Rpd3: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Rpd3

Synonyms -

Cytological map position - 64C1--2

Function - histone deacetylase

Keywords - segmentation, chromatin associated proteins, enhancer of position effect variegation, eye

Symbol - Rpd3

FlyBase ID: FBgn0015805

Genetic map position - 3-

Classification - histone deacetylase

Cellular location - presumably nuclear

NCBI links: | Entrez Gene

Recent literature
Frankel, S., Woods, J., Ziafazeli, T. and Rogina, B. (2015). RPD3 histone deacetylase and nutrition have distinct but interacting effects on Drosophila longevity. Aging (Albany NY) [Epub ahead of print]. PubMed ID: 26647291
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.

Zhang, T., Sheng, Z. and Du, W. (2016). Loss of histone deacetylase HDAC1 induces cell death in Drosophila epithelial cells through JNK and Hippo signaling. Mech Dev [Epub ahead of print]. PubMed ID: 27378074
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.
Morris, O., Liu, X., Domingues, C., Runchel, C., Chai, A., Basith, S., Tenev, T., Chen, H., Choi, S., Pennetta, G., Buchon, N. and Meier, P. (2016). Signal integration by the IkappaB protein Pickle shapes Drosophila innate host defense. Cell Host Microbe 20: 283-295. PubMed ID: 27631699
Pattern recognition receptors are activated following infection and trigger transcriptional programs important for host defense. Tight regulation of NF-κB activation is critical to avoid detrimental and misbalanced responses. This study describes Pickle (CG5118), a Drosophila nuclear IκB that integrates signaling inputs from both the Imd and Toll pathways by skewing the transcriptional output of the NF-κB dimer repertoire. Pickle interacts with the NF-kappaB protein Relish and the histone deacetylase dHDAC1, selectively repressing Relish homodimers while leaving other NF-κB dimer combinations unscathed. Pickle's ability to selectively inhibit Relish homodimer activity contributes to proper host immunity and organismal health. Although loss of pickle results in hyper-induction of Relish target genes and improved host resistance to pathogenic bacteria in the short term, chronic inactivation of pickle causes loss of immune tolerance and shortened lifespan. Pickle therefore allows balanced immune responses that protect from pathogenic microbes while permitting the establishment of beneficial commensal host-microbe relationships.
Woods, J. K., Ziafazeli, T. and Rogina, B. (2016). Rpd3 interacts with insulin signaling in Drosophila longevity extension. Aging (Albany NY) [Epub ahead of print]. PubMed ID: 27852975
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.
Nakajima, E., Shimaji, K., Umegawachi, T., Tomida, S., Yoshida, H., Yoshimoto, N., Izawa, S., Kimura, H. and Yamaguchi, M. (2016). The Histone deacetylase gene Rpd3 is required for starvation stress resistance. PLoS One 11(12): e0167554. PubMed ID: 27907135
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.
Kang, Y., Marischuk, K., Castelvecchi, G.D. and Bashirullah, A. (2017). HDAC inhibitors disrupt programmed resistance to apoptosis during Drosophila development. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 28455414
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.

Kopp, Z. and Park, Y. (2019). Longer lifespan in the Rpd3 and Loco signaling results from the reduced catabolism in young age with noncoding RNA. Aging (Albany NY) 11(1): 230-239. PubMed ID: 30620723
Downregulation of Rpd3 (histone deacetylase) or Loco (regulator of G-protein signaling protein) extends Drosophila lifespan with higher stress resistance. rpd3-downregulated long-lived flies genetically interact with loco-upregulated short-lived flies in stress resistance and lifespan. Gene expression profiles revealed that they regulate common target genes in metabolic enzymes and signaling pathways. Functional analyses of more significantly changed genes indicated that the activities of catabolic enzymes and uptake/storage proteins are reduced in long-lived flies with Rpd3 downregulation. This reduced catabolism exhibited from a young age is considered to be necessary for the resultant longer lifespan of the Rpd3- and Loco-downregulated old flies, which mimics the dietary restriction (DR) effect that extends lifespan in the several species. Inversely, those catabolic activities that break down carbohydrates, lipids, and peptides were high in the short lifespan of Loco-upregulated flies. Long noncoding gene, dntRL (CR45923), was also found as a putative target modulated by Rpd3 and Loco for the longevity. Interestingly, this dntRL could affect stress resistance and lifespan, suggesting that the dntRL lncRNA may be involved in the metabolic mechanism of Rpd3 and Loco signaling.
Rass, M., Oestreich, S., Guetter, S., Fischer, S. and Schneuwly, S. (2019). The Drosophila fussel gene is required for bitter gustatory neuron differentiation acting within an Rpd3 dependent chromatin modifying complex. PLoS Genet 15(2): e1007940. PubMed ID: 30730884
Members of the Ski/Sno protein family are classified as proto-oncogenes and act as negative regulators of the TGF-β/BMP-pathways. A newly identified member of this protein family is fussel (fuss), the Drosophila homologue of the human functional Smad suppressing elements (fussel-15 and fussel-18). Fuss interacts with SMAD4 and overexpression leads to a strong inhibition of Dpp signaling. Fuss is a predominantly nuclear, postmitotic protein, mainly expressed in interneurons and fuss mutants are fully viable without any obvious developmental phenotype. fuss expression was characterized in the adult proboscis, and by using food choice assays it was possible to show that fuss mutants display defects in detecting bitter compounds. This correlated with a reduction of gustatory receptor gene expression providing a molecular link to the behavioral phenotype. In addition, Fuss interacts with Rpd3, and downregulation of rpd3 in gustatory neurons phenocopies the loss of Fuss expression. Surprisingly, there is no colocalization of Fuss with phosphorylated Mad in the larval central nervous system, excluding a direct involvement of Fuss in Dpp/BMP signaling. This work reveals Fuss as a pivotal element for the proper differentiation of bitter gustatory neurons acting within a chromatin modifying complex.
Suda, K., Muraoka, Y., Ortega-Yanez, A., Yoshida, H., Kizu, F., Hochin, T., Kimura, H. and Yamaguchi, M. (2019). Reduction of Rpd3 suppresses defects in locomotive ability and neuronal morphology induced by the knockdown of Drosophila SLC25A46 via an epigenetic pathway. Exp Cell Res: 111673. PubMed ID: 31614134
Mitochondrial dysfunction causes various diseases. Mutations in the SLC25A46 gene have been identified in mitochondrial diseases that are sometimes classified as Charcot-Marie-Tooth disease type 2, optic atrophy, and Leigh syndrome. A homolog of SLC25A46 was identified in Drosophila and designated as dSLC25A46 (CG5755). Previous work has established mitochondrial disease model targeting of dSLC25A46, which causes locomotive dysfunction and morphological defects at neuromuscular junctions, such as reduced synaptic branch lengths and decreased numbers of boutons. To investigate the involvement of epigenetic regulators in mitochondrial diseases, candidate epigenetic regulators that interact with human SLC25A46 were identifed by searching Gene Expression Omnibus (GEO). It was discovered that HDAC1 binds to several SLC25A46 genomic regions in human cultured CD4 (+) cells, and attempts were made to prove this in an in vivo Drosophila model. By demonstrating that Rpd3, Drosophila HDAC1, regulates the histone H4K8 acetylation state in dSLC25A46 genomic regions, this study confirmed that Rpd3 is a novel epigenetic regulator modifying the phenotypes observed with the mitochondrial disease model targeting of dSLC25A46. The functional reduction of Rpd3 rescued the deficient locomotive ability and aberrant morphology of motoneurons at presynaptic terminals induced by the dSLC25A46 knockdown. The present results suggest that the inhibition of HDAC1 suppresses the pathogenic processes that lead to the degeneration of motoneurons in mitochondrial diseases.
Walther, M., Schrahn, S., Krauss, V., Lein, S., Kessler, J., Jenuwein, T. and Reuter, G. (2020). Heterochromatin formation in Drosophila requires genome-wide histone deacetylation in cleavage chromatin before mid-blastula transition in early embryogenesis. Chromosoma 129(1): 83-98. PubMed ID: 31950239
Su(var) mutations define epigenetic factors controlling heterochromatin formation and gene silencing in Drosophila. This study identified SU(VAR)2-1 as a novel chromatin regulator that directs global histone deacetylation during the transition of cleavage chromatin into somatic blastoderm chromatin in early embryogenesis. SU(VAR)2-1 is heterochromatin-associated in blastoderm nuclei but not in later stages of development. In larval polytene chromosomes, SU(VAR)2-1 is a band-specific protein. SU(VAR)2-1 directs global histone deacetylation by recruiting the histone deacetylase RPD3. In Su(var)2-1 mutants H3K9, H3K27, H4K8 and H4K16 acetylation shows elevated levels genome-wide and heterochromatin displays aberrant histone hyper-acetylation. Whereas H3K9me2- and HP1a-binding appears unaltered, the heterochromatin-specific H3K9me2S10ph composite mark is impaired in heterochromatic chromocenters of larval salivary polytene chromosomes. SU(VAR)2-1 contains an NRF1/EWG domain and a C2HC zinc-finger motif. This study identifies SU(VAR)2-1 as a dosage-dependent, heterochromatin-initiating SU(VAR) factor, where the SU(VAR)2-1-mediated control of genome-wide histone deacetylation after cleavage and before mid-blastula transition (pre-MBT) is required to enable heterochromatin formation.
Sanna, S., Esposito, S., Masala, A., Sini, P., Nieddu, G., Galioto, M., Fais, M., Iaccarino, C., Cestra, G. and Crosio, C. (2020). HDAC1 inhibition ameliorates TDP-43-induced cell death in vitro and in vivo. Cell Death Dis 11(5): 369. PubMed ID: 32409664
TDP-43 pathology is a disease hallmark that characterizes both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). TDP-43 undergoes several posttranslational modifications that can change its biological activities and its aggregative propensity, which is a common hallmark of different neurodegenerative conditions. New evidence is provided by the current study pointing at TDP-43 acetylation in ALS cellular models. Using both in vitro and in vivo approaches, it was demonstrated that TDP-43 interacts with histone deacetylase 1 (HDAC1) via RRM1 and RRM2 domains, that are known to contain the two major TDP-43 acetylation sites, K142 and K192. Moreover, this study showed that TDP-43 is a direct transcriptional activator of CHOP promoter and this activity is regulated by acetylation. Finally and most importantly, it was observed both in cell culture and in Drosophila that a HDCA1 reduced level (genomic inactivation or siRNA) or treatment with pan-HDAC inhibitors exert a protective role against WT or pathological mutant TDP-43 toxicity, suggesting TDP-43 acetylation as a new potential therapeutic target. HDAC inhibition efficacy in neurodegeneration has long been debated, but future investigations are warranted in this area. Selection of more specific HDAC inhibitors is still a promising option for neuronal protection especially as HDAC1 appears as a downstream target of both TDP- 43 and FUS, another ALS-related gene.
Mugat, B., Nicot, S., Varela-Chavez, C., Jourdan, C., Sato, K., Basyuk, E., Juge, F., Siomi, M. C., Pelisson, A. and Chambeyron, S. (2020). The Mi-2 nucleosome remodeler and the Rpd3 histone deacetylase are involved in piRNA-guided heterochromatin formation. Nat Commun 11(1): 2818. PubMed ID: 32499524
In eukaryotes, trimethylation of lysine 9 on histone H3 (H3K9) is associated with transcriptional silencing of transposable elements (TEs). In Drosophila ovaries, this heterochromatic repressive mark is thought to be deposited by SetDB1 on TE genomic loci after the initial recognition of nascent transcripts by PIWI-interacting RNAs (piRNAs) loaded on the Piwi protein. This study shows that the nucleosome remodeler Mi-2, in complex with its partner MEP-1, forms a subunit that is transiently associated, in a MEP-1 C-terminus-dependent manner, with known Piwi interactors, including a recently reported SUMO ligase, Su(var)2-10. Together with the histone deacetylase Rpd3, this module is involved in the piRNA-dependent TE silencing, correlated with H3K9 deacetylation and trimethylation. Therefore, Drosophila piRNA-mediated transcriptional silencing involves three epigenetic effectors, a remodeler, Mi-2, an eraser, Rpd3 and a writer, SetDB1, in addition to the Su(var)2-10 SUMO ligase.

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).


Bases in 5' UTR - 233

Bases in 3' UTR - 389


Amino Acids - 529

Interactive Fly, Drosophila Rpd3: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 1 July 99

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.