Abstract

Cell differentiation is achieved by sequential gene expression. Late differentiation marker genes are already regulated at the chromatin level prior to differentiation in embryonic stem cells and many cell line models. Therefore, we hypothesized that ‘stem-ness’ of hematopoietic stem/progenitor cells (HSPCs) are programmed by epigenetic mechanisms and attempted to reveal the molecular mechanisms in hematopoietic gene expression. Histone H3 molecule, which is one of the most basic components of chromatin, has at least three variants: H3.1, H3.2, and H3.3. Previous studies have shown that one of the H3 variants, H3.3, was consistent with open chromatin structure. Here we found that the incorporation of histone variant H3.3 initiates on hematopoietic genes in HSPCs prior to differentiation.

HSPC fractions were purified from C57BL/6J mouse bone marrow, and chromatin immunoprecipitation sequencing (ChIPSeq) analysis was performed using newly-established monoclonal antibodies that specifically recognize endogenous H3.3. Although previous conventional studies have demonstrated that H3.3 deposition dominantly occurred in the “gene body”, our sensitive ChIPSeq analysis revealed that more than half of the H3.3 existed in the inter-genic regions around hematopoietic genes. The region of H3.3 incorporation changed during differentiation, i.e., virtually all genes were marked with H3.3 in embryonic stem cells, while all hematopoietic genes were marked with H3.3 in LSK, and more lineage specific genes were marked when cells are differentiated. Furthermore, our analysis visualized that within the regions incorporated with H3.3, transcriptionally active regions marked by H3K4me3 and repressed regions marked by H3K27me3 are mutually exclusive. These data suggest that in hematopoietic differentiation, H3.3 incorporation initiates around relatively wide ranges of hematopoietic genes, and then either of active or repressive histone modification sequentially occurs.

Interestingly, in leukemic cells, such selective H3.3 incorporation appeared to be disorganized. To identify factors that induce H3.3 incorporation defect in leukemic cells, we used a public database provided by the ENCODE project. We have constructed a system to manage all these datasets and to comprehensively explore the factors closely related to H3.3. Interestingly, correlations of our H3.3 ChIPSeq data with the ENCODE transcription factors’ binding site data were significantly different between analyses of AML and normal cells. By this approach, we identified hematopoietic transcription factors such as CEBPB and YY1 were associated with impaired H3.3 incorporation in AML. In addition, by comparing these transcription factors and single nucleotide variants (SNVs) obtained from Exome-Sequence, we found links between these transcription factors and particular SNVs in common pathways. These data suggest that this H3.3 ChIPSeq analysis should also be useful to extract oncogenic variants from many SNVs obtained by conventional Exome-Sequence analysis.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.