Essential role of <em>Drosophila Hdac1</em> in homeotic gene silencing

Abstract

The identities of body segments along the anteroposterior axis are specified by a subgroup of homeobox-containing genes, including those originally identified in Antennapedia and bithorax complexes in Drosophila and their orthologs in other animal species (1). The spatial and temporal patterns of expression of these genes are strictly controlled during development. Alterations in their levels or domains of expression result in partial or complete homeotic transformations of certain body parts, e.g., the transformations of haltere to wing or antenna to leg (2, 3).

Two groups of antagonizing trans-acting genes are known to exert dosage-sensitive enhancing or suppressing effects on homeotic phenotypes caused by inappropriate homeotic gene expression (4, 5). The chromosomal proteins encoded by several trithorax group (trx-G) and Polycomb group (Pc-G) genes directly regulate transcription of homeotic genes, either positively (trx-G) or negatively (Pc-G) (6, 7). Some of these activities appear to be exerted at the level of nucleosomal organization, thereby affecting the accessibility of cis-regulatory sequences to the transcriptional machinery. For example, the trx-G proteins BRAHMA, MOIRA, OSA, and SNR1 are components of an ATP-dependent, chromatin-remodeling complex of the SWI/SNF family (8, 9). SWI/SNF complexes have been shown to increase the fluidity of the chromatin, thus facilitating the binding of transcriptional factors (10). Such activities of the SWI/SNF complexes can be blocked by a repressor complex, PRC1, which contains at least three Pc-G proteins, POLYCOMB (PC), POLYHOMEOTIC (PH), and POSTERIOR SEX COMBS (PSC) (11).

In addition to remodeling nucleosomes, trx-G and Pc-G proteins are also involved in the maintenance of homeotic gene expression (6, 7), a crucial step in cellular memory of the determined states. Although little is known about the mechanisms of cellular memory at the molecular level, the acetylation and deacetylation of the N-terminal lysine residues of nucleosomal core histones have been implicated. The stable maintenance of an artificially activated transgene controlled by a linked Fab-7 response element, which is critical for regulation of the homeotic gene Abdominal-B (Abd-B) by Pc-G and trx-G proteins, is correlated with hyperacetylation of histone H4 (12, 13). In addition, the Drosophila MI-2 homolog, which is a component of nucleosomal-remodeling histone deacetylase complexes (14), was identified by its direct interaction with the HUNCHBACK (HB) repressor protein (15). HB proteins are essential for establishing the silenced state of homeotic genes in early embryos, and Mi-2 mutations enhance the homeotic phenotypes caused by hb and Pc-G mutations at various developmental stages (15, 16). Furthermore, the Drosophila homolog of YY1 (which recruits HDAC2 in mammals for transcription repression) is encoded by the Pc-G gene pleiohomeotic (17, 18). Although these observations suggest the involvement of histone deacetylation in homeotic gene silencing, evidence supporting a direct role of HDAC is still lacking.

Two HDAC families, which are distinguishable by their sensitivity to trichostatin A (TSA) or dependence on NAD cofactor, have been found in many eukaryotes (19, 20). The TSA-sensitive family has sequence similarity to prokaryotic enzymes involved in acetoin utilization and acetyl-polyamine catabolism (21, 22). Based on sizes and sequence similarities, the TSA-sensitive family can be divided further into RPD3 and HDA1 types, which may form different protein complexes with overlapping functions on histone substrates (19, 23). In support of their roles in gene silencing, the association of RPD3-type HDACs with various repressors or corepressors has been demonstrated (24). Despite the expanding knowledge on HDACs, the physiological functions of individual enzymes are not fully understood and their roles in metazoan development remain largely unexplored (25).

In this study, we examined the roles of four Drosophila HDACs in homeotic gene silencing. We found that HDAC1 (an RPD3-type HDAC) shows specific genetic interactions with Pc-G mutations to enhance ectopic expression of homeotic genes, biochemical associations with Pc-G proteins, and cytological colocalization with Pc-G proteins on salivary gland polytene chromosomes. From these results, we conclude that HDAC1 plays an essential role in homeotic gene silencing.

The major PSC sites detected by a combination of monoclonal antibodies 1F4 and 6E8 are listed. Note that there are some differences between our list and the sites previously published (40). The bold, plain, and italicized letters indicate major, minor, and sites undetected by the earlier work, respectively. The relative staining intensity of HDAC1 at these sites is indicated by the number of “+.” The absence of HDAC1 staining is indicated by “−.”

Acknowledgments

Abbreviations