Role of histone variant H3.3 in transcription and mitotic progression

Abstract

Chromatin structure needs to be dynamic and flexible in order for the eukaryotic cellular processes to function correctly. Incorporation of histone variants into chromatin serves to increase epigenetic plasticity by conferring new structural and functional properties to chromatin. Histone variants are implicated in many cellular processes such as transcription or cell division and their deregulation is involved in tumorigenesis. H3.3 is an evolutionarily well conserved histone variant that differs by only a few amino-acids from its replicationdependent counterparts. With the aim of determining H3.3 function, novel knockin/ conditional knock-out mouse models were established and characterized. In these models, one of the two genes coding for H3.3, H3f3a or H3f3b has been modified to code for an N-terminal FLAG-FLAG-HA tagged H3.3A or H3.3B which can be depleted upon Cre expression. Nucleosome resolution genome-wide mapping FH-H3.3A and FH-H3.3B determined that H3.3A and H3.3B were similarly enriched at promoter regions and their enrichment levels positively correlated with high expression and gene body enrichment. They were also found enriched in telomeres and some repetitive DNA sequences. In a subset of these repetitive regions H3.3A and H3.3B showed differential enrichment properties. As double H3.3-KO mouse generation resulted lethal, mouse embryonic fibroblasts (MEFs) were isolated from FH-H3.3 mice and transformed. Using a combination of Cre recombinase mediated knock-out and RNA interference technology, a new cellular model was established where H3.3 expression was essentially depleted. Although H3.3 enrichment profiles were indicative of a role in active transcription, whole transcriptome analysis upon single H3.3 depletion in livers and an almost complete H3.3 depletion in MEFs yielded very few differentially regulated genes. Interestingly, H3.3 depleted MEFs showed a high increase in mitotic defects and abnormal nuclear structures. Thus, an important yet often understudied role for H3.3 in genomic maintenance during mitotic progression was highlighted.