Abstract
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.
Highlights
Histone post-translational modifications (PTMs) impact nuclear processes either by generally altering chromatin structure to make it more permissive/repressive to incoming transcription/ replication/repair machinery, or by serving as flags recognized by specific proteins that modulate the various DNA-related processes
We find that HAT2 is essential for cell survival, and decrease in HAT2 levels leads to growth and cell cycle defects
We find that the expression of these cyclins are activated in a cell cycle-dependent manner, and this activation is regulated by H4K10 acetylation
Summary
Histone post-translational modifications (PTMs) impact nuclear processes either by generally altering chromatin structure to make it more permissive/repressive to incoming transcription/ replication/repair machinery, or by serving as flags recognized by specific proteins that modulate the various DNA-related processes. Due to the wide range of cellular processes they regulate the functional roles of histone PTMs have been extensively investigated. Though these are highly conserved among most eukaryotes, trypanosomatid histones are divergent in sequence [1,2,3]. Trypanosomatids include several protozoan pathogens that cause communicable diseases such as sleeping sickness, Chagas disease and various forms of Leishmaniases. While histone PTMs have not been globally identified in any Leishmania species so far, they are likely to carry the same repertoire of modifications as Trypanosoma species since the histone sequences are highly conserved between these family members [4]
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