BIOLOGICAL OVERVIEW

Rpd3 is a transcriptional regulator and in yeast, a known histone deacetylase. Histone acetylation is considered an important mechanism activating gene expression, since acetylation of histones spreads out (or opens up) chromatin, thereby making genes more accessible to transcriptional activators. Histone deacetylation acts in the opposite direction: it promotes gene silencing. 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 null mutants show an increase in PEV, and since PEV is considered a phenomenon involving gene silencing (the spreading of heterchromatin), the function of wild type Rpd3 may be to counteract PEV induced gene silencing. Rpd3 has also been implicated as a corepressor of Even-skipped, helping Eve silence Eve targets (Mannervik, 1999). This essay will consider what appears to be the two known, and paradoxically opposite, activities of Rdp3: its effect on PEV to counteract gene silencing and its effect in conjunction with Eve to enhance gene silencing.

PEV is measured in Drosophila by its effect on the expression of white, particularly with regard to a white gene that has changed chromosomal position due to a chromosomal rearrangement. In a well characterized inversion of the white locus in Drosophila, white became relocated next to a breakpoint within centromeric heterochromatin. Thus, white can be permanently inactivated in some cells by the spreading of the adjacent condensed heterochromatin. The resulting mottled eye-color phenotype is sensitive to the dose of several unlinked loci, known as suppressors and enhancers of PEV. These modifiers respectively decrease and increase the frequency of clones in which the white gene is inactive. A homozygous viable P-transposon insertion at 64B, on the third chromosome, enhances the variegation of white. In 5%-10% of eyes, patches of ommatidia show the disorganization characteristic of the phenotype of the roughest mutation, a locus near white, but more distant from heterochromatin in the rearrangement. This suggests that the enhancement of variegation allows the silencing to spread past the white locus and to inactivate the roughest locus in a fraction of the cells (De Rubertis, 1996).

The locus next to the inserted P-element was cloned and found to encode a homolog of yeast RPD3. in situ detection of transcripts 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. These results indicate that enhanced PEV results from the loss of Rpd3 function in the phenotypically relevant tissue. The inactivating mutation affects only the expression of Rpd3 in the eye (De Rubertis, 1996).

Based on the Drosophila results, a test was performed to see whether mutation of yeast RPD3 also effects PEV. In Saccharomyces cerevisiae, genes positioned up to 4.9 kb proximal to a telomere can be transcriptionally silent, a phenomenon called telomeric position-effect. Sure enough, an rpd3 null allele increases telomeric silencing in yeast just as the homologous gene does in Drosophila. RPD3 has been shown to have an analogous function with respect to genomic silencing in both budding yeast and Drosophila. In both organisms, loss of RPD3 function results in increased genomic silencing, which decreases with the distance from heterochromatin (De Robertis, 1996).

The possibility that RPD3 indirectly affects genomic silencing (for example, by repressing the expression of a positive regulator of chromatin condensation) cannot be excluded. Nevertheless, the striking similarity of rpd3 mutant phenotypes in yeast and Drosophila strongly suggests that RPD3 is directly involved in counteracting gene silencing. However, there are precedents in yeast and Drosophila of such opposite actions, and of putative effects of acetylation on transcriptonal repression. In yeast, rpd3 mutations can restore the metastable repression in strains carrying a particular combination of genes, a mutant silencer-binding protein (rap1s) and a mutated silencer element (hmr delta A) (Sussel, 1995). Moreover, in the silenced yeast mating-type loci, lysine 12 of histone H4 is acetylated, and this acetylation establishes transcriptonal silencing (Braunstein, 1996). In this regard, rpd3 mutant strains show increased acetylation of lysines 5 and 12 of histone H4 (Rundlett, 1996). In Drosophila, a particular isoform of histone H4 acetylated on lysine 12 is abundant in heterochromatin, whereas the other isoforms are underrepresented (Turner, 1992). Lysines 5 and 12 of H4 are acetylated when newly synthesized histone is deposited, and then rapidly deacetylated (Sobel, 1995). Here, RPD3, as a hypothetical deposition-related deacetylase, could support silencing (De Rubertis, 1996). Thus a specific RPD3-dependent deacetylation could act as a negative signal in establishing a silencing protein complex. More generally, these results suggest that silenced loci may have specialized chromatin structures that are conserved between yeast and Drosophila (De Rubertis, 1996).

Working out the effects of Rpd3 on segmentation gene expression requires the scientific equivalent of investigative journalism. The Berkeley Drosophila Genome Project identified a P-induced lethal mutation (l(3)04556) that maps 47 bp downstream of the Rpd3 putative transcription start site. Previous work by Perrimon (1996) has shown that embryos derived from l(3)04556 homozygous germline clones exhibit pair-rule patterning defects that are similar to those observed in fushi tarazu mutants. It was first established that most repressors are active in Rpd3 mutant embryos. These results suggest that the Rpd3 mutation might impair expression of Fushi tarazu or Ftz-F1 proteins, known to be gene activators, because these are required for the expression of the even-numbered engrailed stripes. Alternatively, the loss of Rpd3 might lead to a change in the expression pattern of a repressor, which in turn inhibits Ftz activity (Mannervik, 1999).

To distinguish between these possibilities, an examination was made of the expression of odd-skipped, a known repressor of en. odd is initially expressed in a pair-rule pattern of seven stripes, but during gastrulation seven additional secondary stripes are formed to generate a 14-stripe expression pattern. In normal embryos, these stripes are evenly spaced, whereas in Rpd3 mutants they are not. In the mutant embryos there is a partial pair-wise alignment of adjacent odd stripes. A similar change is observed in even-skipped embryos. Previous studies suggest that both ftz and odd stripes are under the control of the Eve repressor (Manoukian, 1992). Differential repression of ftz and odd resolves the two patterns, so that each ftz stripe is normally shifted anterior to each odd-numbered odd stripe. In Rpd3 mutants, the ftz and odd patterns fail to resolve, so that odd-numbered odd stripes mostly coincide with the ftz stripes. It is suggested that this failure in ftz-odd resolution is responsible for the pair-rule phenotype observed in Rpd3 mutant embryos. A prediction of this proposal is that eve mutants should exhibit similar alterations in the ftz and odd expression patterns. Double staining assays reveal that eve mutant embryos exhibit a similar failure to resolve the ftz and odd expression patterns (Mannervik, 1999).

There are several possible explanations for impaired Ftz function in Rpd3 mutants. It is conceivable that the Rpd3 mutation disrupts Ftz-mediated activation. However, the idea that Rpd3 functions as a corepressor of Eve is favored. The similarities in the Rpd3 and ftz mutant phenotypes may be caused by the coincident odd and ftz expression patterns observed in embryos derived from l(3)04556 germline clones. The Odd repressor is thought to block Ftz-mediated activation of en. Evidence is presented that this expansion in Odd might result from an inability of Eve to repress odd expression in Rpd3 mutant embryos. Consistent with this proposal, in vitro translated Eve is shown to interact with a glutathione S-transferase-Rpd3 fusion protein. Because the Eve repressor is required for both the odd- and even-numbered en stripes, it would appear that the Rpd3 mutation does not cause a general loss of Eve function. For example, eve hypomorphs cause the loss of odd-numbered en stripes, whereas null mutations cause a loss of all en stripes. It would therefore appear that Eve fails to repress certain promoters (e.g., odd and possibly ftz) in Rpd3 mutant embryos, but retains repressor function on other promoters (e.g., paired and sloppy-paired). This selectivity in the regulation of different target promoters is consistent with the notion that Eve mediates repression through multiple mechanisms, including the recruitment of corepressors and direct interactions with TBP. Multiple modes of repression may be mediated by other transcriptional repressors, such as Hairy, which appears to interact with different classes of corepressors (Mannervik, 1999 and references).

Given the importance of Rpd3 as a corepressor of both yeast and mammalian transcriptional repressors, it was anticipated that the Rpd3 mutants would exhibit more severe patterning defects. Instead, it would appear that this histone deacetylase does not represent a major pathway of repression in the early embryo. Of course, it is conceivable that the complete loss of Rpd3 products would cause more severe patterning defects. The available Rpd3 mutation is only partial and Rpd is expressed maternally. Unfortunately, it might not be possible to produce germline clones for a null mutation in the Rpd3 gene because the present hypomorphic allele produces very few eggs and mutations in genes that encode associated proteins such as Sin3 and Mi-2 (see Evolutionary Homologs section) fail to produce viable germline clones. An alternative explanation for the relatively mild Rpd3 patterning defects is that there is redundancy among different deacetylases. Indeed, two additional histone deacetylases are maternally expressed and ubiquitously distributed throughout the early embryo (Mannervik, 1999).

rpd3 is identified in a screen for genes that function in leg disc regeneration in Drosophila

Many diverse animal species regenerate parts of an organ or tissue after injury. However, the molecules responsible for the regenerative growth remain largely unknown. The screen reported in this study aimed to identify genes that function in regeneration and the transdetermination events closely associated with imaginal disc regeneration using Drosophila melanogaster. A collection of 97 recessive lethal P-lacZ enhancer trap lines were screened for two primary criteria: first, the ability to dominantly modify wg-induced leg-to-wing transdetermination and second, for the activation or repression of the lacZ reporter gene in the blastema during disc regeneration. Of the 97 P-lacZ lines, six genes (Krüppelhomolog- 1, rpd3, jing, combgap, Aly and S6 kinase) were identified that met both criteria. Five of these genes suppress, while one enhances, leg-to-wing transdetermination and therefore affects disc regeneration. Two of the genes, jing and rpd3, function in concert with chromatin remodeling proteins of the Polycomb Group (PcG) and trithorax Group (trxG) genes during Drosophila development, thus linking chromatin remodeling with the process of regeneration (McClure, 2008).

There are three different mechanisms that organisms use to re-grow and replace lost or damaged body parts, and often, more than one mechanism can function within different tissues of the same organism. Muscle and bone, for example, repair themselves by activating a resident stem cell population, while the liver regenerates by compensatory proliferation of normally quiescent differentiated cells. Appendage/fin regeneration in lower vertebrates occurs by a process termed epimorphic regeneration, which proceeds in three distinct stages: (1) wound healing and migration of the surrounding epithelial cells to form the wound epidermis, (2) formation of the regeneration blastema -- a mass of undifferentiated and proliferating cells of mesenchymal origin and (3) regenerative outgrowth and pattern re-formation. Whether these diverse modes of regeneration share a common molecular and genetic basis is not known (McClure, 2008).

Regeneration in the Drosophila imaginal discs, the primordia of the adult fly appendages, closely parallels epimorphic limb/fin regeneration in lower vertebrates. Cells in the imaginal discs are rigidly determined to form specific adult structures (e.g., legs and wings) by the third larval instar. If the discs are fragmented at this time and cultured in vivo, they will regenerate. Disc regeneration begins 12 h after wounding, when transient heterotypic contacts are made between peripodial (squamous epithelium) and columnar cells (disc proper) near the cut edges of the wound. These initial contacts involve microvilli-like extensions and provide temporary wound closure. Then, approximately 24 h after wounding, homotypic cell contacts (between columnar or between squamous cells) are made involving the close apposition of cell membranes and cellular bridges, which eventually (48 h after wounding) restore the physical continuity of the disc. Before and during wound healing, cell division is randomly distributed throughout the disc. However, once completed (36-48 h after wounding), division is observed only in cells near the wound site. These cells are known as the regeneration blastema. Thus, like appendage regeneration in lower vertebrates, disc regeneration involves wound healing followed by blastema formation (McClure, 2008).

Blastema cells are responsible for the regeneration and repatterning of the entire missing disc fragment. Thus, these cells exhibit remarkable developmental plasticity. For example, in anterior- only leg disc fragments, some blastema cells will switch to posterior identity and establish a novel posterior compartment in the regenerate. This anterior/posterior conversion occurs during heterotypic wound healing, when hedgehog (hh)- expressing peripodial cells induce ectopic engrailed (en) expression in the apposing anterior columnar cells. In addition, the disc blastema, like its vertebrate counterpart, is able to form a normal regenerate (complete leg disc and adult leg) when isolated from the remaining disc fragment. Regenerative plasticity is also observed when a few blastema cells switch fate to that of another disc type (e.g., leg-to-wing), in a phenomenon known as transdetermination. Transdetermination events are closely associated with regenerative disc growth. Clonal analysis, for example, has shown that blastema cells first regenerate the missing disc structures, and only then, are they competent to transdetermine (McClure, 2008).

Little is known about how the regeneration blastema forms in the fragmented leg disc, although ectopic Wingless (Wg/Wnt1) expression is detected along the cut site, both prior to and during blastema formation. Wg is a developmental signal in many different tissues and animals; in flies Wg patterns all of the imaginal discs, functioning as both a morphogen and mitogen to regulate disc cell fate and growth. In lower vertebrates, Wnt ligands are key regulators of blastema formation during epimorphic regeneration. Thus, activation of Wg within the disc blastema is potentially important for regeneration. This idea is consistent with the observation that ubiquitous expression of wg during the second or third larval instars, in unfragmented leg discs, is sufficient to induce a regeneration blastema in the proximodorsal region of the disc, known as the weak point. Moreover, ubiquitous expression of wg mimics the pattern deviations associated with leg disc fragmentation and subsequent regeneration, including the duplication of ventral with concomitant loss of dorsal pattern elements and leg-to-wing transdetermination events. Thus, leg disc regeneration can be examined using two experimental protocols: fragmentation or ubiquitous wg expression. However, it is important to point out that only fragmentation-induced regeneration involves wound healing (McClure, 2008).

Precisely which molecules and signaling pathways are required for the process of regeneration remain poorly understood, partly because the organisms historically used to study regeneration (e.g., newts and salamanders) have been refractory to genetics and molecular manipulations. Recently, however, the use of new genetic techniques together with 'regeneration' model systems -- such as planarians, hydra and zebrafish have given researchers the opportunity to examine the mechanisms of regeneration and to identify the genes, proteins and signaling pathways that regulate different regenerative processes. For example, a large scale RNAi-based screen was performed to survey gene function in planarian tissue homeostasis and regeneration. Out of ~1000 genes examined, RNAi knock-down of 240 displayed regeneration-related phenotypes, including defects in wound healing, blastema formation and blastema cell differentiation. Despite these studies, however, it remains unclear whether regeneration requires only the modulation of genes expressed at the time of injury, the reactivation of earlier developmental genes and/or signaling pathways, or the activation of novel genes specific to the process of regeneration. Thus, a major interest in the field of regenerative biology is the identification of gene products that regulate blastema formation, blastema growth and regenerative cellular plasticity. A genetic screen, using wg-induced leg disc regeneration, aimed at identifying genes that regulate cellular plasticity and regeneration using Drosophila was carried out prothoracic leg discs. A collection of 97 recessive lethal P-element lacZ (PZ) insertion lines were screened for ectopic lacZ expression during wg-induced leg disc regeneration, and six genes were identified that function in wg-induced leg disc regeneration, including genes with functional ties to Wg signaling as well as chromatin remodeling proteins (McClure, 2008).

This study consisted of an enhancer trap screen designed to identify genes with changed gene expression during leg disc regeneration as well as required for regenerative proliferation and growth. The screen identified 19 genes that when heterozygous mutant (PZ/+), dominantly modify wg-induced leg-to-wing transdetermination, which serves as a functional assay for disc regeneration. Of the 19 genes, 37% are transcription factors or involved in transcriptional regulation (tai, Krh1, ken, jing, combgap (cg), rpd3 and Aly), 21% function in cell cycle regulation and growth (oho23B, S6k, polo and cycA), 10.5% play a role in protein secretion (Secβ61 and Syx13), and 31% are of other or unknown function [l(3)01629, CG30947, l(2)00248, l(3)05203, l(3)01344, Nup154]. The identification of transcription factors as the most frequent class of genes that modify wg-induced leg disc regeneration was similarly observed in a DNA microarray screen designed to identify genes enriched in leg disc cells that transdetermine to wing (Klebes, 2005). Together, these findings strongly suggest that transcription factors and their downstream targets play a prominent role in disc cell plasticity (McClure, 2008).

Using lacZ expression analyses, together with whole mount in situ hybridization experiments, the expression patterns of the 19 genes that modified wg-induced leg-to-wing transdetermination were verified. This analysis identified several different expression patterns upon wg-induced regeneration, including a loss of gene expression, ubiquitous expression and genes with expression limited to the regeneration blastema. Such observations indicate that a complex change of gene expression, both negative and positive, mediates the process of epimorphic regeneration. Six (jing, Alyi cg, rpd3, Kr-h1 and S6k) of the 19 modifiers displayed expression limited to the regeneration blastema, indicating that novel markers of regeneration and transdetermination have been identified. The blastema-specific expression patterns of jing, Aly, cg, Kr-h1, rpd3 and S6k raised the intriguing possibility that these genes may be functionally involved in the formation, cell proliferation or maintenance of the blastema during disc regeneration. Indeed, upon ubiquitous wg expression jing/+ animals rarely formed a regeneration blastema, indicating that two wild-type copies of jing are required for the initiation of the regenerative process. In contrast, Aly/+ and cg/+ animals formed a normal blastema, but only after a one-day delay. Therefore, two wild-type copies of the Aly and cg genes are required for the proper timing of regeneration. In addition, it was found that the frequency of blastema formation was reduced in rpd3/+ animals, implicating this gene in the process of regeneration. Interestingly, heterozygous mutations in all four of these genes (jing, Aly, cg and rpd3) strongly suppress wg-induced leg-to-wing transdetermination. It is speculated that the transdetermination frequency declines in these mutant animals because the initiation and/or timing of blastema formation is delayed. This idea is consistent with all previous work which has shown that blastema cells are only competent to transdetermine after they have regenerated the missing disc structures. Heterozygous mutations in Kr-h1 and S6k did not significantly alter the formation of the wg-induced regeneration blastema, however, these genes did affect regeneration-induced transdetermination. Such results suggest that Kr-h1 and S6k specifically function to modulate the cell fate changes that occur as a consequence of regeneration (McClure, 2008).

Investigations into the molecular basis of transdetermination have shown that inputs from the Wg, Decapentapelagic (Dpp) and Hedgehog (Hh) signaling pathways activate key selector genes out of their normal developmental context, such as ectopic Vg activation in the leg disc, which then drives cell-fate switches. Several of the genes identified in this screen have functional ties to Wg, Dpp and Hh signaling pathways. For example, Cg is a zinc-finger transcription factor that is required for proper transcriptional regulation of the Hh signaling effector gene Cubitus interruptus (Ci). In cg mutant wing and leg discs, Ci expression is lowered in the anterior compartment, resulting in the ectopic activation of wg and dpp and significant disc overgrowth. Another gene identified in this screen -- ken, functions in concert with Dpp to direct the development of the Drosophila terminalia. Further characterizations of whether these genes and other modifiers of transdetermination and regeneration affect Wg, Dpp and Hh expression and/or signaling may shed light on the regulation of regeneration and regeneration-induced proliferation and cell fate plasticity (McClure, 2008).

Heart-specific Rpd3 downregulation enhances cardiac function and longevity

Downregulation of Rpd3, a homologue of mammalian Histone Deacetylase 1 (HDAC1), extends lifespan in Drosophila melanogaster. Once revealed that long-lived fruit flies exhibit limited cardiac decline, this study investigated whether Rpd3 downregulation would improve stress resistance and/or lifespan when targeted in the heart. Contested against three different stressors (oxidation, starvation and heat), heart-specific Rpd3 downregulation significantly enhanced stress resistance in flies. However, these higher levels of resistance were not observed when Rpd3 downregulation was targeted in other tissues or when other long-lived flies were tested in the heart-specific manner. Interestingly, the expressions of anti-aging genes such as sod2, foxo and Thor, were systemically increased as a consequence of heart-specific Rpd3 downregulation. Showing higher resistance to oxidative stress, the heart-specific Rpd3 downregulation concurrently exhibited improved cardiac functions, demonstrating an increased heart rate, decreased heart failure and accelerated heart recovery. Conversely, Rpd3 upregulation in cardiac tissue reduced systemic resistance against heat stress with decreased heart function, also specifying phosphorylated Rpd3 levels as a significant modulator. Continual downregulation of Rpd3 throughout aging increased lifespan, implicating that Rpd3 deacetylase in the heart plays a significant role in cardiac function and longevity to systemically modulate the fly's response to the environment (Kopp, 2015).

The data showed that decreased Rpd3 expression in Drosophila has a benefit for stress resistance against the environment. To downregulate the rpd3 gene in whole body, two ways were approached using the heterozygous rpd3 mutant (P{PZ}rpd3[04556]/+) and the UAS/Gal4 system to carry out RNAi (rpd3Ri/armG4). Both flies showed higher survivorship under oxidative stress compared to the control flies. However, the flies differed in increased survivorship percent (rpd3-/+: 31% and rpd3Ri/armG4: 22%). Considering that the downregulation yield of the rpd3 gene was different between the two approaches (rpd3-/+: 54% and rpd3Ri/armG4: 40%), it is possible that more downregulation of the rpd3 gene may induce higher resistance to stress. In the heart-specific Rpd3 downregulation, a similar pattern was observed between the rpd3Ri/tinG4 and rpd3RiS/tinG4 flies. The 21bp target sequence of rpd3RiS transgene was less effective at rpd3 downregulation compared to the 482bp sequences of rpd3Ri transgene when tested in the whole body. Thus, the rpd3RiS/tinG4 flies showed a 23% increase in survivorship compared to a 35% increase in rpd3Ri/tinG4 flies (Fig. 3A). Those data let to a speculation that the content of rpd3 downregulation determines the consequent stress-resistance enhancement (Kopp, 2015).

It was found that heart-specific Rpd3 downregulation systemically increases expression of anti-aging genes such as Sod2 and dFOXO. It was also shown that more downregulation of the rpd3 gene in a heart induces higher expression of anti-aging genes. This may provide an explanation of how Rpd3 downregulation in the heart enhances stress resistance mechanism, particularly since dFOXO is considered to activate sod2 gene. In response to cellular stresses, such as nutrient deprivation or increased levels of reactive oxygen species, dFOXO is activated and inhibits growth through acting on target genes such as Thor (d4E-BP). As a translational repressor, 4E-BP activity is shown to be critical for survival under dietary restriction and oxidative stress, and is linked to lifespan. This dFOXO/4E-BP signaling is also revealed to play a key role in the coordination of organismal and tissue aging through an organism-wide regulation of proteostasis in response to muscle aging. Interestingly, this Drosophila forkhead transcription factor (dFOXO) activates d4E-BP transcription, which is upregulated under stressed conditions. Consistent with increased foxo expression in flies with heart-specific Rpd3 downregulation, the data also showed that Thor was significantly upregulated with heart-specific Rpd3 downregulation. When induced by stress, fat body antimicrobial peptide (AMP) genes are activated in response to nuclear dFOXO activity. Upregulation of both foxo and DptB (one of target AMP) genes in heart-specific Rpd3 downregulation illustrates that Rpd3 downregulation in the heart modulates gene expression in other tissues such as fat body for stress adaption. One possible mechanism of this modulation is that heart-specific Rpd3 downregulation produces secreted proteins through Rpd3 deacetylase activity from heart, which thus regulates gene expression in other tissues (Kopp, 2015).

A positive correlation between stress resistance and lifespan extension was shown in several long-lived mutant flies. Previous findings have also suggested that enhanced stress resistance may extend lifespan in Drosophila. The data indicated that downregulating the rpd3 gene in the whole body or heart enhances both stress resistance and lifespan with improved cardiac function. However, insufficient heart-specific Rpd3 downregulation in older aged flies failed to prolong lifespan or improve cardiac condition, implying that throughout lifetime, Rpd3 in the heart influences both cardiac function and lifespan. Currently, although a conclusion of whether improved cardiac function from heart-specific Rpd3 modulation directly impacts longevity mechanism cannot yet be made, it is reported that enhanced cardiac capability could extend the lifespan of Drosophila (Kopp, 2015).

GENE STRUCTURE

Bases in 3' UTR - 389

PROTEIN STRUCTURE

Amino Acids - 529

date revised: 1 March 2024

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