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 cycle-dependent 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.

染色质修饰影响几个过程。在调查的杜氏利什曼原虫组蛋白乙酰转移HAT2，使用体外生化实验和HAT2杂合基因敲除，我们发现了组成核HAT2乙酰化组蛋白H4K10在体外和体内。HAT2是必不可少的。HAT2耗尽的细胞显示出生长和细胞周期缺陷，并在宿主细胞中存活不良。实时PCR和DNA芯片分析以及救援实验表明，细胞周期蛋白CYC4和CYC9的下调分别是造成HAT2耗尽细胞的S期和G2 / M缺陷的原因。利什曼原虫基因以单向簇的形式排列，并且簇状的基因被协调转录为长的多顺反子单元，通常来自发散链开关区（dSSRs），其在相反链上双向启动转录。在研究在HAT2缺失的细胞中CYC4和CYC9表达水平降低而其多顺反子转录单位中其他基因没有被协同下调的机制中，我们使用报告基因分析发现CYC4和CYC9具有自己的特异性启动子。使用H4乙酰基K10抗体的染色质免疫沉淀测定和RNA的实时PCR分析表明，这些基因特异性启动子以细胞周期依赖性方式被激活。核运行分析证实，CYC4和CYC9在特定的细胞周期阶段从其自身的启动子被转录激活。因此，基因调节分为两层。多顺反子单元的转录主要起始于dSSR，这很可能是组成性发生的。基因的子集具有其自己的启动子，其中至少一些以细胞周期依赖性方式被激活。第二级调节对H4K10乙酰化水平更为敏感，从而导致HAT2缺失细胞中的表达下调。该报告提出了第一个数据，指出锥虫中启动子的细胞周期特异性激活，从而揭示了该寄生虫家族中基因调控的新方面。并且这很可能是组成性发生的。基因的子集具有其自己的启动子，其中至少一些以细胞周期依赖性方式被激活。第二级调节对H4K10乙酰化水平更为敏感，从而导致HAT2缺失细胞中的表达下调。该报告提出了第一个数据，指出锥虫中启动子的细胞周期特异性激活，从而揭示了该寄生虫家族中基因调控的新方面。并且这很可能是组成性发生的。基因的子集具有其自己的启动子，其中至少一些以细胞周期依赖性方式被激活。第二级调节对H4K10乙酰化水平更为敏感，从而导致HAT2缺失细胞中的表达下调。该报告提出了第一个数据，指出锥虫中启动子的细胞周期特异性激活，从而揭示了该寄生虫家族中基因调控的新方面。