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Chromatin regulation by the histone acetyltransferase MOF in Drosphila
Like in mammals, sex determination in Drosophila melanogaster involves an unequal distribution of sex chromosomes, with male flies carrying an X and a Y chromosome, as compared to two Xs in females. To prevent the deleterious effects of chromosomal aneuploidy, flies have evolved a dosage compensation system, which upregulates transcription from the single male X chromosome to match transcript levels produced from the two female Xs. This transcriptional activation is achieved by the dosage compensation complex (DCC), a ribonucleoprotein complex consisting of five male specific lethal proteins (MSL) and two non coding RNAs on the X (roX). The DCC is physically tethered to hundreds of target loci along the male X chromosome, where it promotes hyper acetylation of X-linked chromatin at Lysine 16 of histone H4 (H4K16ac). This histone mark is associated with an open, permissive chromatin structure, and its enrichment on the male X chromosome is thought to be required for the twofold increase in X-linked transcription during dosage compensation. However, the exact mechanism by which X-linked transcription is activated in males is still unknown. Responsible for hyper-acetylation of the male X chromosome is the histone acetyltransferase males absent on the first (MOF), which is part of the DCC. Recent studies have shown that MOF plays an additional role in autosomal gene regulation, as it has been found at thousands of autosomal gene promoters as part of the non specific lethal (NSL) complex. However, to what extent H4K16ac at autosomal genes is MOF-dependent, and how MOF is differentially distributed between the two complexes is currently unknown. During the course of my PhD, I used genetic, biochemical, and genomewide approaches to address a wide range of questions, concerning MOF functions in autosomal gene regulation and dosage compensation; the DCC recruitment process to X-linked target genes; and the mechanism of transcriptional upregulation of X-linked genes during dosage compensation. Besides other contributions, investigating the role of the H3K36 specific methyltransferase HypB/Set2 during MSL targeting and dosage compensation, as well as the role of MOF for NSL function at autosomal promoters, I was addressing these questions in the context of two main projects. During the first one of these, I have been able to show that MOF is responsible for genomewide H4K16ac in male and female flies, and that MOF is an essential gene in females. I demonstrated that the Drosophila specific unstructured N-terminus of the MOF protein is required for assembly of the DCC on the male X chromosome, and at the same time constrains MOFs HAT activity. The N-terminus therefore controls MOFs function in X chromosome compensation. I was furthermore able to reveal the biological role of the chromobarrel domain, which is conserved from yeast to human. Unexpectedly, disruption of the MOF chromobarrel domain, which has been shown previously to be required for MOF interaction with roX RNAs, led to a dramatic loss of H4K16ac from all chromosomes. Accordingly, I showed that the chromobarrel domain serves to trigger H4K16ac after the recruitment of MOF to its chromatin targets, revealing for the first time a biological role of this domain in vivo. In a parallel project, to work towards unraveling of the dosage compensation mechanism, I wanted to identify the step in the RNA PolII transcription cycle at which dosage compensation operates in flies. To this end, I generated genomewide profiles of RNA PolII in 3rd instar larva salivary glands from male and female flies, and from male flies with disrupted dosage compensation. Strikingly, we find that the density of PolII is approximately twofold elevated on the male X chromosome as compared to autosomes, including X-linked promoters. This data suggests that dosage compensation operates via enhanced transcription initiation, which constitutes a major advance in our understanding of the dosage compensation process.
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570 Biowissenschaften, Biologie
570 Life sciences