Histone deacetylase 1


DEVELOPMENTAL BIOLOGY

Embryonic

groucho and rpd3 interact genetically. gro is a maternally required gene with critical roles in multiple developmental processes, including anterior/posterior and dorsal/ventral pattern formation, neurogenesis, and sex determination. To determine whether or not Rpd3 could possibly interact with Gro to help mediate these processes, the distribution of this protein was examined in ovaries and embryos. Immunostaining of wild-type ovaries indicates that Rpd3 protein is ubiquitously present in the nuclei of both nurse and follicle cells throughout oogenesis. In addition, Rpd3 protein is detected in all the nuclei of the syncytial blastoderm embryo. Rpd3 protein levels drop significantly and remain low during gastrulation and at later stages of embryogenesis. However, a high level of spatially restricted expression is observed in the head region of stage 9-10 embryos (Chen, 1999).

HDAC inhibitors disrupt programmed resistance to apoptosis during Drosophila development

It has been previously shown that the ability to respond to apoptotic triggers is regulated during Drosophila development, effectively dividing the fly life cycle into stages that are either sensitive or resistant to apoptosis. This study shows that the developmentally programmed resistance to apoptosis involves transcriptional repression of critical pro-apoptotic genes by histone deacetylases (HDACs). Administration of HDAC inhibitors (HDACi), like Trichostatin A (TSA) or Suberoylanilide Hydroxamic Acid (SAHA), increases expression of pro-apoptotic genes and is sufficient to sensitize otherwise resistant stages. Conversely, reducing levels of pro-apoptotic genes confers resistance to otherwise sensitive stages. Given that resistance to apoptosis is a hallmark of cancer cells, and that HDACi have been recently added to the repertoire of FDA-approved agents for cancer therapy, these results provide new insights for how HDACi help kill malignant cells and also raise concerns for their potential unintended effects on healthy cells (Kang, 2017).

Effects of Mutation or Deletion

Drosophila Rpd3 was identified in a genetic screen for mutations affecting position-effect variegation (PEV) of white gene expression in the eye (De Rubertis, 1996). Rpd3 mutants increase PEV, and since PEV is considered a phenomenon involving gene silencing, the function of wild type Rpd3 may be to counteract PEV induced gene silencing. in situ detection of transcripts has revealed a significant reduction of transcript level in eye-antennal discs of homozygous Rpd3 mutant flies, whereas other discs from the same animals showed no clear difference. This result indicates that enhanced PEV results from the loss of Rpd3 function in the phenotypically relevant tissue. The inactivating mutation effects only the expression of Rpd3 in the eye (De Rubertis, 1996).

It is now thought that the 'Rpd3 mutation' described De Rubertis (1996) is actually in the adjacent gene Src64B. There is a small exon of Src64B between the P element insert described by De Rubertis and Rpd3. It therefore appears that the enhancement observed in that mutant is actually the result of disruption of Src64B and not the result of mutation of Rpd3. Mottus (2000, see below) describes analysis of 3 point mutations all of which dominantly suppress PEV and 2 deletions in Rpd3 which do not display a dominant effect on PEV. At the time the Mottus paper was written, the genome project was not yet completed and the possible involvement of Src64B was not yet suspected (R. Mottus, 2006 personal communication to the editor of The Interactive Fly).

A functional interaction between the histone deacetylase Rpd3 and the corepressor Groucho in Drosophila development

Embryos heterozygous for a P element insertion in rpd3 show reduced levels of Rpd3 staining in the head, whereas homozygous embryos display no detectable expression in the head. In addition, this P1633 insertion fails to complement a deletion that removes the rpd3 gene entirely, Df(3L)10H. Therefore, P1633 likely represents a null or strong hypomorphic allele of rpd3 (Chen, 1999).

To look for evidence for an interaction between maternally expressed gro and rpd3, the frequency of unhatched embryos produced by females carrying various combinations of gro and rpd3 alleles was scored (in this experiment two gro alleles were used: groE48, a strong hypomorphic allele and groBX22, a null allele). Approximately 3%-4% of embryos produced by mothers singly heterozygous for either groBX22, groE48, P1633, or Df(3L)10H fail to hatch. This frequency is not significantly greater than that observed for embryos derived from wild-type mothers. However, embryos laid by females doubly heterozygous for gro and rpd3 alleles [groBX22/P1633, groE48/P1633, groBX22/Df(3L)10H, and groE48/Df(3L)10H] show a dramatic increase in embryonic lethality---16%-30% of the embryos fail to hatch. These synergistic effects on embryonic lethality suggest that gro and rpd3 function together during Drosophila oogenesis and/or embryogenesis (Chen, 1999).

Cuticles prepared from the unhatched progeny derived from females singly heterozygous for either gro or rpd3 are indistinguishable from wild type. However, those unhatched embryos produced by mothers doubly heterozygous for gro and rpd3 often display cuticles with striking abnormalities in anterior/posterior pattern formation. The majority of the unhatched embryos (>70%) generated by the groE48/P1633 and groBX22/P1633 trans-heterozygous mothers display replacement of anterior embryonic segments by a mirror-image duplication of the three to five posterior-most segments. Mirror-image duplicated structures include denticle belts and posterior spiracles. The remaining unhatched embryos were normal or only had minor defects in head structures. The unhatched embryos collected from the groBX22/Df(3L)10H, and groE48/Df(3L)10H trans-heterozygous mothers display more severe cuticle phenotypes. Most (50%-60%) have no cuticle, whereas 5%-10% show a mirror-image duplication of the posterior spiracle and disordered denticle belts. The remaining unhatched embryos display cuticles that are nearly wild type or have minor defects in head structures (Chen, 1999).

The cuticle phenotype of embryos derived from mosaic females containing P1633 homozygous germ-line clones was examined. The majority of the embryos (>65%) lacking maternally expressed rpd3 fail to hatch, and most of those unhatched embryos (>80%) exhibit variable pair-rule segmentation defects. Observed in particular was the partial or complete fusion of adjacent denticle belts resulting in embryos with five to eight thoracic and abdominal segments. In addition, variable, often severe, defects were observed in the head skeleton. Gro has been shown to interact physically with the pair-rule gene products Hairy and Runt and to be required for their function as transcriptional repressors. Therefore, the pair-rule phenotype observed in embryos lacking maternal Rpd3 suggests that like Gro, Rpd3 may also be involved in the repression mediated by certain pair-rule gene products (Chen, 1999).

Mutational analysis of a histone deacetylase in Drosophila melanogaster: Missense mutations suppress gene silencing associated with position effect variegation

For many years a correlation has been been noted between acetylation of histones and an increase in transcriptional activity. One prediction, based on this correlation, is that hypomorphic or null mutations in histone deacetylase genes should lead to increased levels of histone acetylation and result in increased levels of transcription. It was therefore surprising when it was reported (De Rubertis, 1996), in both yeast and fruit flies, that mutations that reduce or eliminate a histone deacetylase result in transcriptional silencing of genes subject to telomeric and heterochromatic position effect variegation (PEV). Here, six new Rpd1 mutations are described, referred to as HDAC1 in this paper. A suite of phenotypes accompanying the mutations are observed consistent with the notion that HDAC1 acts as a global transcriptional regulator. However, in contrast to recent findings, specific missense mutations in the structural gene of HDAC1 suppress the silencing of genes subject to PEV. It is proposed that the missense mutations reported here are acting as antimorphic mutations that 'poison' the deacetylase complex and a model is proposed that accounts for the various phenotypes associated with lesions in the deacetylase locus (Mottus, 2000).

P-UTR is a lethal P-element insertion in Rpd1/HDAC1. Some P-UTR/HDAC1328 adult male flies eclose but only survive for a few days. These animals display very strong suppression of PEV and several other phenotypes. To further examine these phenotypes HDAC1303/P-UTR flies were generated. In this cross, under carefully maintained culture conditions, adult males eclose at ~40% of the number expected and females at ~30% of the number expected. Neither sex survives for more than several days, and the females produce a small number of eggs, which appear to be unfertilized. These animals display a suite of defects, including very strong suppression of wm4; wings that are severely notched; bristles that are smaller than normal, malformed, often curved, and duplicated; allila that are larger than normal, and a reduction in the number of sex combs on the legs of the males, from a mean of 10.7 ± 0.9 to a mean of 7.7 ± 1.0. This suggests that mutations in the histone deacetylase HDAC1 cause defects in a variety of cellular systems and is consistent with the proposed role of HDAC1 as a global transcriptional regulator. It also suggests that the Su(var) HDAC1s retain at least some of their functions, because P-UTR is lethal when homozygous, yet appreciable numbers of adults can be recovered when P-UTR is heterozygous with members of the Su(var) HDAC1s (Mottus, 2000).

Because P-UTR and the Su(var) HDAC1s are recessive lethal, it appears that HDAC1 function is essential for survival in D. melanogaster, unlike in S. cerevisiae, where null alleles of the RPD3 gene are viable but display a suite of phenotypes. To further characterize the requirements for HDAC1, the developmental time at which HDAC1 is required for survival in D. melanogaster was determined. Because P-UTR is a very strong hypomorph and it could not be determined whether or not the Su(var) HDAC1s are complete null alleles of the gene and/or whether residual gene activity would mask the earliest requirement for HDAC1, null alleles of HDAC1 were generated. Null alleles (HDAC1def8 and HDAC1def24) of HDAC1 die during the larval stage. Surprisingly, inspection of the stock cultures reveals that a large percentage of the homozygous mutant larvae survive until very late in third instar. These larvae are readily identifiable because in the stock cultures the mutations are balanced over TM6Tb. Larvae bearing the balancer chromosome can be distinguished from larvae homozygous for the HDAC1 mutations because Tb alters the morphology of the larval spiracles. This suggests three possible scenarios: (1) maternal HDAC1 is perduring until very late in development; (2) HDAC1 is required during embryogenesis and not required again until late in third instar and maternal HDAC1 provides sufficient activity for this early function; or (3) HDAC1 is not required for the early stages of Drosophila development. Based on a recently published report investigating the phenotypes associated with P-UTR, the second of the above three possibilities is favored. Lethal phase analyses of the Su(var) HDAC1s were conducted. HDAC1313 and HDAC1326 also died during the larval period. Inspection of the stock cultures revealed a large number of homozygous mutant larvae at the third instar stage, and therefore these alleles cause death at approximately the same time as the null alleles. However, only ~50% of larvae bearing HDAC1303 and HDAC1328 die during the larval period while ~50% survive into pupation. This is consistent with the sequencing data that has demonstrated that these mutations are caused by identical base pair substitutions. Thus, with regard to lethality, it appears that HDAC1313 and HDAC1326 are indistinguishable from null alleles while HDAC1303 and HDAC1328 retain some HDAC1 activity (Mottus, 2000).

An unexpected observation from the lethal phase analysis is that the Su(var) HDAC1s appears to have a dominant semilethal affect on males regardless of their genotype. In the lethal phase analysis, three of the four genotypes produced are expected to survive and one of the classes (+/TM3) does not carry any chromosomes with a mutation in HDAC1. In the crosses with the null alleles HDAC1def8 and HDAC1def24, males and females in the classes that are expected to live appear in approximately the same numbers. However, in the Su(var) HDAC1 crosses, males of genotypes expected to survive, including males that have completely wild-type HDAC1 genes, survive at significantly lower rates than expected. For example, males in the cross involving HDAC1313 only survive at ~50% the level of their genotypically identical female siblings in the same cross. Males in crosses involving the other Su(var) HDAC1s also survive at significantly lower levels than females. Because in these crosses the mothers carried the Su(var) HDAC1 mutations, one explanation for this observation may be that these mutations may be exerting a dominant maternal effect on the dosage compensation mechanism. In Drosophila, dosage compensation occurs as a result of hypertranscription of the male X chromosome. The male X chromosome adopts a special conformation that is believed to be necessary for enhanced transcription. Accordingly, if histone deacetylation is an essential step in establishing the specialized chromatin structure required in the male, the Su(var) HDAC1s may be defective in this process. Alternatively, although most genes on the male X chromosome are transcribed at double the normal rate, there are loci that are not subject to dosage compensation and therefore need to be silenced or repressed on the specialized male X chromosom. In the Su(var) HDAC1 strains these loci may escape repression, resulting in reduced male viability in the sons of mutant mothers (Mottus, 2000).

How can one explain the apparently contradictory effects on PEV and telomeric position effects (TPEV) of the various kinds of mutations in the histone deacetylase genes in yeast and Drosophila? It may be that histone deacetylases belong to a growing class of genes that have the following characteristics: (1) they are members of a closely related gene family; (2) they encode multidomain proteins, and (3) null mutations have little or no obvious phenotypic effect while point mutants have profound, often dominant effects. One recent example of this class of genes in lower eukaryotes is the FUS3/KSS1 gene pair of S. cerevisiae. Normally, these closely related proteins function in separate pathways. Single deletion strains of either gene are still proficient for mating because when Fus3p is deleted, and only when it is deleted, Kss1p acts as an impostor and replaces Fus3p. However, deletion of both proteins renders the strain sterile. Examples of this class of gene are certainly not limited to lower eukaryotes. For example, gene knockout experiments in mice have revealed a surprising number of genes in which the phenotype of the homozygous null mutation is either not detectable or very minor. A cursory examination of the Mouse Knockout Database identifies at least 13 such genes. In contrast to the mild phenotypes of knockout alleles, analysis of mutations in some of these genes has shown that point mutations can have very profound, often dominant, effects. One example is the SRC oncogene, a member of a closely related family of proteins. The knockout causes only minor dental abnormalities, yet almost all known point mutations have severe phenotypic consequences, including cancer (Mottus, 2000 and references therein).

It is now apparent that most, if not all, of the biological activities in the cell are carried out by large, multiprotein complexes. A single type of complex may have multiple targets or functions that are dependent on the specific members of the complex at a particular time during the cell cycle or at a particular location in the cell. If one of the proteins of the complex is absent, as in a null mutation, and that protein is a member of a closely related family, then another member(s) of the family may substitute for the missing protein. Because they are closely related, the impostor can provide partial activity and, as a consequence, a null mutation may have no obvious phenotype. In contrast, point mutations that only alter a single domain may allow the aberrant protein to be incorporated into its complex(es). In cases in which the mutation occurs in a domain required for a specific function, the complex would then be completely inactive for that particular function. Accordingly, a point mutation may have a dominant negative effect and display a much more severe phenotype than a null mutation (Mottus, 2000 and references therein).

This model may accommodate observations of the various Drosophila HDAC1 mutations. In eukaryotes, the HDACs are a closely related family of proteins that form complexes with other proteins including other HDACs. For example, in yeast, two different HDACs, RPD3 and HDA1, have been isolated and characterized, and sequence analysis of the yeast genome suggests there may be at least three additional HDACs. Two large multiprotein complexes, HDA and HDB, containing histone deacetylase activity have been isolated and analysis of HDA has shown that it contains at least two HDACs. Similarly, in mammals, five different HDACs have been identified and a complex containing the human RPD3-like deacetylase, HDAC1, also contains HDAC2. In Drosophila, two more HDACs have now been identified, HDAC2 and HDAC3 (Johnson, 1998 and Mannervik, 1999). It seems likely that more candidate deacetylases will be identified as the genome sequencing projects proceed. Accordingly, the biochemical and sequence analyses of HDACs in yeast and mammals suggest that HDACs are members of a related gene family and, more importantly to this model, function as members of large protein complexes (Mottus, 2000).

The foregoing provides the framework for a model that may explain the apparently contradictory results observed with different kinds of mutations in this histone deacetylase and their effects on PEV and TPEV. In the rpd3 null mutant in yeast, TPEV is enhanced, that is, the expression of the reporter gene is repressed. It is postulated that in the absence of RPD3, other HDACs, with differing specificities, substitute for RPD3 in the multiprotein complex, resulting in an incorrect histone deacetylation pattern. The phenotypic consequence of the incorrect deacetylation pattern is enhancement of TPEV, possibly due to excess deacetylation at the site of the reporter gene by the impostor deacetylase. In Drosophila the only mutation in HDAC1 that enhances PEV is P-1.8, an insertion of a P element 1.8 kb 5' to the coding region. In situ hybridization with a probe for the HDAC1 mRNA demonstrates that in the eye disc transcription of HDAC1 is markedly reduced or absent but in the leg disc the HDAC1 transcript accumulates to normal levels. One possible explanation for this observation is that the P element has inserted into an eye disc specific enhancer element, resulting in little or no transcription in the eye disc. Thus, HDAC1 may be effectively absent in the eye disc. In its absence, other HDACs could substitute for HDAC1, producing an incorrect deacetylation pattern, the consequence of which is enhancement of PEV. In contrast, the Su(var) HDAC1s described here are capable of producing a protein with only a single amino acid change in which a specific function has likely been compromised, possibly the deacetylase activity. Because only a single amino acid has been changed, the protein would still associate with its complex, bind its other components efficiently, and be targeted to the correct site. However, the complex would be unable to deacetylate its target histones, leading to hyperacetylation and decreased silencing. In this way a point mutation would act as a dominant negative mutation and would suppress PEV. In contrast, null mutations, such as the deficiencies described here, have no observable effect on PEV because in heterozygotes, wild-type HDAC1, produced from the nondeleted homolog, can associate normally with the histone deacetylase complexes. The other HDACs can only substitute for HDAC1 in its complete absence as is the case with Kss1p and Fus3p in yeast described above (Mottus, 2000).

This model relies on the supposition that an aberrant form of HDAC1 is being produced in the Su(var) HDAC1 strains. It is thought such a protein is made for the following reasons. (1) Conceptual translation of the protein produces a full-length product with only a single amino acid change. (2) When the members of the Su(var) HDAC1s are crossed to P-1.8, the strain bearing the P-element insertion 1.8 kb 5' to the HDAC1 gene, flies bearing both mutations are viable and fertile and show a weak-to-moderate suppression of PEV. Because the P insert line is effectively a null in the eye disc, the suppression observed in the heterozygotes is interpreted as evidence that the Su(var) HDAC1s are producing a product. (3) In the complementation and recombination studies, heterozygotes bearing both the P-UTR chromosome and the Su(var) HDAC1s survive at an appreciable frequency. In these flies, PEV in the In(1)wm4 strain is very strongly suppressed and the eyes are virtually indistinguishable from wild-type strains. Because P-UTR is lethal as a homozygote and this lethality is only associated with lesion in HDAC1, the observation that such flies survive suggests that the Su(var) HDAC1s are producing a product that retains sufficient activity in the essential function of HDAC1 to rescue the lethality associated with the P-UTR chromosome. (4) The observation that the Su(var) HDAC1s displays a dominant maternal effect reduction in the viability of males, regardless of their phenotype, a reduction that was not observed in crosses with the deficiency strains, implies that the Su(var) HDACs are producing a protein product because this observed maternal effect is not seen in the absence of any product (Mottus, 2000).

Essential role of Drosophila Hdac1 in homeotic gene silencing

Deacetylation of the N-terminal tails of core histones plays a crucial role in gene silencing. Rpd3 and Hda1 represent two major types of genes encoding trichostatin A-sensitive histone deacetylases. Drosophila Rpd3, referred to here by its alternative name HDAC1, interacts cooperatively with Polycomb group repressors in silencing the homeotic genes that are essential for axial patterning of body segments. The biochemical copurification and cytological colocalization of HDAC1 and Polycomb group repressors strongly suggest that HDAC1 is a component of the silencing complex for chromatin modification on specific regulatory regions of homeotic genes (Chang, 2001).

In Drosophila, five potential genes encoding TSA-sensitive HDACs have been identified. Of these five, Rpd3 (Hdac1) and Hdac3 are Rpd3 types; Hdac2 and Hdac4 are Hda1 types, and CG10899 appears to diverge significantly from both types. Because the expressions of homeotic genes are oppositely controlled by Pc-G and trx-G proteins in a dosage-sensitive manner, it is possible to assess the roles of these HDACs in homeotic gene regulation by examining the genetic interactions between Pc-G or trx-G mutations and HDAC mutations. In a preliminary study, deficiencies that delete four of five potential HDAC genes (a deficiency for Hdac3 is not available currently) have been examined for genetic interactions with Pc. Only Df(3R)10H, which deletes Hdac1, shows a significant genetic interaction with a Pc mutation, resulting in a more than 2-fold increase in ectopic sex comb teeth on the second and third legs of male adults. In addition, this deficiency substantially reduces the frequency of mesothoracic transformation typically found in trx-G mutants. These results are consistent with a negative role for Hdac1 in homeotic gene regulation (Chang, 2001).

Df(3R)10H deletes not only Hdac1, but several other genes as well. To show that deletion of the Hdac1 gene is responsible for the genetic interactions with Pc, genetic interactions between Pc-G mutants and Hdac1P-UTR, a semilethal mutant with a P element inserted at +47 of Hdac1 were examined. Hdac1P-UTR also shows dosage-sensitive genetic interactions with Pc and Psc mutations, indicating that Hdac1 is important in regulating the function of homeotic genes. In contrast to the results with Pc and Psc, no genetic interactions were observed between Hdac1P-UTR and extra sex combs (esc) or Enhancer of zeste [E(z)] mutations. The differences in the genetic interactions with the Pc-G mutations might reflect the presence of two physically distinct complexes formed by these proteins, because the PC and PSC proteins copurify in one Pc-G complex and the ESC and E(Z) proteins copurify in a different Pc-G complex. It is interesting to note that Df(3R)10H and Hdac1P-UTR by themselves did not cause leg transformation. Thus, the homeotic effects of Hdac1 appear to manifest themselves only in combination with Pc-G mutations as noted for several other Pc-G, including Enhancer of Polycomb, Suppressor 2 of zeste, and Mi-2 (Chang, 2001).

Further support for the role of Hdac1 in homeotic gene regulation was obtained by analyzing several newly characterized Hdac1 mutations. As observed for Hdac1P-UTR, two missense mutations (Hdac1303 and Hdac1313) and one small deletion (Hdac1def8) enhance the Pc mutant phenotype. Surprisingly, one missense mutation, Hdac1326, suppresses the Pc phenotype significantly. This unexpected suppression probably results from a stronger effect on ectopic expression of posterior homeotic genes, causing repression of more anterior ones (Chang, 2001).

To demonstrate that the effect of Hdac1 mutations is exerted at the level of expression of homeotic genes, the expressions were examined of Sex combs reduced (Scr) and Ultrabithorax (Ubx) proteins in wild-type and Pc mutant imaginal discs. Scr proteins normally are expressed at high levels in the first leg discs, but are not expressed in the second and third leg discs. In Pc4 mutant heterozygotes, however, Scr proteins also can be detected at low levels in second and third leg discs. Consistent with the increase in ectopic sex comb teeth, dramatic increases in the levels of Scr proteins are observed in the second and third leg discs from Pc4 mutant heterozygotes that were also heterozygous for any of the Hdac1 alleles except Hdac1326. In addition, Ubx proteins are marginally detectable only in the peripodial membranes of imaginal wing discs of wild-type or Pc4 mutant heterozygous larvae. In larvae heterozygous for both Pc4 and an Hdac1 mutation, high levels of Ubx proteins are observed in the medial sections of the wing discs proper. In contrast to the lack of ectopic Scr expression in Pc4 heterozygotes carrying the Hdac1326 allele, a much stronger effect on ectopic Ubx expression is observed; Ubx protein levels in both first and second leg discs are increased substantially. It is highly likely that the expanded Ubx expression reduces Scr expression, resulting in suppressed Pc phenotype (i.e., reduced numbers of ectopic sex comb teeth) in Pc4/Hdac1326 trans-heterozygotes. These results strongly suggest that Hdac1 acts cooperatively with Pc to repress homeotic genes during larval and pupal development (Chang, 2001).

Experiments also were performed to explore the role of Hdac1 in regulating the embryonic expressions of two homeotic genes, Abd-B and Ubx. Abd-B proteins normally are expressed in a graded fashion in the posterior part of ventral nerve cord, starting from parasegment 10 (PS10). Although this pattern is not altered in homozygous Hdac1303 mutants, significant levels of Abd-B proteins are observed in more anterior parasegments of homozygous Psce24 mutants. Much higher levels of ectopic Abd-B proteins are found in Psce24 Hdac1303 double mutants, indicating a synergistic effect of Hdac1 and Psc on Abd-B repression. Consistent (but less striking) effects also are observed on Ubx protein levels. The anterior boundary of the Ubx expression domain is PS5, with the exception of a small cluster of cells in the middle of PS4 that also express Ubx proteins. Although homozygous Psce24 mutants only show sporadic low levels of Ubx expression in more anterior parasegments, Psce24 Hdac1303 double mutants show significantly higher levels of ectopic Ubx expression in more cells. In PS5, more cells with higher levels of Ubx proteins are observed in the double mutants than in either of the single mutants. In contrast, Ubx expression is reduced substantially in the abdominal parasegments of the double mutants compared with that in the single mutants, presumably reflecting Ubx repression by more extensive ectopic expression of Abd-B and possibly ABD-A. These data indicate that Hdac1 is essential for homeotic gene silencing in embryos (Chang, 2001).

The genetic interactions between Hdac1 and Pc-G mutations suggest that the encoded proteins might be physically associated. This idea was tested by examining whether HDAC1 and PC proteins can be copurified from cultured Drosophila cells. A permanent S2 cell line was established that expresses a PC protein with a FLAG-epitope tag at the C-terminal end under the control of a metallothionein promoter. Nuclear extracts prepared from induced cells were passed over a FLAG antibody column. After the addition of FLAG peptide, tagged PC and its associated proteins were eluted. Using 3H-labeled, acetylated core histones as substrates to assay these fractions, it was found that HDAC activity elutes with the same profile as PC. In addition, this activity is sensitive to the HDAC-specific inhibitor TSA (Chang, 2001).

To determine the identity of the HDAC associated with PC, immunoblotting was performed with an affinity-purified antibody against the C-terminal part of HDAC1. HDAC1 was detected in the eluted fraction. In addition, substantial amounts of PSC and PH also were copurified, consistent with previous findings that they are components of large PC protein complexes. Much lower amounts of another Pc-G protein, Sex combs on mid-leg (Scm), were detected in these preparations. Thus, these results indicate that HDAC1 is associated with the PC protein complexes in cultured cells (Chang, 2001).

To examine whether HDAC1 is associated with PC complexes in embryos, HDAC1 proteins were immunoprecipitated from embryonic nuclear extracts. PC was detected when an HDAC1 antibody was used for the immunoprecipitation. These results further support the idea that the associations between PC and HDAC1 proteins are physiologically relevant (Chang, 2001).

Given the genetic and biochemical interactions between Hdac1 and Pc, it might be anticipated that a fraction of HDAC1 would colocalize with Pc-G complexes on polytene chromosomes. Approximately 100 common binding sites have been identified for several Pc-G proteins. At least 70% of these sites (identified by staining with PSC mAbs) also stain with the HDAC1 antibody, including the Antennapedia complex at 84AB and the bithorax complex at 89E. These results suggest that HDAC1 proteins act together with a substantial fraction of the Pc-G complex. However, the relative intensities of the signals for PSC and HDAC1 at these sites do not always correlate, suggesting a regulatory, rather than a constitutive function. Furthermore, HDAC1 is much more widely distributed along the chromosomes than is PSC, consistent with its role in global gene regulation and/or chromatin structure (Chang, 2001).

The colocalizations of HDAC1 and PSC were examined further on polytene chromosomes from a transgenic line that carries a Ubx upstream cis-regulatory region (i.e., bxd-14) inserted at 62A. This insert contains a functional PRE and creates a new Pc-G-binding site. Staining with both PSC and HDAC1 antibodies reveals that a new PSC site coincides with a new HDAC1-binding site. This new binding site is beside an HDAC1 site present in the wild-type chromosome, creating a broader signal of HDAC1 at this site. These results strongly suggest that HDAC1 and Pc-G proteins are recruited to this ectopic PRE (Chang, 2001).

These results do not imply that there is a direct physical interaction between HDAC1 and any Pc-G proteins that have been characterized to date. It is possible that an adapter-like molecule might be involved. In several organisms, direct interactions or cytological colocalization between HDAC1 and SIN3A proteins have been demonstrated. However, there is currently no evidence that SIN3A is required for homeotic gene repression. The observations that HDAC1 is not detected at about 30% of PSC sites and that HDAC1 intensity does not always correlate with that of remaining PSC sites suggests that HDAC1 is not constantly associated with the PC complexes. This is consistent with a catalytic rather than a structural function. Different levels of HDAC1 staining might reflect varying degrees of repression at these sites. Because more acetyl groups need to be removed when a gene becomes repressed from an active state, it is also possible that higher levels of HDAC1 are required for the initiation of a repressed state than for the maintenance of a repressed state. Thus, the significance of the relative levels of HDAC1 should be interpreted with caution (Chang, 2001).

The histone deacetylase inhibitor trichostatin A influences the development of Drosophila melanogaster

The consequences of the deacetylase inhibitor trichostatin A (TSA) on the development of Drosophila melanogaster was examined. When fed to flies, TSA causes lethality and delays development at concentrations as low as 5 microM, has stronger effects on males than females, and acts synergistically with mutations in the gene encoding the RPD3 deacetylase to cause notched wings, but does not appear to affect a SINA signaling pathway that is normally repressed by the SIN3 corepressor. These findings suggest that deacetylated histones play an important role in normal developmental progression and establish parameters for genetic screens to dissect the role of deacetylases in this process (Pile, 2001).

The SIN3 corepressor and RPD3 histone deacetylase are components of the evolutionarily conserved SIN3/RPD3 transcriptional repression complex. The SIN3/RPD3 complex and the corepressor SMRTER are required for Drosophila G2 phase cell cycle progression. Loss of the SIN3, but not the p55, SAP18, or SAP30, component of the SIN3/RPD3 complex by RNA interference (RNAi) causes a cell cycle delay prior to initiation of mitosis. Loss of RPD3 reduces the growth rate of cells but does not cause a distinct cell cycle defect, suggesting that cells are delayed in multiple phases of the cell cycle, including G2. Thus, the role of the SIN3/RPD3 complex in G2 phase progression appears to be independent of p55, SAP18, and SAP30. SMRTER protein levels are reduced in SIN3 and RPD3 RNAi cells, and loss of SMRTER by RNAi is sufficient to cause a G(2) phase delay, demonstrating that regulation of SMRTER protein levels by the SIN3/RPD3 complex is a vital component of the transcriptional repression mechanism. Loss of SIN3 does not affect global acetylation of histones H3 and H4, suggesting that the G2 phase delay is due not to global changes in genome integrity but rather to derepression of SIN3 target genes (Pile, 2002).

Sir2 mediates longevity in the fly through a pathway related to calorie restriction: Sir2 blocks the life-span-extending effect of calorie reduction or rpd3 mutations

Calorie restriction can extend life span in a variety of species including mammals, flies, nematodes, and yeast. Despite the importance of this nearly universal effect, little is understood about the molecular mechanisms that mediate the life-span-extending effect of calorie restriction in metazoans. Sir2 is known to be involved in life span determination and calorie restriction in yeast mother cells. In nematodes increased Sir2 can extend life span, but a direct link to calorie restriction has not been demonstrated. Sir2 is directly involved in the calorie-restriction life-span-extending pathway in Drosophila. An increase in Drosophila Sir2 (dSir2) extends life span, whereas a decrease in dSir2 blocks the life-span-extending effect of calorie reduction or rpd3 mutations. These data led to the proposal of a genetic pathway by which calorie restriction extends life span and provides a framework for genetic and pharmacological studies of life span extension in metazoans (Rogina, 2004).

The Rpd3/Sir2 histone deacetylases have been implicated in both life span determination and calorie restriction in yeast. Rpd3 and Sir2 can effect the activity of a variety of genes and physiological systems by deacetylating histones and other proteins such as p53. A decrease in Rpd3 or an increase in Sir2 extends mother cell life span in yeast, and the effect of Sir2 on yeast life span is linked to calorie restriction. A similar mechanism may operate in metazoans, because an increase in Sir2 extends life span in nematodes, and a decrease in Rpd3 extends life span in flies (Rogina, 2002). The increase in life span associated with decreased Rpd3 in flies is thought to occur through a mechanism related to calorie restriction (Rogina, 2002), The finding of an increase in Drosophila Sir2 (dSir2) transcription in both long-lived rpd3 mutant flies and long-lived calorie-restricted normal flies implicates dSir2 as a potential member of the calorie-restriction life-span-extending pathway (Rogina, 2002). Further evidence of a role for Sir2 in the determination of life span is the finding that the Sir2 agonist resveratrol extends life span in yeast, nematodes, and flies in a Sir2- and calorie-restriction-dependent manner. These data suggest that Sir2 may be one of the primary elements of the calorie-restriction-induced life span extension in flies and other metazoans (Rogina, 2004).

To test whether dSir2 is involved in longevity determination in the fly, the life span was examined of flies in which the level of dSir2 had been increased by using molecular genetic techniques. Flies were constructed that ubiquitously overexpressed dSir2 by combining, in individual flies, the Drosophila tubulin promoter fused to the gene for the yeast GAL4 activator protein (tubulin-GAL4 driver) with a native dSir2 gene that has a P element with GAL4-binding sites (EP-UAS) inserted just upstream. Flies carrying the tubulin-GAL4 driver and each of the different EP-UAS-dSir2 genes, dSir2EP2300, dSir2EP2384, or dSir2EYO3602, had a >4-fold increase in dSir2 mRNA expression over the endogenous level. Consistent with the hypothesis that an increase in dSir2 in flies will increase life span, up to a 57% increase in average life span was seen in the tubulin-GAL4/dSir2EP2300, tubulin-GAL4/dSir22384, and tubulin-GAL4/dSir2EYO3602 flies, with an increase across all lines of 29% for females and 18% for males (Rogina, 2004).

To determine whether a threshold level of dSir2 expression is required for life span extension in the fly, flies were examined in which the armadillo-GAL4 driver was combined with the dSir2EP2300 chromosome. The armadillo-GAL4 driver is a weaker driver than the tubulin-GAL4 driver: compared with control flies, armadillo-GAL4/dSir2EP2300 flies showed only a 10%-20% increase in dSir2 mRNA levels and no life span extension, suggesting that a significant increase in dSir2 mRNA is required to cause an extension in life span (Rogina, 2004).

Knowing that ubiquitous overexpression of dSir2 increases life span, it was of interest to determine which tissues normally express dSir2 in adults and whether an effect in a single tissue could mediate the life span extension caused by dSir2 overexpression. Using anti-dSir2 antibodies, it was found that, similar to embryos and larvae, in adults dSir2 protein is found at high levels in the nuclei of neurons and in the nuclei and cytoplasm of fat body cells. The finding of a prominent expression of dSir2 in the nervous system of normal animals led to an examination of whether an increase in dSir2 in neurons may be one of the primary mediators of the Sir2-related life span extension. Neuronal dSir2 overexpression in flies carrying the pan-neuronal promoter ELAV-GAL4 driver:dSir2EP2300 extended the average life span by 52% in females and 20% in males (Rogina, 2004).

ELAV-GAL4 drives expression in embryos and larvae as well as adults. A different system for overexpressing dSir2 was used to test whether increased expression of dSir2 only in adult neurons might lead to life span extension. The RU-486 Gene-Switch system allows for the comparison of genetically identical animals from the same cohort, one group receiving RU-486, which induces expression of the EP-UAS gene, and the other group receiving only diluent. In two independent trials the maximum life span of adult dSir2EP2300/ELAV-GeneSwitch flies receiving RU-486 was increased by 9% and 16%, respectively, in females and was decreased by 1% and increased by 10%, respectively, in males. In females, respective 5% and 12% increases in median life span were also seen. The smaller increase in life span in flies with the ELAV-GeneSwitch driver, relative to those with the ELAV driver, is consistent with a lower level of dSir2 induction with the ELAV-GeneSwitch driver at the dose of RU-486 used (200 µM). In addition, preliminary studies suggest that RU-486 itself may have some mild deleterious effects on life span in flies, especially on males. The greater life span extension obtained by using the standard ELAV driver could be due partly to increased dSir2 activity before eclosion in adult ELAV-GAL4/dSir2EP2300 flies. Regardless, the results demonstrate that pan-neuronal overexpression of dSir2 during only the adult stage is sufficient to produce a modest extension of maximal life span (Rogina, 2004).

To further explore the possibility that a subset of neurons may be important in dSir2-mediated life span extension, the life span of flies containing the D42-GAL4 motoneuron-specific driver and dSir2EP2300 was examined. No life span extension was seen in the D42/dSir2EP2300 flies (Rogina, 2004).

Decreases in physical activity, reproductive status (especially in females), or calorie intake are known to increase life span in the fly. Although quantitative studies were not performed, visual inspection suggested no obvious decrease in physical activity or fertility in the long-lived dSir2-overexpressing flies compared with their matched controls. Furthermore, the fact that life span was found to be significantly increased in both males and females suggests that a potential decrease in female reproduction is unlikely to be the primary cause of the observed life span extension (Rogina, 2004).

Considered together, the results of experiments driving dSir2 demonstrate that overexpression of dSir2 correlates well with increased life span in flies. In four different driver-GAL4/UAS-dSir2 lines in which dSir2 was substantially overexpressed either ubiquitously or in neurons, the life span of flies was extended significantly. However, when the driver caused only a small ubiquitous increase in dSir2 or an increase only in motor neurons, life span was not extended. Furthermore, an intermediate increase in dSir2 in the adult nervous system caused by the ELAV-GeneSwitch driver caused an intermediate increase in life span (Rogina, 2004).

Flies given low-calorie food, in addition to showing an increase in life span, showed an increase in dSir2 mRNA expression (Rogina, 2002), To determine whether dSir2 is directly in the calorie-restriction life-span-extending pathway in flies, the life span of flies that had reduced or no dSir2 expression on a diet of low-calorie food was compared with that of genetically identical flies from the same cohort on a diet of normal or high-calorie food. Flies with either no dSir2 gene function (e.g., dSir24.5/dSir25.26) or with severely decreased dSir2 gene function (e.g., dSir2KG00871/dSir2KG00871) showed no life span extension on a diet of low-calorie food relative to genetically identical flies on a diet of normal or high-calorie food. The inability of dSir2 mutant flies to increase their life span in response to a low-calorie diet demonstrates that a sufficient level of dSir2 must be available for the activation of life span extension by calorie reduction and, furthermore, that dSir2 is an important element in the calorie-reduction life-span-extending pathway (Rogina, 2004).

If dSir2 mediates the effect of calorie-restriction-induced life span extension, it would also be expected that calorie restriction would not further increase life span in flies in which dSir2 activity is already elevated. The life span of two of the long-lived dSir2-overexpressing lines, tubulin-GAL4/dSir2EP2300 and ELAV-GAL4/dSir2EP2300, was examined under normal and low-calorie food conditions. Under the husbandry conditions used, a decrease in calorie content in the food typically increases the life span of normal flies by 35%-40% or more (Rogina, 2002), However, when the long-lived ELAV-GAL4/dSir2EP2300 and tubulin-GAL4/dSir2EP2300 flies were placed on a diet of low-calorie food, no further increase in life span was seen with the ELAV-GAL4/dSir2EP2300 flies, whereas the tubulin-GAL4/dSir2EP2300 flies showed a reduction in life span toward normal. Control flies for ELAV-GAL4/dSir2EP2300 placed on a diet of low-calorie food demonstrated a 28% and 22% increase in median life span for males and females, respectively. The lack of a cumulative effect of calorie reduction and dSir2 overexpression on life span suggests that life span extensions are mediated through similar or related pathways (Rogina, 2004).

The data presented in this study on dSir2, along with previous work on life span and calorie reduction of rpd3 mutants (Rogina, 2002), indicate that the life-span-extending effects of nutrient reduction in the fly are mediated through dSir2 and Rpd3. The observation of an increase in dSir2 mRNA in long-lived rpd3 mutants (Rogina, 2002) led to the postulate that the effect of Rpd3 may depend partially on an increase in dSir2. Together these data suggest a model for how calorie reduction extends life span in the fly. In this model, the stimulus of calorie reduction triggers a decrease in Rpd3 activity and a subsequent increase in dSir2 activity. The increase in dSir2, either alone or in conjunction with additional changes initiated by the decrease in Rpd3 activity, results in life span extension (Rogina, 2004).

Further confirmation that dSir2 and Rpd3 are in the same life-span-extending pathway was obtained by examining the life span of flies carrying a long-lived rpd3 mutation (rpd3def24) and a dSir2 mutation (dSir217 or dSir2EP2300). dSir2 mutations do not reduce life span in otherwise normal flies (Newman, 2002). Therefore, if dSir2 were not in the same pathway as Rpd3, the life span would not be altered from the extended life span of rpd3 mutant flies. As predicted by the model, flies with both a dSir2 mutation and rpd3def24 mutation were not long-lived, whereas their counterparts, flies mutant for only rpd3def24, remained long-lived. dSir2 and Rpd3, therefore, seem to be in the calorie-reduction life-span-extending pathway in flies. The finding that long-lived rpd3 mutations increase dSir2 levels (Rogina, 2002) suggests that dSir2 is downstream of Rpd3 (Rogina, 2004).

The search for elements that extend life span in metazoans has identified the involvement of the insulin-signaling, nutrient-sensing, and Sir2 pathways. Although the Sir2 pathway has been linked to calorie availability in yeast, it has not been shown to function in the calorie restriction pathway in metazoans. The data presented in this study demonstrate a direct link between the life-span-extending effects of dSir2 and calorie restriction in the fly. Five different GAL-4 drivers (tubulin, ELAV, armadillo, ELAV-Gene-Switch, and D42-motoneuron) were used to drive expression of endogenous dSir2 genes with three separate nearby insertions of UAS elements. In four strains in which dSir2 expression was increased substantially, either ubiquitously or in neuronal cells, the life span of the flies was extended substantially, up to 57% when dSir2 mRNA expression was increased 4-fold. Conversely, in two other similarly constructed strains in which dSir2 expression was not elevated or was only marginally elevated, life span was not altered relative to that of control flies. Thus, in six fly strains constructed by using different combinations of drivers and dSir2 responders, increased longevity correlated very well with elevation of dSir2. Furthermore, life span cannot be extended by calorie restriction in flies that lack dSir2 activity, nor can life span be further increased by calorie restriction in flies in which dSir2 activity is already raised. The recent findings that a Sir2 agonist, resveratrol (shown to increase the activity of yeast, nematode, fly, and human Sir2) extends life span in yeast, nematodes, and flies in a manner that is Sir2-dependent and associated with calorie restriction provide additional evidence for a primary role of Sir2 activity in determining life span in metazoans. Together, these observations make a strong case that calorie restriction extends life span in flies by increasing dSir2 activity (Rogina, 2004).

The data presented here, in conjunction with previous work on Rpd3 (Rogina, 2002), show that dSir2 and Rpd3 are important components in the calorie-restriction life-span-extending pathway of flies. A decrease in dSir2 prevents the life-span-extending effect of calorie restriction, and the life-span-extending effect of calorie restriction is not cumulative with the life-span-extending effect of increased dSir2. Similarly, the life-span-extending effect of Rpd3 mutations is not cumulative with the effect of calorie restriction (Rogina, 2002). Long-lived flies with reduced Rpd3 activity have elevated dSir2 mRNA (Rogina, 2002). This study shows that, in flies with decreases in both Rpd3 and dSir2 activity, life span is not extended, indicating that an increase in dSir2 activity in response to a decrease in Rpd3 activity is necessary for life span extension. Together these data suggest that dSir2 is downstream of Rpd3 in the calorie-restriction life-span-extending pathway in flies. This model provides a useful framework and testable model for examining the relationship of Sir2, calorie reduction, and longevity by using genetic, molecular, and pharmaceutical approaches. The documentation of a molecular genetic pathway responsible for effecting calorie-restriction-related life span extension will be useful for identifying biochemical mediators and drug interventions that can mimic calorie restriction. Given the conservation of elements of the calorie restriction/Rpd3/Sir2 pathway in extending life span in yeast and now flies, agents that stimulate the activity of Sir2 are potential tools for extending life span in metazoans (Rogina, 2004).

Mutations in the extra sex combs and Enhancer of Polycomb genes increase homologous recombination in somatic cells: Heterozygous deficiency for Rpd3 masks the radiation-resistant phenotype of both esc and E(Pc) mutants

Heterozygous mutant alleles of E(Pc) and esc increase homologous recombination from an allelic template in somatic cells in a P-element-induced double-strand break repair assay. Flies heterozygous for mutant alleles of these genes showed increased genome stability and decreased levels of apoptosis in imaginal discs and a concomitant increase in survival following ionizing radiation. It is proposed that this was caused by a genomewide increase in homologous recombination in somatic cells. A double mutant of E(Pc) and esc had no additive effect, showing that these genes act in the same pathway. Finally, it was found that a heterozygous deficiency for the histone deacetylase, Rpd3, masked the radiation-resistant phenotype of both esc and E(Pc) mutants. These findings provide evidence for a gene dosage-dependent interaction between the Esc/E(z) complex and the Tip60 histone acetyltransferase complex (see Tip60). It is proposed that esc and E(Pc) mutants enhance homologous recombination by modulating the histone acetylation status of histone H4 at the double-strand break (Holmes, 2006; full text of article).

Eukaryotes use both homologous recombination and nonhomologous end-joining to repair DSBs; survival is compromised severely when both are inactivated. In a homologous recombination assay, every cell in the adult head had a double-stranded break (DSB) generated at the white locus by excision of the P element. This was true for flies heterozygous for either the esc6 or the E(Pc)1 mutation and for wild-type flies. These breaks were repaired either by homologous recombination using the allelic white gene or by nonhomologous end-joining. Heterozygous mutants of any of three genes, E(Pc), Pcl, or esc, increased the likelihood that homologous recombination was used to repair a DSB made by P-element excision in cells of the developing eye-antennal imaginal disc. Thus, although nonhomologous end-joining was not measured directly, it was possible to conclude that the increase in pigmentation indicated an increase in the repair of DSBs by homologous recombination at the expense of nonhomologous end-joining and that heterozygosity for null alleles of either gene changed the balance between these two pathways. Further studies will be needed to determine if there is a defect in nonhomologous end-joining that is compensated by an increase in homologous recombination, or if nonhomologous end-joining is unaffected and homologous recombination is enhanced (Holmes, 2006).

The repair bias toward homologous recombination was observed when either paired or unpaired allelic templates were used. This suggested that the effects of the esc and E(Pc) mutants were not related to any chromosome-pairing-dependent activities of these genes (Holmes, 2006).

Alterations in DSB repair capacity or DSB pathway choice were not restricted to DSBs made by P-element excision at the white locus. Heterozygous mutants of either esc or E(Pc) showed a significant increase in genome integrity and a significant decrease in apoptosis following exposure to ionizing radiation. Not surprisingly, these animals were somewhat resistant to high doses of ionizing radiation (Holmes, 2006).

The increase in homologous recombination and the increase in genome integrity seen in esc6 heterozygous animals was suppressed by a P{esc+} transgene. This demonstrated that the effects were specific to the esc mutations and were not caused by genetic background effects. While the effect of the E(Pc)1 mutant was not suppressed with a transgene, it was observed that a large deficiency that included the E(Pc) gene had a similar effect to that of E(Pc)1 in the homologous recombination assay (Holmes, 2006).

Notably, the increase in homologous recombination was not seen in animals heterozygous for either single mutant of Psc. Furthermore, Psc, Pc, or Scm heterozygous animals are not resistant to ionizing radiation. The esc, Pcl, E(Pc), and Psc genes produce proteins that are localized to three different complexes. The proteins produced from the esc and Pcl genes are members of the esc/E(z) histone H3 methyltransferase complex, the E(Pc) protein is found in the Tip60/NuA4 histone acetyltransferase complex, and the Psc, Pc, and Scm proteins are components of the PRC1 complex. Interestingly, the esc and E(Pc) mutants alter the choice of repair pathway by a common mechanism since the esc6/E(Pc)1 double mutant lacked an additive effect in the homologous recombination assay over either single mutant. This suggests that the effects that were observed on the choice of repair pathway are not linked to the methylation activity of the Esc/E(z) complex or to the methyl-H3-binding activity of PCR1, but rather are linked to some other function that connects the Esc/E(z) and Tip60/NuA4 complexes (Holmes, 2006).

Since the Esc/E(z) complex often associates with a histone chaperone and the Rpd3 histone deacetylase and the E(Pc) protein is a component of a histone acetyl transferase known to be required for DSB repair in yeast, the hypothesis was tested that histone acetylation forms a link between these two complexes, and it was found that a heterozygous mutation for Rpd3 blocked the ability of heterozygous esc6 or E(Pc)1 alleles to confer resistance to ionizing radiation. This suggests that the Rpd3 gene product acts upstream of the esc and E(Pc) proteins in a pathway that influences the choice of which mechanism is used to repair DSBs (Holmes, 2006).

In yeast, the acetylation status of histone H4 plays a crucial role in determining whether a DSB is repaired by nonhomologous end-joining or by homologous recombination, and this role is distinct from its role in regulation of gene expression. Recent work in yeast and Drosophila shows that the NuA4/Tip60 histone acetyltransferase complex [which includes the yeast E(Pc) ortholog] is recruited to DSB sites and acetylates the tail lysines of histone H2A and H4. Histone acetylation is thought to neutralize the positively charged histone tail, thereby reducing the affinity between DNA and histones and loosening the compaction of the chromatin. The homologous recombination or nonhomologous end-joining repair machinery can then gain access to the damage and facilitate repair (Holmes, 2006).

Furthermore, nonhomologous end-joining requires the acetylation of all four lysine residues on the H4 histone tail, whereas homologous recombination requires only partial acetylation of these lysines. Likewise, mutations in the nonessential NuA4 subunit, Yng2, result in global hypoacetylation of histone H4 and are synthetic lethal with the YKU70 gene, further supporting the argument that nonhomologous end-joining requires complete acetylation of histone H4. Finally, yeast with hypoacetylated histone H4 not only are proficient in homologous recombination but also show enhanced recombination in a sister-chromatid exchange assay. These data suggest a compensatory relationship between nonhomologous end-joining and homologous recombination in Saccharomyces cerevisiae (Holmes, 2006).

The yeast data, together with the current results, suggest a model for the gene dose-dependent interaction among Rpd3, Esc, and E(Pc) in Drosophila. It is proposed that, similar to S. cerevisiae, nonhomologous end-joining in Drosophila somatic cells is dependent upon full acetylation of histone H4 tails, but that homologous recombination is not. In this model, a heterozygous E(Pc) mutation would cause a decrease in E(Pc) protein levels, resulting in a decrease in activity of the Tip60 histone acetyltransferase at the DSB and a shift toward homologous recombination. Likewise, a heterozygous esc mutation would result in less of the esc/E(z) complex. Since the Rpd3 protein is found in several different complexes aside from the Esc/E(z) complex, a decrease in the amount of Esc/E(z) complex would result in more free Rpd3 protein in the cell. The excess free Rpd3 protein could deacetylate more of the histone H4 at the DSB and thus shift DSB repair toward homologous recombination. In either instance, a decrease in the levels of Rpd3 protein in the presence of mutations in either esc or E(Pc) might be expected to result in more complete histone H4 acetylation and to restore the normal balance between homologous recombination and nonhomologous end-joining (Holmes, 2006).

It is becoming increasingly clear that PcG proteins have functions beyond the regulation of homeotic genes. Many recent studies have identified deregulation of different PcG proteins (Ezh2, Pcl, Bmi-1) in tumorigenesis, suggesting increased proliferation as a possible mechanism. If Esc or E(Pc) proteins were highly expressed in mammalian cancer, one might expect more frequent nonhomologous end-joining and, consequently, genome instability. Conversely, loss of one copy of either gene may provide a survival advantage under challenge with ionizing radiation or radiomimetic drugs. It is thus possible that the increased proliferation of tumor cells with mutations in these genes is, in part, not a direct result of increased expression of these genes, but rather a secondary effect of genome instability caused by decreases in gene conversion and increases in error-prone nonhomologous end-joining (Holmes, 2006).

Epigenetic blocking of an enhancer region controls irradiation-induced proapoptotic gene expression in Drosophila embryos

Drosophila embryos are highly sensitive to gamma-ray-induced apoptosis at early but not later, more differentiated stages during development. Two proapoptotic genes, reaper and hid, are upregulated rapidly following irradiation. However, in post-stage-12 embryos, in which most cells have begun differentiation, neither proapoptotic gene can be induced by high doses of irradiation. The sensitive-to-resistant transition is due to epigenetic blocking of the irradiation-responsive enhancer region (IRER), which is located upstream of reaper but is also required for the induction of hid in response to irradiation. This IRER, but not the transcribed regions of reaper/hid, becomes enriched for trimethylated H3K27/H3K9 and forms a heterochromatin-like structure during the sensitive-to-resistant transition. The functions of histone-modifying enzymes Hdac1(Rpd3) and Su(var)3-9 and PcG proteins Su(z)12 and Polycomb are required for this process. Thus, direct epigenetic regulation of two proapoptotic genes controls cellular sensitivity to cytotoxic stimuli (Zhang, 2008).

Mutations in String/CDC25 inhibit cell cycle re-entry and neurodegeneration in a Drosophila model of Ataxia telangiectasia

Mutations in ATM (Ataxia telangiectasia mutated) result in Ataxia telangiectasia (A-T), a disorder characterized by progressive neurodegeneration. Despite advances in understanding how ATM signals cell cycle arrest, DNA repair, and apoptosis in response to DNA damage, it remains unclear why loss of ATM causes degeneration of post-mitotic neurons and why the neurological phenotype of ATM-null individuals varies in severity. To address these issues, a Drosophila model of A-T was generated. RNAi knockdown of ATM in the eye caused progressive degeneration of adult neurons in the absence of exogenously induced DNA damage. Heterozygous mutations in select genes modified the neurodegeneration phenotype, suggesting that genetic background underlies variable neurodegeneration in A-T. The neuroprotective activity of ATM may be negatively regulated by deacetylation since mutations in a protein deacetylase gene, RPD3, suppressed neurodegeneration, and a human homolog of RPD3, histone deacetylase 2, bound ATM and abrogated ATM activation in cell culture. Moreover, knockdown of ATM in post-mitotic neurons caused cell cycle re-entry, and heterozygous mutations in the cell cycle activator gene String/CDC25 inhibited cell cycle re-entry and neurodegeneration. Thus, it is hypothesized that ATM performs a cell cycle checkpoint function to protect post-mitotic neurons from degeneration and that cell cycle re-entry causes neurodegeneration in A-T (Rimkus, 2008).

The data indicate that ATM knockdown by RNAi causes degeneration of Drosophila post-mitotic photoreceptor neurons. The neurodegeneration phenotype of ATM knockdown flies is similar to that observed in A-T patients. Neurodegeneration in the fly model occurred in the absence of exogenously induced DNA damage, it occurred independently of developmental defects, and it was progressive, increasing in severity as flies aged. Thus, ATM knockdown flies appear to be an appropriate model to study the cellular mechanisms underlying neurodegeneration in A-T (Rimkus, 2008).

A-T is a monogenic disease resulting from mutation of the ATM gene; however, the genetic screen identified second site genes that affect an A-T phenotype, neurodegeneration. Remarkably, independent lines of evidence, from the literature and from the current studies, support the relevance of each of the modifier genes to the mechanism underlying neurodegeneration in A-T (Rimkus, 2008).

ATM is recruited to DSBs by the trimeric MRE11-RAD50-NBS1 (MRN) DNA repair complex, which possesses ATP- dependent nuclease (MRE11) and DNA-tethering (RAD50) activities (Abraham, 2005). Three of the six genes identified in the screen (Stg, RAD50, and PP2A-B') are known components of the ATM signaling pathway that responds to DNA damage in mammals. A role for RAD50 in promoting neurodegeneration is not specific to the eye, since mutation of RAD50 suppress the lethality of Elav-ATMi flies. In addition, mutation of the gene encoding the NBS1 subunit of MRN suppresses the GMR-ATMi rough eye phenotype, suggesting that suppression by RAD50 and NBS1 mutants is due to reduced activity of the MRN complex. Nevertheless, the mechanism underlying suppression of neurodegeneration is unclear since reduced levels of the MRN complex would intuitively be expected to enhance GMR-ATMi phenotypes. One possibility is that the MRN complex is deregulated in the absence of ATM and carries out activities that are lethal to neurons. Finally, PP2A has been shown to dephosphorylate several ATM signaling pathways substrates, including ATM. Mutation of PP2A-B', which encodes a regulatory subunit of the PP2A complex, may enhance neurodegeneration in ATM knockdown flies by affecting the phosphorylation state of ATM substrates (Rimkus, 2008).

Two of the identified genes, MEKK4 and Delta, have potential links to ATM. Mutation of MEKK4 may enhance neurodegeneration in GMR-ATMi flies by allowing cell cycle progression. Published studies suggest a model whereby ATM and MEKK4 pathways collaborate to prevent cell cycle re-entry of post-mitotic neurons by maintaining the latency of CDC25 proteins. Delta encodes a ligand for the Notch receptor, which regulates cell cycle progression and differentiation in many tissues, including the eye. Thus, there may be cross-talk between the ATM and Notch signaling pathways in neurons (Rimkus, 2008).

Finally, studies in cultured cells revealed a direct link between HDAC2, the human homolog of Drosophila RPD3, and ATM. HDAC2 was found to directly associate with ATM and regulate its kinase activity in the absence of exogenously induced DNA damage. Thus, HDAC2 is likely the TSA-sensitive deacetylase that negatively regulates ATM kinase activity. HDAC2 may function by counteracting acetylation of ATM or downstream components of the ATM signaling pathway. It is important to note that although deacetylation of ATM by HDAC2 may regulate ATM activity, HDAC2 is not necessarily an important factor in A-T since the majority of mutations in A-T patients are nonsense or frameshift mutations that result in complete loss or truncation of ATM protein. Nevertheless, the demonstrated physical and functional interactions between HDAC2 and ATM indicate that HDAC2 is an important component of the ATM signaling paradigm and that information garnered from studies of ATM knockdown flies can advance understanding of ATM function in humans (Rimkus, 2008).

Results from the genetic screen predict that A-T patients with mild neurodegeneration will carry heterozygous mutations in suppressor genes, such as CDC25 family members, whereas A-T patients with severe neurodegeneration will carry heterozygous mutations in enhancer genes, such as MEKK4. It will be interesting to see if genes that enhance neurodegeneration in ATM knockdown flies also enhance neurodegeneration in mice. For example, do ATM-/- MEKK4+/- mice exhibit progressive degeneration of cerebellar neurons? If so, this would make mice a practical model for studying the neurodegenerative aspects of A-T (Rimkus, 2008).

The data indicate a causal relationship between cell cycle re-entry and neurodegeneration in the Drosophila model of A-T presented in this study. (1) ATM knockdown in photoreceptor neurons resulted in cell cycle re-entry and neurodegeneration, implicating ATM in both processes. (2) Heterozygous mutation of cell cycle regulatory genes Stg/CDC25, Cdk2, dE2F1, and dE2F2 modified the neurodegeneration phenotype of ATM knockdown flies, highlighting the importance of cell cycle regulation in neurodegeneration. (3) Inhibition of cell cycle re-entry by mutation of Stg/CDC25 also inhibited degeneration of ATM knockdown neurons. In contrast, inhibition of neurodegeneration by expression of P35 did not inhibit cell cycle re-entry. (4) Inhibition of neurodegeneration by expression of P35 caused the accumulation of neurons in S/G2/M phases of the cell cycle, indicating that the neurons that re-entered the cell cycle are the ones that degenerated (Rimkus, 2008).

These findings add to a growing literature linking cell cycle re-entry and neurodegeneration. The observations that terminally differentiated neurons are resistant to oncogenic transformation and that brain tumors of neuronal origin rarely occur suggest that cell cycle re-entry of post-mitotic neurons results in death rather than proliferation. In fact, it has been shown in a variety of systems, including flies and humans, that when neurons re-enter the cell cycle, the result is degeneration rather than proliferation and that ectopic cell cycle activation in neurons is sufficient to trigger degeneration. Furthermore, up-regulation of cell cycle genes, such as proliferating cell nuclear antigen, cyclin A, and cyclin B, has been shown to occur in post-mitotic Purkinje and granule cells of A-T patients; and neurons of ATM-/- mice have been found to undergo DNA replication. Similarly, studies in both fly and mammalian models of Alzheimer’s disease support a causative link between cell cycle re-entry and neurodegeneration. Thus, failure of cell cycle regulation may be a common cause of neurodegenerative disorders, including A-T (Rimkus, 2008).

It is important to keep in mind that equally plausible and nonexclusive models have been put forth for why neurodegeneration occurs in A-T. The oxidative stress model proposes that neurodegeneration occurs as a consequence of increased oxidative stress, and the DNA damage model proposes that neurodegeneration occurs as a consequence of the accumulation of DNA damage. While these models are described as functioning independently, it is unlikely that this is the case. For example, oxidative stress could lead to cell cycle re-entry through several different pathways, and in response to DNA damage, neurons may re-enter the cell cycle before undergoing cell death (Rimkus, 2008).

This paper has described a powerful experimental model in Drosophila to study the molecular mechanisms that underlie neurodegeneration in the human disease A-T. ATM knockdown in flies caused post-mitotic neurons to re-enter the cell cycle and die by programmed cell death. This finding suggests that ATM performs a cell cycle checkpoint function in post-mitotic neurons, as it does in response to DNA damage in proliferating nonneuronal cells and neuroblasts. Heterozygous mutation of Stg/CDC25 suppressed neurodegeneration in ATM knockdown flies and inhibited cell cycle re-entry, suggesting that cell cycle re-entry is causative for neurodegeneration in A-T. In the future, further genetic, cell biological, and molecular analysis of the Drosophila A-T model will allow addressing of unresolved issues, such as the extent to which oxidative stress and DNA damage contribute to neurodegeneration in the absence of ATM, what factors trigger cell cycle re-entry in the absence of ATM, and what factors link cell cycle re-entry to programmed cell death as opposed to cell division in the absence of ATM (Rimkus, 2008).

RPD3 histone deacetylase and nutrition have distinct but interacting effects on Drosophila longevity

Single-gene mutations that extend longevity have revealed regulatory pathways related to aging and longevity. RPD3 is a conserved histone deacetylase (Class I HDAC). Previous studies have shown that Drosophila Rpd3 mutations increase longevity. This study tested the longevity effects of RPD3 on multiple nutrient levels. Dietary restriction (DR) has additive effects on RPD3-mediated longevity extension, but the effect may be modestly attenuated relative to controls. RPD3 and DR therefore appear to operate by distinct but interacting mechanisms. Since RPD3 regulates transcription, the mRNA levels for two proteins involved in nutrient signaling, 4E-BP and Tor, were examined in rpd3 mutant flies. 4E-BP mRNA was reduced under longevity-increasing conditions. Epistasis between RPD3 and 4E-BP with regard to longevity was then tested. Flies only heterozygous for a mutation in Thor, the 4E-BP gene, have modestly decreased life spans. Flies mutant for both rpd3 and Thor show a superposition of a large RPD3-mediated increase and a small Thor-mediated decrease in longevity at all food levels, consistent with each gene product having distinct effects on life span. However, DR-mediated extension was absent in males carrying both mutations and lessened in females. These results support the view that multiple discrete but interacting mechanisms regulate longevity (Frankel, 2015).

Loss of histone deacetylase HDAC1 induces cell death in Drosophila epithelial cells through JNK and Hippo signaling

Inactivation of HDAC1 and its homolog HDAC2 or addition of HDAC inhibitors in mammalian systems induces apoptosis, cell cycle arrest, and developmental defects. Although these phenotypes have been extensively characterized, the precise underlying mechanisms remain unclear, particularly in in vivo settings. This study shows that inactivation of Rpd3, the only HDAC1 and HDAC2 ortholog in Drosophila, induced apoptosis and clone elimination in the developing eye and wing imaginal discs. Depletion of Rpd3 by RNAi cell-autonomously increased JNK activities and decreased activities of Yki, the nuclear effecter of Hippo signaling pathway. In addition, inhibition of JNK activities largely rescued Rpd3 RNAi-induced apoptosis, but did not affect its inhibition of Yki activities. Conversely, increasing the Yki activities largely rescued Rpd3 RNAi-induced apoptosis, but did not affect its induction of JNK activities. Furthermore, inactivation of Mi-2, a core component of the Rpd3-containing NuRD complex strongly induced JNK activities; while inactivation of Sin3A, a key component of the Rpd3-containing Sin3 complex, significantly inhibited Yki activities. Taken together, these results reveal that inactivation of Rpd3 independently regulates JNK and Yki activities and that both Hippo and JNK signaling pathways contribute to Rpd3 RNAi-induced apoptosis (Zhang, 2016).

Rpd3 interacts with insulin signaling in Drosophila longevity extension

Histone deacetylase (HDAC) 1 regulates chromatin compaction and gene expression by removing acetyl groups from lysine residues within histones. HDAC1 affects a variety of processes including proliferation, development, metabolism, and cancer. Reduction or inhibition of Rpd3, yeast and fly HDAC1 orthologue, extends longevity. However, the mechanism of rpd3's effects on longevity remains unclear. This study reports an overlap between rpd3 and the Insulin/Insulin-like growth factor signaling (IIS) longevity pathways. rpd3 reduction downregulates expression of members of the IIS pathway, which is associated with altered metabolism, increased energy storage, and higher resistance to starvation and oxidative stress. Genetic studies support the role of IIS in rpd3 longevity pathway, as illustrated with reduced stress resistance and longevity of flies double mutant for rpd3 and dfoxo, a downstream target of IIS pathway, compared to rpd3 single mutant flies. The data suggest that increased dfoxo is a mediator of rpd3 's effects on fly longevity and intermediary metabolism, and confer a new link between rpd3 and IIS longevity pathways (Woods, 2016).

The Histone deacetylase gene Rpd3 is required for starvation stress resistance

Epigenetic regulation in starvation is important but not fully understood yet. This study identified the Rpd3 gene, a Drosophila homolog of histone deacetylase 1, as a critical epigenetic regulator for acquiring starvation stress resistance. Immunostaining analyses of Drosophila fat body revealed that the subcellular localization and levels of Rpd3 dynamically changed responding to starvation stress. In response to starvation stress, the level of Rpd3 rapidly increased, and it accumulated in the nucleolus in what appeared to be foci. These observations suggest that Rpd3 plays a role in regulation of rRNA synthesis in the nucleolus. The RT-qPCR and ChIP-qPCR analyses clarified that Rpd3 binds to the genomic region containing the rRNA promoters and activates rRNA synthesis in response to starvation stress. Polysome analyses revealed that the amount of polysomes was decreased in Rpd3 knockdown flies under starvation stress compared with the control flies. Since the autophagy-related proteins are known to be starvation stress tolerance proteins, autophagy activity was examined, and it was reduced in Rpd3 knockdown flies. Taken together, it is concluded that Rpd3 accumulates in the nucleolus in the early stage of starvation, upregulates rRNA synthesis, maintains the polysome amount for translation, and finally increases stress tolerance proteins, such as autophagy-related proteins, to acquire starvation stress resistance (Nakajima, 2016).


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Histone deacetylase 1: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 30 October 2020

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