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. 2017 Oct 2;16(19):1781-1789.
doi: 10.1080/15384101.2017.1281483. Epub 2017 Jan 27.

Probe the function of histone lysine 36 methylation using histone H3 lysine 36 to methionine mutant transgene in mammalian cells

Dong Fang  1 , Haiyun Gan  1 , Heping Wang  2 , Hui Zhou  1 , Zhiguo Zhang  1
Affiliations

Probe the function of histone lysine 36 methylation using histone H3 lysine 36 to methionine mutant transgene in mammalian cells

Dong Fang et al. Cell Cycle. .

Abstract

Chondroblastoma is a cartilaginous tumor that typically arises under 25 y of age (80%). Recent studies have identified a somatic and heterozygous mutation at the H3F3B gene in over 90% chondroblastoma cases, leading to a lysine 36 to methionine replacement (H3.3K36M). In human cells, H3F3B gene is one of 2 genes that encode identical H3.3 proteins. It is not known how H3.3K36M mutant proteins promote tumorigenesis. We and others have shown that, the levels of H3K36 di- and tri-methylation (H3K36me2/me3) are reduced dramatically in chondroblastomas and chondrocytes bearing the H3.3K36M mutation. Mechanistically, H3.3K36M mutant proteins inhibit enzymatic activity of some, but not all H3K36 methyltransferases. Chondrocytes harboring the same H3F3B mutation exhibited the cancer cell associated phenotypes. Here, we discuss the potential effects of H3.3K36M mutation on epigenomes including H3K36 and H3K27 methylation and cellular phenotypes. We suggest that H3.3K36M mutant proteins alter epigenomes of specific progenitor cells, which in turn lead to cellular transformation and tumorigenesis.

Keywords: Asf1; Cancer cells; Chondroblastoma; Chromosomes; Epigenetic; H3K36me3; Histone methylation; Tumorigenesis.

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Figures

Figure 1.
Figure 1.
Expression of H3.3K36M mutant protein in distinct cells leads to different effects on RNA splicing. Expression of H3.3K36M mutant protein in 293T cells (B) but not T/C28a2 cells (A) leads to increase in splicing defects. (C) The chondroblastoma tissue samples with H3.3K36M mutation showed the similar RNA splicing pattern as the wild type bone tissues. The annotation of the splice junction in different RNA-seq data sets was indicated in the figure. The genes from Ensembl, GENECODE, UCSC, and RefSeq were combined and used as a reference gene model. Known: the junction is part of the gene model; complete_novel: complete new junction; partial_novel: one of the splice junction (SJ, either 5SJ or 3SJ) is new, while the other splice junction is annotated (known).
Figure 2.
Figure 2.
Expression of double mutants containing the H3.3K36M and each Asf1 binding deficient mutant results in reduced H3K36me2/me3 levels in T/C28a2 cells. T/C28a2 cells were infected with virus expressing FLAG-tagged wild type H3.3, or H3.3 mutants with relevant mutations indicated in the figure (A-D). Cells were collected for chromatin fractionation assays to separate proteins into soluble and chromatin fractions. Proteins in whole cell extracts (WCE), chromatin and soluble fractions were analyzed by Western blot.
Figure 3.
Figure 3.
H3.3R129E and H3.3K36MR129E mutant proteins were enriched on chromatin. (A) Soluble histones or mononucleosomes were purified from HEK293T cells with vector (negative control), HEK293T cells expressing FLAG-tagged WT H3.3, H3.3K36M, H3.3R129E, or H3.3K36MR129E mutant. Proteins from Input and immunoprecipitated (IP) samples were analyzed by Western blotting using the indicated antibodies. (B) ChIP-qPCR results of incorporations of different mutant histone H3.3 proteins and reduction of H3K36me3 at different loci. ChIP-qPCR is performed by using T/C28a2 cells after infection of different mutant histone H3.3 expressing virus. Data represents the average and standard deviations of 3 independent experiments. (*P < 0.05, **P < 0.01).
Figure 4.
Figure 4.
H3K27me3 increased dramatically at intergenic regions with reduced of H3K36me2 in H3.3K36M cell compare with WT cell. (A) Normalized tag distribution profiles of H3K27me3 for intergenic regions with reduced H3K36me2 in 2 H3.3K36M mutant cell lines compare with WT cells. The reads are normalized to reads per kilobase per 10 million mapped reads. (B) The normalized reads density of H3K27me3 ChIP-Seq was plotted from 10 kb upstream to 10Kb downstream from TSS sites in WT and H3.3K36M cells. The reads are normalized to reads per kilobase per 10 million mapped reads. (C-E) The same calculation as in B, but the whole human genes were split into equal number of 3 groups according to their expression levels. H: high expression, M: medium expression, and L: Low expression.
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