H3.3 is a histone variant, which marks transcription start sites as well as telomeres and heterochromatic sites on the genome. H3.3 presence is thought to positively correlate with transcriptional status of its target genes. Using a conditional genetic strategy against H3.3B combined with short hairpin RNAs against H3.3A, we essentially depleted all H3.3 gene expression in mouse embryonic fibroblasts. Following nearly complete loss of H3.3 in cells, our transcriptomic analyses show very little impact on global gene expression as well as on histone variant H2A.Z localization. Instead, fibroblasts display slower cell growth and an increase in cell death coincident with large-scale chromosome misalignment in mitosis and large polylobed or micronuclei in interphase cells. Thus we conclude that H3.3 may additionally have an important under-explored role in chromosome segregation, nuclear structure and maintenance of genome integrity.

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