De novo H3.3 genetic variants and their impact on histone interactions and chromatin

Abstract

The H3 histone family is composed of multiple histone variants, including H3.1 and H3.3. The H3.1 histone variant is categorized as a ‘replication-coupled’ histone because it is highly expressed from multiple genes in S-phase and is deposited on DNA during DNA replication. H3.3 is expressed at basal levels and deposited throughout interphase and is hence categorized as a ‘replication-independent’ histone. H3.1 and H3.3 differ by five amino acids but have slightly different roles. H3.3 replaces histones displaced from active transcription sites and is deposited over heterochromatic repetitive DNA regions, such as pericentromeric regions and telomeres, to maintain proper nucleosome densities. H3.3 is encoded by two genes, H3-3A and H3-3B. Missense genetic variants on H3-3A were first identified in pediatric gliomas, where they alter the epigenomic landscape. More recently, a worldwide consortium led by Dr. Elizabeth Bhoj (Children’s Hospital of Philadelphia, CHOP) identified >90 patients with 67 uncharacterized germline missense genetic variants on either of the two H3.3 genes. These patients are characterized by broadly defined progressive neurological dysfunction and congenital anomalies. The impact of these de novo H3.3 genetic variants on protein-protein interactions, which in turn impact chromatin and transcriptional programs, is unknown. The goal of this project was to identify genetic variant-specific changes in H3.3 protein interactions, but more importantly, also common proteomic and epigenomic changes in groups of genetic variants. I thus explored the impact of eleven different genetic variants on H3.3 associations using proximity-dependent biotin identification of associating proteins (BioID). Many of the genetic variants commonly disrupted H3.3 associations with proteins that have a PWWP domain. Intriguingly, H3.3T45I and H3.3L48R had marked opposite effects on the associations with PWWP domain-containing proteins. Nearly all PWWP domains bind histone H3 methylated on lysine 36 (H3K36me), and indeed, the H3.3 genetic variants with altered PWWP associations also had altered H3K36me levels because they affected H3K36 methyltransferase activities. This study shows for the first time the impact of a group of Bryant-Li-Bhoj (BLBS) genetic variants that converge on H3K36. It also shows how H3.3T45I and H3.3L48R—both outside the histone tail—impact H3K36 methylation through mechanisms that are distinct from those of the better-studied H3.3G34R/V genetic variants that also occur in cancer and are on the histone tail.