Abstract
The histone H3 variant H3.3 is a highly conserved and dynamic regulator of chromatin organization. Therefore, fully elucidating its nucleosome incorporation mechanisms is essential to understanding its functions in epigenetic inheritance. We previously identified the RNase P protein subunit, Rpp29, as a repressor of H3.3 chromatin assembly. Here, we use a biochemical assay to show that Rpp29 interacts with H3.3 through a sequence element in its own N terminus, and we identify a novel interaction with histone H2B at an adjacent site. The fact that archaeal Rpp29 does not include this N-terminal region suggests that it evolved to regulate eukaryote-specific functions. Oncogenic H3.3 mutations alter the H3.3-Rpp29 interaction, which suggests that they could dysregulate Rpp29 function in chromatin assembly. We also used KNS42 cells, an H3.3(G34V) pediatric high-grade glioma cell line, to show that Rpp29 1) represses H3.3 incorporation into transcriptionally active protein-coding, rRNA, and tRNA genes; 2) represses mRNA, protein expression, and antisense RNA; and 3) represses euchromatic post-translational modifications (PTMs) and promotes heterochromatic PTM deposition (i.e. histone H3 Lys-9 trimethylation (H3K9me3) and H3.1/2/3K27me3). Notably, we also found that K27me2 is increased and K36me1 decreased on H3.3(G34V), which suggests that Gly-34 mutations dysregulate Lys-27 and Lys-36 methylation in cis The fact that Rpp29 represses H3.3 chromatin assembly and sense and antisense RNA and promotes H3K9me3 and H3K27me3 suggests that Rpp29 regulates H3.3-mediated epigenetic mechanisms by processing a transcribed signal that recruits H3.3 to its incorporation sites.
Highlights
The histone H3 variant H3.3 is a highly conserved and dynamic regulator of chromatin organization
We reported that Rpp29 depletion increases H3.3 chromatin deposition and sense (S) and AS RNA levels, which suggests that an RNase P variant represses H3.3 chromatin assembly and transcription
We previously reported that Rpp29 co-localizes with histone H3.3 at an activated transgene array [38]
Summary
In support of this hypothesis, RNase P variants and subunits have been shown to regulate RNA pol I and III transcription sites [43,44,45]; to degrade noncoding RNA, including AS transcripts, in yeast [46]; to regulate piRNA and tRNA gene chromatin [47]; and to promote homology-directed DNA double-strand break repair (i.e. catalytic RNA, Rpp, and Rpp29) [48] It is not known whether H3.3 is involved in any of these noncanonical RNase P functions. These results suggest that Rpp regulates H3.3mediated epigenetic mechanisms by processing a transcribed signal that recruits H3.3 to its incorporation sites
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