Functional characterization of histone replacement variant MacroH2A1 (H2afy) in acute myeloid leukemia (AML)

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Changes of the epigenetic landscape can lead to massively altered expression patterns either directly by loss of regulatory control or through indirect, additive effects, ultimately leading to transcriptional changes. Epigenetic alterations include DNA methylation, chromatin-associated RNAs, histone tail modifications, non-histone proteins and histone variants. Among core histones, the H2A family exhibits highest sequence divergence resulting in the largest number of variants known. Amongst all H2A variants, macroH2A variants exhibit the most unique structural organization as they harbor a non-histone region at the C-terminus, named the macrodomain, making them the largest known histones. MacroH2A1 (H2afy) is highly expressed in AML patient blasts and also in hematopoietic stem cells. We have investigated the functional role of H2afy in AML and normal HSC through CRISPR-mediated inactivation and newly generated conditional mouse models. Our preliminary data provides first evidence for a functional relevance of H2afy in cell competition and maintenance of acute myeloid leukemia stem cells (AML-LSC) while it appears dispensable for normal hematopoietic stem- and progenitor cells (HSPCs). In this project we aim to determine to which extent macrohistone variant H2afy influences LSC and HSC function and how this is influenced by changes in transcription, binding partners or accessibility of specific chromatin regions. We will (1) assess for the impact of macrohistone variants on leukemic stem cell (LSC) function using MLLr-driven models of AML using rescue experiments with truncated H2afy to define relevant domains, CRISPR-mediated genome editing to define H2afy-dependent effectors and xenograft models to validate the findings in human AML. Also we aim to (2) determine the impact of genetic H2afy splicing isoforms on maintenance of normal hematopoietic stem and progenitor cells (HSPCs) and (3) analyze the mechanistic impact of macrohistone variant H2afy expression on transcription, chromatin conformation, histone modifications and binding partners in AML-LSC. Taken together, experiments described in our research proposal will produce a detailed characterization of the functional role and mechanistic impact of H2A histone variant H2afy on AML leukemia stem cells. These experiments will incorporate detailed functional analyses and genome wide characterization to provide a better understanding of biological heterogeneity induced by the presence or expression of the histone replacement variant macroH2A1 (H2afy) and prospectively explore potential therapeutic use.