Figure 4

The ontology of transcriptome changes with loss of H3f3b indicates changes in histone, centromere, mitotic, and DNA synthesis genes. (A) RNA isolated from control and H3f3b knockout (KO) mouse embryonic fibroblasts (MEFS) (wildtype (WT)1,2 versus KO 1,2) was used for expression microarray studies. Gene expression changes are reported as green and red bars for down and upregulated genes respectively based on the indicated cutoffs for fold changes. (B) Immunoblot for H3.3 protein demonstrating strong reductions in total H3.3 and changes in other histone mark protein levels in the two KO MEF lines used for array and ChIP-Seq studies. (C) Average gene expression levels of downregulated histones consistent between conditional and constitutive array. (D) An ontological cluster of downregulated centromeric genes was evident consistently in H3f3b KO MEFs. (E) Data from arrays on WT and KO MEFs indicated a very large ontological cluster of mitotic regulatory genes is downregulated in the absence of H3f3b. (F) DNA synthesis genes downregulated as measured by microarray. (G) KO1 MEFs had significantly larger and rounder nuclei compared to littermate MEF WT1. (H) Cell cycle analysis by flow cytometry for DNA content on WT MEFs 46 and 48 versus KO MEFs 49 and 52 revealed, when quantitated, as shown in (I) a 40% increase in G2/M phase in KO cells. Error bars in (B-E, G, I) are standard deviations. *2610039 is an abbreviation for the 2610039C10Rik gene whose protein product has a mitotic ontology.