The Deposition of H3.3 Mediated by Transcription Factors Determines Hematopoietic Cell Fate

Abstract 1193

Cell differentiation is achieved by sequential gene expression. Late differentiation marker genes are already regulated at the chromatin level prior to differentiation, even in pluripotent stem cells. Therefore, we hypothesized that “stem-ness” in hematopoiesis is already programmed in hematopoietic stem/progenitor cells (HSPCs) and attempted to reveal the molecular mechanisms of epigenetic regulations in hematopoietic gene expression. Histone H3 molecule, which is one of the most basic component of chromatin, has at least three variants:H3.1, H3.2, and H3.3. In previous study, it is known that one of H3 variants H3.3 was consistent with open chromatin structure. Only limited organisms which have complex differentiation mechanisms such as mammals have all three variants, suggesting that these histone variants significantly correlate with differentiation. Therefore, we focused on these H3 variants incorporation patterns in HSPCs, and found that the deposition of histone variant H3.3, that is a marker of open chromatin regions, specifically occurred on hematopoietic genes in HSPCs prior to differentiation.

HSPC fractions were purified from C57BL/6J mouse bone marrow, and chromatin immunoprecipitation sequence analyses were performed utilizing monoclonal antibodies that specifically recognize H3.3. Although previous studies demonstrated that H3.3 deposition dominantly occurred in the “gene body”, our informatics analysis revealed that more than half of H3.3 existed in the inter-genic regions around hematopoietic genes. The region of H3.3 incorporation changed during differentiation, i.e., almost all of genes were marked with H3.3 in embryonic stem cells, while almost all of hematopoietic genes were marked with H3.3 in LSK, and more lineage specific genes were marked with H3.3 when the differentiation occurred. To explore the factor dependency of the deposition of H3.3, we examined H3.3 incorporation around binding sites of more than 20 different types of transcription factors. The binding sites of Oct3/4, Klf4, Sox2, and Myc, also known as iPS factors, were strongly correlated with H3.3 incorporation sites in embryonic stem cells, while in HSPCs, the incorporated regions of H3.3 significantly co-localized with the binding sites of several hematopoietic key transcription factors such as Scl and Runx1. When we knocked down Oct3/4 in embryonic stem cells, these H3.3 incorporations and differentiation marker genes expressions were diminished, on the other hand, when we knocked down H3.3 in embryonic stem cells, well-ordered differentiation marker genes expressions were suppressed. These data suggest that H3.3 incorporations by key transcription factors are the essences of the “stem-ness”. Interestingly, in leukemic cells, this selective H3.3 incorporation was not observed, suggesting that H3.3 incorporation could reflect the orderly progression of normal hematopoiesis. In conclusion, we found that the incorporation of H3.3 is the earliest epigenetic event involved in determining hematopoietic cell fate.