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).

GENE STRUCTURE

Bases in 3' UTR - 389

PROTEIN STRUCTURE

Amino Acids - 529

date revised: 1 July 99

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