An Essential Role for MYST5 (CG1894) in Histone H4 Acetylation and RNA Polymerase II Pause Release Open Access

Bryant, Donald (2013)

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The post-translational modification of histone tails is critical for transcriptional regulation in eukaryotes. One major type of covalent modification shown to be important for transcriptional regulation is the acetylation of lysine residues on the N-terminal tails of histones, and the enzymes that carry out this reaction are referred to as histone acetyltransferases (HATs). Here, I demonstrate that the D. melanogaster protein CG1894 (MYST5) is a member of the MYST (MOZ, Ybf2/Sas3, Sas2, TIP60) family of histone acetyltransferases and is essential for the acetylation of lysines 5 and 8 on the N-terminal tail of histone H4 (H4K5 and H4K8). Through genome-wide mapping studies, I show that MYST5 binding correlates with active transcription and that MYST5 binds the promoters of a subset of genes that are highly transcriptionally paused and enriched with a "stalled" RNA polymerase II. Moreover, I demonstrate that MYST5 colocalizes with Drosophila Myc, Mof (males absent on the first), and GAF (GAGA associated factor), which have all been implicated in the regulation of polymerase pausing. Finally, I show that loss of MYST5 results in a reduction in the levels of elongating polymerase. Collectively, these data indicate that MYST5 plays an active role in transcription by contributing to the regulation of RNA polymerase II pause release.

Table of Contents

Table of Contents

Introduction - 1

Materials and Methods - 7
Fly Stocks - 7
Antibodies - 7
Western Analyses - 8
Polytene Chromosome and Immunofluorescence Analyses - 8
Chromatin-immunoprecipitation and DNA Library Preparation - 9
ChIP-seq Data Processing - 10
ChIP-seq Data Analysis - 11

Results - 13
CG1894 is a MYST histone acetyltransferase that binds the promoters
of actively expressed genes - 14
MYST5 binds a highly paused subset of genes - 18
MYST5 colocalizes with factors that function in pausing - 21
MYST5 is essential for H4K5 and H4K8 acetylation, associates with
active transcription, and functions downstream of the Jil-1 kinase
during transcription - 27
MYST5 is essential for polymerase pause release but not for binding
of Fs(1)h - 30

Discussion - 34

Supplementary Figures - 41

References - 45

Table of Figures

Figure 1 - A basic view of eukaryotic transcription - 4
Figure 2 - The genomic binding patterns of MYST5 correlate with active
transcription - 17
Figure 3 - MYST5 associates with genes that are highly paused - 19
Figure 4 - MYST5 may interact with other pausing factors at a
subset of genes - 25
Figure 5 - MYST5 is essential for H4K5 and H4K8 acetylation
and functions downstream of Jil-1 during active transcription - 29
Figure 6 - MYST5 is necessary for proper pause release but
not Fs(1)h binding - 33
Figure 7 - Model of MYST5's role during transcription - 36
Supplementary Figure 1 - CG1894 is a MYST histone acetyltransferase - 41
Supplementary Figure 2 - MYST5 binds genes known to be paused and
genes with high pausing indices - 42
Supplementary Figure 3 - MYST5 may identify subsets of genes bound
by GAF, Myc, and Mof that are involved in developmental processes - 43
Supplementary Figure 4 - Many histone and polymerase modifications
remain unchanged in larvae null for MYST5 - 44
Table 1 - Gene ontology analysis of genes bound by MYST5 - 23

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