Cell cycle stage-specific transcriptional activation of cyclins mediated by HAT2-dependant H4K10 acetylation of promoters in Leishmania donovani

Chromatin modifications affect several processes. In investigating the Leishmania donovani histone acetyltransferase HAT2, using in vitro biochemical assays and HAT2-heterozygous genomic knockout we found the constitutively nuclear HAT2 acetylated histone H4K10 in vitro and in vivo. HAT2 was essential. HAT2-depleted cells displayed growth and cell cycle defects, and poor survival in host cells. Real time PCR and DNA microarray analyses, as well as rescue experiments, revealed that downregulation of cyclins CYC4 and CYC9 were responsible for S phase and G2/M defects of HAT2-depleted cells respectively. Leishmania genes are arranged in unidirectional clusters, and clustered genes are coordinately transcribed as long polycistronic units, typically from divergent strand switch regions (dSSRs) which initiate transcription bidirectionally on opposite strands. In investigating the mechanism by which CYC4 and CYC9 expression levels are reduced in HAT2-depleted cells without other genes in their polycistronic transcription units being coordinately downregulated, we found using reporter assays that CYC4 and CYC9 have their own specific promoters. Chromatin immunoprecipitation assays with H4acetylK10 antibodies and real time PCR analyses of RNA suggested these gene-specific promoters were activated in cell cycledependent manner. Nuclear run-on analyses confirmed that CYC4 and CYC9 were transcriptionally activated from their own promoters at specific cell cycle stages. Thus, there are two tiers of gene regulation. Transcription of polycistronic units primarily initiates at dSSRs, and this most likely occurs constitutively. A subset of genes have their own promoters, at least some of which are activated in a cell-cycle dependent manner. This second tier of regulation is more sensitive to H4K10 acetylation levels, resulting in downregulation of expression in HAT2-depleted cells. This report presents the first data pointing to cell cycle-specific activation of promoters in trypanosomatids, thus uncovering new facets of gene regulation in this parasite family.