HSC4 protein coelutes with the complex on a sizing column. It is possible that HSC4 remains bound to one of its targets and becomes incorporated into the complex with that protein. It is also possible that HSC4 has a catalytic role. HSC4 is related to a variety of ATPases, including actin. A bacterial homolog of HSC4, DnaK, is thought to rearrange the protein complex involved in phage lambda DNA replication. Other actin-related proteins have been found in the SWI/SNF and RSC chromatin remodeling complexes, and
it has been suggested that they catalyze conformational changes in
those complexes or their substrates. HSC4 could have a similar
function in PRC1 repression (Mollaaghababa, 2001).
In Drosophila, relocation of a euchromatic gene near centromeric or telomeric heterochromatin often leads to its mosaic silencing. Nevertheless, modifiers of centromeric silencing do not affect telomeric silencing, suggesting that each location requires specific factors. Previous studies suggest that a subset of Polycomb-group (PcG) proteins could be responsible for telomeric silencing. This study presents the effect on telomeric silencing of 50 mutant alleles of the PcG genes and of their counteracting trithorax-group genes. Several combinations of two mutated PcG genes impair telomeric silencing synergistically, revealing that some of these genes are required for telomeric silencing. In situ hybridization and immunostaining experiments on polytene chromosomes reveal a strict correlation between the presence of PcG proteins and that of heterochromatic telomeric associated sequences (TASs), suggesting that TASs and PcG complexes could be associated at telomeres. Furthermore, lines harboring a transgene containing an X-linked TAS subunit and the mini-white reporter gene can exhibit pairing-sensitive repression of the white gene in an orientation-dependent manner. Finally, an additional binding site for PcG proteins was detected at the insertion site of this type of transgene. Taken together, these results demonstrate that PcG proteins bind TASs in vivo and may be major players in Drosophila telomeric position effect (TPE) (Boivin, 2003).
Among the 50 mutant alleles of PcG and trxG genes tested, <10 behave as dominant modifiers of TPE. By contrast, combination analyses reveal that 10 alleles that have no effect alone have synergistic effects on TPE.
Interestingly, the subgroup of dominant suppressors that act alone on TPE (Pc, ph, Psc, and Scm) are members of the PRC1 complex that has been purified from embryonic nuclear extracts. Some other PcG mutations, such as Asx, E(z), Pcl, or Sce, act as suppressors in combination, suggesting that the products of these genes participate with a specific telomeric PcG complex. Strikingly, this subgroup of eight PcG genes was already highlighted in a genetic interaction study showing that Pc, Scm, Psc, Pcl, Sce, and Asx are lethal when heterozygous with ph2, a temperature-sensitive mutation, all combinations leading to similar phenotypes in the dying embryos (Boivin, 2003).
It has been shown that telomeric inserts are less accessible than euchromatic inserts to restriction enzymes and to DAM methylase. In addition, the accessibility of telomeric inserts to DAM methylase increases in a ph410 background and this is correlated to derepression of the white gene. This result is similar to that obtained with the ph PRE-mini-white transgenes suggesting that PcG products adopt a similar chromatin-based mechanism to repress their euchromatic and telomeric targets (Boivin, 2003).
PREs were initially identified by their ability to prevent ectopic activation of a Hox reporter gene construct. This capacity depends on the dose of the PcG proteins. Placed in a transgene, PREs can also induce mosaic expression of the flanking reporter gene, a phenotype resembling that of PEV and TPE. Moreover, PRE-mediated repression often exhibits pairing sensitivity, defined as the lower expression of the flanking reporter gene in a homozygous state than in a heterozygous one. This study shows that a 1.2-kb fragment of the 1.8-kb X-chromosome TAS induces variegation or pairing-sensitive repression in an orientation-dependent manner and creates new binding sites for the PcG proteins as detected by immunostaining on polytene chromosomes. These results demonstrate that this TAS fragment mimics some properties of a PRE and thus reinforce the parallels that can be made between telomeric silencing and PcG-mediated euchromatic repression. TASs from the left tip of chromosome 2 (2L-TAS) retain aspects of telomeric silencing in ectopic positions. At this telomere, TASs are composed of repeats of 457 bp that present only limited homology with TASs present at the X, 2R, and 3R telomeres. Analysis of the sequence of one repeat (457 bp) revealed nine GAF-binding sites but no PHO-binding site. Several transgenic lines have been establised carrying different constructs made up of 6 kb of 2L-TAS (~13 repeats) adjacent to the mini-white reporter gene and flanked by Su(Hw) insulator sequences. Depending on the orientation of the TASs inside the transgene, some lines present reduced expression of the mini-white gene when compared to lines carrying a similar transgene without TASs or with TASs in the opposite orientation. Such orientation-dependent silencing has been described for the Fab7 PRE of the Ubx gene, but does not appear to be a general property of PREs since another PRE from Ubx (Mcp) has been shown to function in both orientations . From this study, the more efficient orientation for the 1.2-kb X-TAS-induced repression appears to be the same as that described for the 2L-TASs: repression appears to be stronger from the centromere-proximal side (Boivin, 2003).
Repression induced by the 2L-TAS when inserted within a transgene is weakly sensitive to Su(z)25. Surprisingly, no effect of PcG mutations on the repression induced by the 1.2-kb X-TASs could be detected, except a slight suppressor effect of Su(z)25 on P-CoT-1 in a homozygous state. At the moment, no explanation is available for why the repression induced by the 1.2-kb X-TASs in a euchromatic environment is not sensitive to modification of the dose of PcG proteins that could otherwise affect TPE (Boivin, 2003).
Increasing the distance between the 2L-TAS and the mini-white gene with 2.4 kb of unrelated DNA in another transgene did not change the silencing capacity of 2L-TAS. In this study, the 1.2 kb of X-TAS is located >5 kb away from the mini-white gene, thus showing the silencing capability of TASs over an extended distance. Similar results were obtained with transgenes containing the Fab7 PRE. According to chromatin-immunoprecipitation experiments, PcG products can spread as far as 10-15 kb from PREs and repression could be expected to occur over such a distance (Boivin, 2003).
In fact, what was observed with the 1.2 kb of X-TAS in the pCoT- transgenes resembles what has been observed with PREs from the Bithorax complex. Using Fab7-mini-white transgenes, it has been shown that some insertion sites present pairing sensitivity (as observed with P-CoT-2 and P-CoT-3), while others present variegation with darker spots (as observed with P-CoT-1). The Fab7 PRE has been shown to convey a derepressed state through meiosis after being activated in the embryonic stage by the UAS/GAL4 system. In the case of TPE, the derepressed state observed in a PcG mutant background is not transmitted to the next generation. A fundamental difference between these studies is that the suppressor effect observed in the case of TPE is due to the lack of one PcG partner. It is hyperactivation (forced activation) induced by GAL4 via the UAS sequences that abolishes the repressor capacity of the Fab7 PRE. This activation likely involves fundamental changes in chromatin conformation and/or epigenetic marks (such as hyperacetylation) that may be different from the effect of a decrease in the dosage of a repressor. To compare TPE and the Fab7 PRE it would thus be interesting to test transmission through meiosis of the derepressed state of the UAS-Fab7 transgene induced by a PcG mutation rather than upon activation by GAL4. Different PREs thus share properties but also present particularities that likely depend on their sequence. Indeed, the dissection of Mcp, another PRE from the Bithorax complex, revealed that repression in cis and pairing-sensitive repression could be separated. This shows that PREs may combine several regulatory properties and future dissection of the different TASs will tell which functions telomeric PREs combine (Boivin, 2003).
The wave of differentiation that traverses the Drosophila eye disc requires rapid transitions in gene expression that are controlled by a number of signaling molecules also required in other developmental processes. A mosaic genetic screen has been used to systematically identify autosomal genes required for the normal pattern of photoreceptor differentiation, independent of their requirements for viability. In addition to genes known to be important for eye development and to known and novel components of the Hedgehog, Decapentaplegic, Wingless, Epidermal growth factor receptor, and Notch signaling pathways, several members of the Polycomb and trithorax classes of genes, encoding general transcriptional regulators, were identified. Mutations in these genes disrupt the transitions between zones along the anterior-posterior axis of the eye disc that express different combinations of transcription factors. Different trithorax group genes have very different mutant phenotypes, indicating that target genes differ in their requirements for chromatin remodeling, histone modification, and coactivation factors (Janody, 2004).
Very similar phenotypes were observed in clones mutant for Pc or E(z), which encode components of two distinct complexes implicated in transcriptional repression. Although likely null alleles for both genes were used, the phenotype of E(z) clones appeared slightly stronger, with a greater likelihood of derepressing hth in posterior regions of the eye disc. The E(z) protein has been shown to act as a histone methyltransferase for H3 K27 within a complex that also includes Extra sex combs (Esc), Suppressor of zeste 12 [Su(z)12], and the histone-binding protein NURF-55. esc appears to act only early in embryonic development, while E(z) and Su(z)12 are continuously required to repress inappropriate homeotic gene expression in wing imaginal discs. The core PRC1 complex contains Pc, as well as Ph, Psc, and dRing1, and can prevent SWI/SNF complexes from binding to a chromatin template. Pc, Psc, and ph are all required to prevent homeotic gene misexpression in wing discs; however, Psc and ph act redundantly with closely related adjacent genes. The two complexes are thought to be linked through binding of the Pc chromodomain to K27-methylated H3. The stronger phenotype of E(z) mutations in the eye disc might suggest that methylation of H3 K27 can recruit other proteins in addition to Pc (Janody, 2004).
In the eye disc, loss of E(z) or Pc leads to misexpression of the homeotic gene Ubx, but this does not seem to account for the entire phenotype. Although Ubx is sufficient to turn on tsh ectopically, misexpression of hth and tsh can occur in E(z) or Pc clones in which Ubx is not misexpressed. This suggests that hth and tsh are either direct targets of Pc/E(z)-mediated repression or targets of a downstream gene other than Ubx, possibly one of the homeotic genes not examined. Tsh misexpression would be sufficient to explain the suppression of photoreceptor differentiation in clones close to the morphogenetic furrow, since it is able to maintain expression of Hth and Ey and, in combination with them, to repress eya. Misexpression of Tsh can also account for overgrowth of Pc or E(z) mutant cells at the posterior margin of the eye disc (Janody, 2004).
trithorax group genes were initially identified as suppressors of Polycomb phenotypes and are therefore thought to contribute to the activation of homeotic gene expression. Some members of the group encode components of the Brahma chromatin-remodeling complex, others encode components of the mediator coactivation complex, and still others encode histone methyltransferases. In addition to their distinct biochemical functions, members of the trithorax group act on different sets of target genes during eye development and can also have different effects on the same target genes. Components of the Brahma complex are strongly required for cell growth and/or survival; brm and mor, but not osa, are also absolutely required for photoreceptor differentiation. However, these three genes do not seem to be required for the restricted expression in anterior-posterior domains of the eye disc of the transcription factors examined. In contrast, the mediator complex subunits encoded by skd and kto are not required for cell proliferation, although they are strongly required for photoreceptor differentiation. trx, which encodes a histone methyltransferase, is required primarily for the normal development of marginal regions of the disc. No significant effect on photoreceptor differentiation were seen in clones mutant for kismet1, which encodes chromodomain proteins, or ash21, which encodes a PHD protein. These differences are unlikely to be due to different expression patterns of the trithorax group genes, since Trx, Skd, Kto, and Osa are ubiquitously expressed in the eye disc (Janody, 2004).
The effects of these genes on the rapid transitions between domains of expression of different transcription factors are of particular interest. In the most anterior region of the eye disc, hth expression is enhanced by skd and kto. The domain just posterior to this also expresses tsh and ey, and activation of both of these genes requires trx. However, skd and kto have opposite effects on the two genes, enhancing tsh expression and preventing the maintenance of ey expression in posterior cells. Since Hth and Tsh can positively regulate each other's expression, it is possible that only one of these genes is directly dependent on skd and kto. Next, dac and h are activated transiently and eya is activated and sustained. The establishment of both dac and eya is delayed in trx mutant clones, and h expression is reduced. This delay in establishing the preproneural domain may be due to the failure to activate ey and tsh earlier in development, since Ey and Tsh combine to activate eya. The effect of Pc or E(z) mutations on eya, dac, and h appears very similar to the effect of trx mutations. However, in Pc or E(z) clones, the delay in eya and dac expression is likely to be caused by the failure to repress tsh and hth, since the combination of these two proteins has been shown to repress genes expressed in the preproneural domain. skd and kto clones also show a reduction in h and anterior eya expression, but an inappropriate maintenance of dac and dpp. These mediator complex components may thus contribute both to the activation of genes such as h in the preproneural domain and to the activation of unknown genes that shut off the expression of ey, dac, and dpp. Alternatively, skd and kto could be directly involved in the repression of these genes. Finally, trx is important to prevent misexpression of hth in cells near the posterior and lateral margins. Although Dpp normally represses hth, in trx mutant clones dpp and hth are both inappropriately expressed in marginal cells. This may reflect a role for trx in the process of morphogenetic furrow initiation, perhaps contributing to the ability of dpp to control gene expression (Janody, 2004).
Further study will be needed to determine which genes are direct targets of each trithorax group protein. However, the results point to a strong specificity of these general transcriptional regulators, suggesting that they may be specialized to mediate the effects of particular signaling pathways or to control specific subsets of downstream genes (Janody, 2004).
The Drosophila trithorax group gene kismet (kis) was identified in a screen for extragenic suppressors of Polycomb (Pc) and subsequently shown to play important roles in both segmentation and the determination of body segment identities. One of the two major proteins encoded by kis (Kis-L) is related to members of the SWI2/SNF2 and CHD families of ATP-dependent chromatin-remodeling factors. To clarify the role of Kis-L in gene expression, its distribution on larval salivary gland polytene chromosomes was examined. Kis-L is associated with virtually all sites of transcriptionally active chromatin in a pattern that largely
overlaps that of RNA Polymerase II (Pol II). The levels of elongating Pol II
and the elongation factors SPT6 and CHD1 are dramatically reduced on polytene
chromosomes from kis mutant larvae. By contrast, the loss of Kis-L
function does not affect the binding of PC to chromatin or the recruitment of
Pol II to promoters. These data suggest that Kis-L facilitates an early step
in transcriptional elongation by Pol II (Srinivasan, 2005).
The Drosophila kismet gene was identified in a screen for
dominant suppressors of Polycomb, a repressor of homeotic
genes. kismet mutations suppress the
Polycomb mutant phenotype by blocking the ectopic
transcription of homeotic genes. Loss of zygotic kismet
function causes homeotic transformations similar to those
associated with loss-of-function mutations in the homeotic
genes Sex combs reduced and Abdominal-B. kismet is also
required for proper larval body segmentation. Loss of
maternal kismet function causes segmentation defects
similar to those caused by mutations in the pair-rule gene
even-skipped. The kismet gene encodes several large nuclear
proteins that are ubiquitously expressed along the anteriorposterior
axis. The Kismet proteins contain a domain
conserved in the trithorax group protein Brahma and
related chromatin-remodeling factors, providing further
evidence that alterations in chromatin structure are
required to maintain the spatially restricted patterns of
homeotic gene transcription (Daubresse, 1999).
The genetic interactions between kis and Pc provided the first
clue that kis plays an important role in the determination of
body segment identity. kis mutations suppress
the adult Pc phenotype by preventing the ectopic transcription
of homeotic genes. Thus, kis is a member of the trithorax group
of homeotic gene activators. Mosaic analyses reveal that loss
of kis function causes homeotic transformations, including the
transformation of first leg to second leg and the fifth abdominal
segment to a more anterior identity. These phenotypes are
identical to those associated with loss-of-function Scr and
Abd-B mutations, respectively. Taken together, these findings
suggest that kis acts antagonistically to Pc to activate the
transcription of both Scr and Abd-B.
It is intriguing that kis mutations alter the fate of only the
fifth abdominal segment, since the identities of the fifth through
ninth abdominal segments are determined by a single homeotic
gene, Abd-B (Daubresse, 1999).
Variations in the levels of Abd-B protein result in the
differences between these abdominal segments, with Abd-B
expression being lowest in the fifth abdominal segment. Parasegment-specific
cis-regulatory regions, termed infra-abdominal (iab) regions
control Abd-B expression. Each iab region is
named for the segment that it affects (iab-5 through iab-9).
Mutations in both iab-5 and kis affect
the identity of only the fifth abdominal segment, suggesting
that the Kis protein may interact specifically with the iab-5
cis-regulatory element of Abd-B (Daubresse, 1999).
kis probably interacts not only with Scr and Abd-B,
but with other homeotic genes as well. For example, the
isolation of kis mutations as enhancers of loss-of-function
Deformed (Dfd) mutations suggests that
kis is probably also required to activate transcription of this
ANTC homeotic gene. Furthermore, kis duplications strongly
enhance the transformation of wing to haltere in Pc
heterozygotes, a phenotype caused by the ectopic transcription
of Ubx in the wing imaginal disc.
However, kis mutations do not cause haltere-to-wing
transformations due to decreased Ubx transcription. A possible
explanation for the lack of homeotic transformations in kis
clones in segments other than the prothoracic and fifth
abdominal segment is that the mutations used in these studies
are not null alleles. kis1 is a strong
loss-of-function mutation.
It has not been characterized at the molecular level, however,
and may not completely eliminate kis function. It is also
possible that sufficient levels of Kis protein persist in
homozygous mutant tissue following mitotic recombination to
support normal development. Further genetic studies,
including the analysis of conditional kis alleles, will be
necessary to distinguish between these possibilities (Daubresse, 1999).
Germline clonal analysis has revealed an unanticipated role for kis
in segmentation. Embryos from mosaic kisS females exhibit a
deletion or alteration of every other segment, while mutant
embryos from mothers bearing germline clones of
the stronger kis1 allele usually develop only half
of the normal number of segments. This variation
in phenotypic severity is closely correlated with
the extent to which en expression is disrupted. The
phenotypes associated with loss of maternal kis
function resemble those caused by mutations in
pair-rule segmentation genes that cause the
deletion of the odd-numbered parasegments. kis
thus appears to be necessary for the expression (or
function) of one or more pair-rule genes. Recent
genetic studies have suggested that kis may also be
involved in the Notch signaling pathway.
Thus it appears that kis plays roles in addition to
the regulation of homeotic genes (Daubresse, 1999).
What pair-rule genes might require kis for their
activity? Based on the kis mutant phenotype,
perhaps the best candidates are eve and hairy (h),
both of which are required for the formation of
odd-numbered parasegments. Unlike eve, h and
most other segmentation genes, kis is uniformly
expressed in the early embryo. This raises the
possibility that Kis functions as an essential
cofactor or modifier of Eve or other pair-rule
proteins. It is also possible that loss of kis function might result
in pair-rule genes being transcribed outside of their normal
expression domains. Additional work will be
necessary to determine the molecular basis of the segmentation
defects resulting from loss of maternal kis function (Daubresse, 1999).
Reversible acetylation of histone tails plays an important role in chromatin remodelling and regulation of gene activity. While modification by histone acetyltransferase (HAT) is usually linked to transcriptional activation, evidence is provided for HAT function in several types of epigenetic repression. Chameau (Chm), a new Drosophila member of the MYST HAT family, dominantly suppresses position effect variegation (PEV), is required for the maintenance of Hox gene silencing by Polycomb group (PcG) proteins, and can partially substitute for the MYST Sas2 HAT in yeast telomeric position effect (TPE). Finally, in vivo evidence is provided that the acetyltransferase activity of Chm is required in these processes, since a variant protein mutated in the catalytic domain no longer rescues either PEV modification, telomeric silencing of SAS2-deficient yeast cells, or lethality of chm mutant flies. These findings emphasize the role of an acetyltransferase in gene silencing, which supports, according to the histone code hypothesis, the observation that transcription at a particular locus is determined by a precise combination of histone tail modifications rather than by overall acetylation levels (Grienenberger, 2002).
To examine whether Chm and PcG proteins act together to maintain Hox gene repression, the effect of a reduction of chm dosage was tested on homeotic transformations that result from mutations affecting either PcG transregulators or a PRE cis-regulatory element. The first PcG dominant phenotype examined was a T2 into T1 transformation. In the second leg disc of Pc male heterozygotes, derepression of Sex comb reduced leads to the formation on the second leg of a sex comb, a structure normally found on the first leg only. The mutation of one copy of chm significantly enhances this phenotype. chm and PcG gene interactions in the specification of adult abdomen identities were tested. In parasegment 9 (PS9) of males heterozygous for PcXT109 or for the ph410 allele of polyhomeotic (ph), inappropriate expression of Abdominal-B (Abd-B) produces a mild transformation of the fourth abdominal segment into the fifth (A4 into A5), as evidenced by patches of pigmentation in the anterior part of A4. This phenotype, which is never observed in a wild-type context, occurs at low frequency in males heterozygous for chm (less than 1%). Double heterozygotes for chm and for ph or Pc exhibit increased A4 into A5 transformation and/or increased number of transformed individuals, compared to single PcG mutants. Finally, the homeotic transformation induced by a PRE mutation was examined. McpB116 affects Abd-B silencing in PS9, giving rise to incomplete A4 into A5 transformations. This homeotic phenotype is stronger in double heterozygotes for chm14 and McpB116 and becomes further enhanced by the mutation of one copy of Pc. In the various genetic contexts reported here, chm was therefore found to genetically interact with PcG genes and the Mcp element in a positive manner. These synergistic effects strongly suggest that Chm collaborates with PcG proteins for PRE-mediated repression at Hox gene loci (Grienenberger, 2002).
Direct evidence for a role of Chm in Hox gene silencing was obtained from the examination of Ubx expression in imaginal discs. Whereas Ubx is not detected in the columnar epithelium of a wild-type wing disc, derepression is observed in few cells from discs heterozygous for Pc, and a more extended activation occurs in discs heterozygous for both chm and Pc. These results confirm that Chm and PcG proteins act together to repress Hox genes. No misexpression of Ubx could be detected, however, in discs from chm homozygous larvae. Thus, chm can be classified as an enhancer of PcG mutations instead of a novel PcG gene (Grienenberger, 2002).
grappa (gpp) is the Drosophila ortholog of the Saccharomyces
cerevisiae gene Dot1, a histone methyltransferase that modifies the
lysine (K)79 residue of histone H3. gpp is an essential gene identified
in a genetic screen for dominant suppressors of pairing-dependent silencing, a
Polycomb-group (Pc-G)-mediated silencing mechanism necessary for
the maintenance phase of Bithorax complex (BX-C) expression.
Surprisingly, gpp mutants not only exhibit Pc-G phenotypes, but
also display phenotypes characteristic of trithorax-group mutants.
gpp dominantly enhances the phenotypic effects of mutations in Sex
combs extra, Polycomblike, Sex combs on the midleg, and Polycomb.
Mutations in gpp also disrupt telomeric silencing but do not affect
centric heterochromatin. These apparent contradictory phenotypes may result from
loss of gpp activity in mutants at sites of both active and inactive
chromatin domains. Unlike the early histone H3 K4 and K9 methylation patterns,
the appearance of methylated K79 during embryogenesis coincides with the
maintenance phase of BX-C expression, suggesting that there is a unique
role for this chromatin modification in development (Shanower, 2005).
Drosophila imaginal disc cells can switch fates by
transdetermining from one determined state to another. The
expression profiles of cells induced by ectopic Wingless expression to
transdetermine from leg to wing were examined by dissecting transdetermined cells and
hybridizing probes generated by linear RNA amplification to DNA microarrays.
Changes in expression levels implicated a number of genes: lamina
ancestor, CG12534 (a gene orthologous to mouse augmenter of liver
regeneration), Notch pathway members, and the Polycomb and trithorax groups of
chromatin regulators. Functional tests revealed that transdetermination was
significantly affected in mutants for lama and seven different
PcG and trxG genes. These results validate the described methods for
expression profiling as a way to analyze developmental programs, and they show that
modifications to chromatin structure are key to changes in cell fate. These
findings are likely to be relevant to the mechanisms that lead to disease when
homologs of Wingless are expressed at abnormal levels and to the manifestation
of pluripotency of stem cells (Klebes, 2005).
When prothoracic (1st) leg discs are fragmented and cultivated in vivo, cells
in a proximodorsal region known as the 'weak point' can switch fate and
transdetermine. These 'weak point' cells give rise to cuticular wing structures.
The leg-to-wing switch is regulated, in part, by the expression
of the vestigial (vg) gene, which encodes a transcriptional activator that is a
key regulator of wing development. vg
is not expressed during normal leg development, but it is expressed during
normal wing development and in 'weak point' cells that transdetermine from leg
to wing. Activation
of vg gene expression marks leg-to-wing transdetermination (Klebes, 2005).
Sustained proliferation appears to be a prerequisite for fate change, and
conditions that stimulate growth increase the frequency and enlarge the area of
transdetermined tissue. Transdetermination was discovered when fragments
of discs were allowed to grow for an extensive period of in vivo culture. More
recently, ways to express Wg ectopically have been used to stimulate cell
division and cell cycle changes in 'weak point' cells (Sustar,
2005), and have been shown to induce transdetermination very efficiently.
Experiments were performed to
characterize the genes involved in or responsible for transdetermination that
is induced by ectopic Wg. Focus was placed on leg-to-wing transdetermination because
it is well characterized, it can be efficiently induced and it can be monitored
by the expression of a real-time GFP reporter. These attributes make it
possible to isolate transdetermining cells as a group distinct from dorsal leg
cells, which regenerate, and ventral leg cells in the same disc, which do not
regenerate; and, in this work, to directly define their expression profiles.
This analysis identified unique expression properties for each of these cell
populations. It also identified a number of genes whose change in expression
levels may be significant to understanding transdetermination and the factors
that influence developmental plasticity. One is lamina ancestor (lama), whose
expression correlates with undifferentiated cells and is shown to control the area
of transdetermination. Another has sequence similarity to the mammalian
augmenter of liver regeneration (Alr; Gfer -- Mouse Genome Informatics), which
controls regenerative capacity in the liver and is upregulated in mammalian
stem cells. Fifteen regulators of chromatin structure [e.g.
members of the Polycomb group (PcG) and trithorax group (trxG)] are
differentially regulated in transdetermining cells, and mutants in seven of
these genes have significant effects on transdetermination. These studies
identify two types of functions that transdetermination requires -- functions
that promote an undifferentiated cell state and functions that re-set chromatin
structure (Klebes, 2005).
The importance of chromatin structure to the transcriptional state of
determined cells makes it reasonable to assume that re-programming cells to
different fates entails reorganization of the Polycomb group (PcG) and
trithorax group (trxG) protein complexes that bind to regulatory elements. Although
altering the distribution of proteins that mediate chromatin states for
transcriptional repression and activation need not involve changes in the
levels of expression of the PcG and trxG proteins, the array
hybridization data was examined to determine if they do. The PcG Suppressor of zeste
2 [Su(z)2] gene had a median fold repression of 2.1 in eight TD
to DWg/VWg comparisons, but the
cut-off settings did not detect significant enrichment or repression of most
of the other PcG or trxG protein genes with either clustering analysis or the
method of ranking median ratios. Since criteria for assigning biological
significance to levels of change are purely subjective, the
transdetermination expression data was re-analyzed to identify genes whose median ratio
changes within a 95% confidence level. Fourteen percent of the genes satisfied
these conditions. Among these genes, 15/32
PcG and trxG genes (47%) had such statistically significant
changes.
Identification of these 15 genes with differential expression suggests that
transdetermination may be correlated with large-scale remodeling of chromatin
structure (Klebes, 2005).
To test if the small but statistically significant changes in the
expression of PcG and trxG genes are indicative of a functional role in
determination, discs from wild-type, Polycomb
(Pc), Enhancer of Polycomb [E(Pc)], Sex comb on
midleg (Scm), Enhancer of zeste [E(z)],
Su(z)2, brahma (brm) and osa (osa) larvae were examined.
The level of Wg induction was adjested to reduce the frequency of
transdetermination and both frequency of transdetermination and
area of transdetermined cells was determined. The frequency of leg discs
expressing vg increased significantly in E(z), Pc, E(Pc), brm
and osa mutants, and the frequency of leg to wing transdetermination in
adult cuticle increased in Scm, E(z), Pc,
E(Pc) and osa mutants. Remarkably, Su(z)2
heterozygous discs had no vg expression, suggesting that the loss of
Su(z)2 function limits vg expression (Klebes, 2005).
Members of the PcG and trxG are known to act as heteromeric complexes by
binding to cellular memory modules (CMMs). The functional tests demonstrate
that mutant alleles for members of both groups have the same functional
consequence (they increase transdetermination frequency). The findings are
consistent with recent observations that the traditional view of PcG members
as repressors and trxG factors as activators might be an oversimplification,
and that a more complex interplay of a varying composition of PcG and trxG
proteins takes place at individual CMMs.
Furthermore the opposing effects of Pc and Su(z)2 functions are consistent
with the proposal that Su(z)2 is one of a subset of PcG genes that is required
to activate as well as to suppress gene expression. In
addition to measuring the frequency of transdetermination,
the relative area of vg expression was examined in the various PcG and trxG
heterozyogous mutant discs. The relative area decreased in E(Pc),
brm and osa mutant discs, despite the increased frequency of
transdetermination in these mutants. There is no evidence to explain these
contrasting effects, but the roles in
transdetermination of seven PcG and trxG genes that were identified by these results support the proposition that
the transcriptional state of determined cells is implemented through the
controls imposed by the regulators of chromatin structure (Klebes, 2005).
The determined states that direct cells to particular fates or lineages can
be remarkably stable and can persist after many cell divisions in alien
environments, but they are not immune to change. In Drosophila, three
experimental systems have provided opportunities to investigate the mechanisms
that lead to switches of determined states. These are: (1) the classic
homeotic mutants; (2) the PcG and trxG mutants that affect the capacity of
cells to maintain homeotic gene expression, and (3) transdetermination. During
normal development, the homeotic genes are expressed in spatially restricted
regions, and cells that lose (or gain) homeotic gene function presumably
change the transcriptional profiles characteristic of the particular body
part. In the work reported here, techniques of micro-dissection, RNA
amplification and array hybridization were used to monitor the transcription profiles of
cells in normal leg and wing imaginal discs, in leg disc cells that regenerate
and in cells that transdetermine from leg to wing. The results validate the
idea that changing determined states involves global changes in gene
expression. They also identify genes whose function may be unrelated to the
specific fates of the cells characterized, but instead may correlate with
developmental plasticity (Klebes, 2005).
Overlap between the transcriptional profiles in the wing and
transdetermination lists (15 genes) and with genes in subcluster IV
(high expression in wing discs) is extensive. The
overlap is sufficient to indicate that the TD leg disc cells have changed to a
wing-like program of development, but interestingly, not all wing-specific
genes are activated in the TD cells. The reasons could be related to the
incomplete inventory of wing structures produced (only ventral wing)
or to the altered state of the TD cells. During normal
development, vg expression is activated in the embryo and continues
through the 3rd instar. Although the regulatory sequences responsible for
activation in the embryo have not been identified, in 2nd instar wing discs,
vg expression is dependent upon the vgBE enhancer, and in 3rd instar
wing discs expression is dependent upon the vgQE enhancer.
Expression of vg in TD cells depends on activation by the vgBE
enhancer, indicating that cells that respond to Wg-induction do not
revert to an embryonic state. Recent studies of the cell cycle characteristics
of TD cells support this conclusion (Sustar, 2005),
but the role of the vgBE enhancer in TD cells and the incomplete inventory of
'wing-specific genes' in their expression profile probably indicates as well
the stage at which the TD cells were analyzed: they were not equivalent to
the cells of late 3rd instar wing discs (Klebes, 2005).
Investigations into the molecular basis of transdetermination have led to a
model in which inputs from the Wg, Dpp and Hh signaling pathways alter the
chromatin state of key selector genes to activate the transdetermination
pathway. The analyses were limited to a period 2-3 days after the
cells switched fate, because several cell doublings were necessary to produce
sufficient numbers of marked TD cells. As a consequence, these studies did not
analyze the initial stages. Despite this technical limitation, this study
identified several genes that are interesting novel markers of
transdetermination (e.g., ap, CG12534, CG14059 and CG4914), as well as
several genes that function in the transdetermination process (e.g.,
lama and the PcG genes). The results from
transcriptional profiling add significant detail to a general model proposed
for transdetermination (Klebes, 2005).
(1) It is reported that ectopic wg expression results in
statistically significant changes in the expression of 15 PcG and trxG genes.
Moreover, although the magnitudes of these changes were very small for most of
these genes, functional assays with seven of these genes revealed remarkably
large effects on the metrics used to monitor transdetermination -- the
fraction of discs with TD cells, the proportion of disc epithelium that TD
cells represent, and the fraction of adult legs with wing cuticle. These
effects strongly implicate PcG and trxG genes in the process of
transdetermination and suggest that the changes in determined states
manifested by transdetermination are either driven by or are enabled by
changes in chromatin structure. This conclusion is consistent with the
demonstrated roles of PcG and trxG genes in the self-renewing capacity of
mouse hematopoietic stem cells, in Wg signaling and in the maintenance of determined states.
The results now show that the PcG and trxG functions are also crucial to
pluripotency in imaginal disc cells, namely that pluripotency by 'weak point'
cells is dependent upon precisely regulated levels of PcG and trxG proteins,
and is exquisitely sensitive to reductions in gene dose (Klebes, 2005).
The data do not suggest how the PcG and trxG genes affect
transdetermination, but several possible mechanisms deserve consideration. A
recent study (Sustar, 2005) reported that transdetermination correlates with an extension of the S phase
of the cell cycle. Several proteins involved in cell cycle regulation
physically associate with PcG and trxG proteins, and
Brahma, one of the proteins that affects the metrics of transdetermination,
has been shown to dissociate from chromatin in late S-phase and to
reassociate in G1. It is possible that changes in the S-phase of TD cells are
a consequence of changes in PcG/trxG protein composition (Klebes, 2005).
Another generic explanation is that transdetermination is dependent or
sensitive to expression of specific targets of PcG and trxG
genes. Among the 167 Pc/Trx response elements (PRE/TREs) predicted to exist in
the Drosophila genome, one is in direct proximity to the vg gene.
It is possible that upregulation of vg in TD cells is mediated
through this element. Another factor may be the contribution of targets of Wg
signaling, since targets of Wg signaling have been shown to be
upregulated in osa and brm mutants.
These are among a number of likely possible targets, and identifying the sites
at which the PcG and trxG proteins function will be necessary if an
understand is to be gained of how transdetermination is regulated. Importantly, understanding the
roles of such targets and establishing whether these roles are direct will be
essential to rationalize how expression levels of individual PcG and
trxG genes correlate with the effects of PcG and
trxG mutants on transdetermination (Klebes, 2005).
(2) The requirement for lama suggests that proliferation of TD
cells involves functions that suppress differentiation. lama
expression has been correlated with neural and glial progenitors prior to, but
not after, differentiation, and it is observed that lama is expressed in
imaginal progenitor cells and in early but not late 3rd instar discs.
lama expression is re-activated in leg cells that transdetermine. The
upregulation of unpaired in TD cells may be relevant in this context,
since the JAK/STAT pathway functions to suppress differentiation and to promote
self-renewal of stem cells in the Drosophila testis. It is
suggested that it has a similar role in TD cells (Klebes, 2005).
(3) A role for Notch is implied by the expression profiles of several
Notch pathway genes. Notch may contribute directly to transdetermination
through the activation of the vgBE enhancer [which has a binding site for
Su(H)] and of similarly configured sequences that were found to be present in
the regulatory regions of 45 other TD genes. It will be important to test whether Notch signaling
is required to activate these co-expressed genes, and if it is, to learn what
cell-cell interactions and 'community effects' regulate activation of the
Notch pathway in TD cells (Klebes, 2005).
(4) The upregulation in TD cells of many genes involved in growth and
division, and the identification of DNA replication element (DRE) sites in the regulatory region of
many of these genes supports the observation that TD cells become
re-programmed after passing through a novel proliferative state
(Sustar, 2005),
and suggests that this change is in part implemented through DRE-dependent
regulation (Klebes, 2005).
There was an interesting correlation between
transdetermination induced by Wg mis-expression and the role of Wg/Wnt
signaling for stem cells. Wg/Wnt signaling functions as a mitogen and
maintains both somatic and germline stem cells in the Drosophila
ovary,
and mammalian hematopoetic stem cells. Although
the 'weak point' cells in the Drosophila leg disc might lack the
self-renewing capacity that characterizes stem cells, they respond to Wg
mis-expression by manifesting a latent potential for growth and
transdetermination. It seems likely that many of the genes are conserved that are involved in
regulating stem cells and that lead to disease states when relevant
regulatory networks lose their effectiveness (Klebes, 2005).
The prevalence of transcription factors among the
genes whose relative expression levels differed most in the tissue
comparisons was intriguing. It is commonly assumed that transcription factors function
catalytically and that they greatly amplify the production of their targets,
so the expectation was that the targets of tissue-specific transcription
factors would have the highest degree of tissue-specific expression. In these
studies, tissue-specific expression of 15 transcription factors among the 40
top-ranking genes in the wing and leg data sets (38%) is consistent with the
large number of differentially expressed genes in these tissues, but these
rankings suggest that the targets of these transcription factors are expressed
at lower relative levels than the transcription factors that regulate their
expression. One possible explanation is that the targets are expressed in both
wing and leg disc cells, but the transcription factors that regulate them are
not. This would imply that the importance of position-specific regulation lies
with the regulator, not the level of expression of the target. Another
possibility is that these transcription factors do not act catalytically to
amplify the levels of their targets, or do so very inefficiently and require a
high concentration of transcription factor to regulate the production of a
small number of transcripts. Further analysis will be required to distinguish
between these or other explanations, but it is noted that the prevalence of
transcription factors in such data sets is neither unique to wing-leg
comparisons nor universal (Klebes, 2005).
The Polycomb Group (PcG) of epigenetic regulators maintains the repressed state of Hox genes during development of Drosophila, thereby maintaining the correct patterning of the anteroposterior axis. PcG-mediated inheritance of gene expression patterns must be stable to mitosis to ensure faithful transmission of repressed Hox states during cell division. Previously, two PcG mutants, polyhomeotic and Enhancer of zeste, were shown to exhibit mitotic segregation defects in embryos, and condensation defects in imaginal discs, respectively. polyhomeoticproximal but not polyhomeoticdistal is necessary for mitosis. To test if other PcG genes have roles in mitosis, embryos derived from heterozygous PcG mutant females were examined for mitotic defects. Severe defects in sister chromatid segregation and nuclear fallout, but not condensation are exhibited by Polycomb, Posterior sex combs and Additional sex combs. By contrast, mutations in Enhancer of zeste (which encodes the histone methyltransferase subunit of the Polycomb Repressive Complex 2) exhibit condensation but not segregation defects. It is proposed that these mitotic defects in PcG mutants delay cell cycle progression. Possible mitotic roles for PcG proteins are discussed, and suggest that delays in cell cycle progression might lead to failure of maintenance (O'Dor, 2006).
The data for ph mutations confirm the original observation that ph503 mutations exhibit mitotic defects. These original observations have been confirmed in several ways. First, the observation that strains out-crossed to wild-type flies show similar frequencies of defects compared to heterozygous mutants show that the phenotypes arise from ph mutations rather than background effects. Second, embryos derived from homozygous ph409 mothers show similar frequencies of mitotic defects to those derived from heterozygous mothers. These results suggest that for ph, the severity of the phenotype reaches a plateau when the amount of Ph is reduced below a threshold which must be greater than 50% of the wild-type amount. Third, only php (ph409) is necessary for normal mitosis, because mutations in phd (ph401) have no effect on mitosis. This observation is consistent with data that has shown that only one isoform of Ph-P coimmunoprecipitates with Barren or Topoisomerase II. This observation supports the conclusion that Ph-P and Ph-D have different functions. Fourth, because homozygous ph409 flies are viable, the ph phenotypes reported here represent those of maternal germline nulls (O'Dor, 2006).
The results show that early embryos of PcG and Asx mutants exhibit highly penetrant and expressive mitotic phenotypes in syncytial embryos, consistent with problems in cell cycle progression. Two classes of phenotypes are observed: segregation defects and condensation defects, but no mutant exhibits both phenotypes. In these experiments, with the exception of ph, embryos were scored derived from heterozygous mothers, in which 50% of the wild-type product remain. Therefore, the possibility cannot be ruled out that more severe mitotic phenotypes would be observed in embryos derived from homozygous mothers, resulting in less distinct differences between E(z) and other mutants. Consistent with this caveat, when homozygous E(z)5 (l(3)1902) mutant imaginal disks were examined, both condensation defects and chromosome breakage consistent with problems in segregation were observed, so E(z) may function in both condensation and segregation. The data show that embryos derived from homozygous ph-proximal mutants do not have condensation defects, so at a minimum, E(z) has at least one role in mitosis different from that of ph. To accurately compare the roles of different PcG and ETP genes in mitosis, it will be necessary to examine mutations derived from homozygous mutant mothers, or from germline clones (O'
moira mutations suppress the derepression phenotypes caused by mutations in another Pc group gene, Polycomblike. moira mutant clones in the haltere differentiate large bristles, characteristic of the anterior wing margin, and often lead to absence or duplication of halteres. Homozygous mor mutations in the posterior wing result in a distorted wing shape; the venation is disrupted and large socketed bristles appear along the posterior wing margin. Leg clones result in the femur and tibia being short and twisted and enlargement of the tarsal segment. Clones of the head cause the shape of the head to be abnormal in the dorsal region and sometimes cause the ocellus to be abnormal or absent. Embryos homozygous for moira mutations have defects in head structures, including truncated lateralgraten and defects in the mouth hooks and dorsal bridge. The first and second midgut constrictions are shifted posterior to their wild-type positions (Brizuela, 1997).