Abstract
BackgroundThe histone variant H3.3 plays key roles in regulating chromatin states and transcription. However, the role of endogenous H3.3 in mammalian cells and during development has been less thoroughly investigated. To address this gap, we report the production and phenotypic analysis of mice and cells with targeted disruption of the H3.3-encoding gene, H3f3b.ResultsH3f3b knockout (KO) mice exhibit a semilethal phenotype traceable at least in part to defective cell division and chromosome segregation. H3f3b KO cells have widespread ectopic CENP-A protein localization suggesting one possible mechanism for defective chromosome segregation. KO cells have abnormal karyotypes and cell cycle profiles as well. The transcriptome and euchromatin-related epigenome were moderately affected by loss of H3f3b in mouse embryonic fibroblasts (MEFs) with ontology most notably pointing to changes in chromatin regulatory and histone coding genes. Reduced numbers of H3f3b KO mice survive to maturity and almost all survivors from both sexes are infertile.ConclusionsTaken together, our studies suggest that endogenous mammalian histone H3.3 has important roles in regulating chromatin and chromosome functions that in turn are important for cell division, genome integrity, and development.
Highlights
The histone variant H3.3 plays key roles in regulating chromatin states and transcription
Production and validation of H3f3b knockout mice To address H3.3 function in development and chromosome biology, we used homologous recombination to produce a conditional allele of H3f3b, H3f3bFl, in which the entire coding region contained within exons 2 to 4 is flanked by loxP sites
H3f3bFl/WT mouse embryonic stem cells were injected into blastocysts to produce chimeras, which were bred with wildtype mice
Summary
The histone variant H3.3 plays key roles in regulating chromatin states and transcription. The role of endogenous H3.3 in mammalian cells and during development has been less thoroughly investigated. To address this gap, we report the production and phenotypic analysis of mice and cells with targeted disruption of the H3.3-encoding gene, H3f3b. H3.3 is not strictly associated with actively transcribed genes and can be found at repressed and poised. Another H3 variant, CENP-A is essential for centromere function where a role for histone H3.3 is implicated [8]. Nucleosome displacement by the RNA polymerase complex within actively transcribed genes necessitates a higher rate of H3.3-dependent histone replacement throughout different phases of the cell cycle [2,12]. In addition to its role as a replacement for H3.1 and H3.2, H3.3 has an antagonistic relationship with histone H1 as a roughly 50% knockdown of H3.3 using RNAi in fly cells substantially increased H1 genomic binding resulting in increased nucleosomal length [14]
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