A CHAF1B-Dependent Molecular Switch in Hematopoiesis and Leukemic Pathogenesis

Children with Down syndrome (DS) are at substantial risk of developing acute leukemia, which suggests that there are leukemia promoting genes on chromosome 21. Chromatin Assembly Factor 1B (CHAF1B), which resides in the DS critical region, is a member of the heterotrimeric CAF1 chromatin assembly complex that is responsible for depositing H3/H4 heterodimers at the replication fork during S-phase. We previously demonstrated that CHAF1B is overexpressed in acute megakaryocytic leukemia cells with trisomy 21 and have now discovered that it is also elevated in AML cell lines and primary tumor samples. This elevated expression is correlated with poor prognosis but the mechanism by which CHAF1B may promote leukemogenesis is unknown. In this study, we demonstrate that CHAF1B acts as regulator of transcription, maintaining an undifferentiated state by competing with CEBPA, FLI1, and RUNX2 for occupancy at the promoters and enhancers of myeloid differentiation genes.

To study CHAF1Bin the context of normal hematopoiesis, we generated Chaf1b floxed mice and found that homozygous deletion was associated with rapid pancytopenia and bone marrow failure due to a cell cycle arrest and cell death. These effects were associated with downregulation of cell adhesion and metabolic pathways, as well as upregulation of genes related to sensitivity to stress and inflammatory stimuli. On the other hand, consistent with a pro-leukemic function, overexpression of CHAF1B promoted serial replating in vitro and was associated with the upregulation of genes involved in metabolism and proliferation. However, CHAF1B overexpression alone was not sufficient to promote leukemia in vivo .

Since CHAF1B expression is elevated in cell lines with MLL rearrangements and primary AML samples, we next studied its requirements in the murine MLL-AF9 AML model. First, we found that overexpression of CHAF1B enhanced the formation of MLL-AF9 leukemic cells (LCs) in vitro and significantly accelerated disease in vivo . In contrast to the cell death and cell cycle arrest observed in HSPCs following Chaf1b deletion, there were no changes in proliferation or survival of LCs at 48 hours following Chaf1b deletion. Instead, we found Chaf1b deletion led to terminal myeloid differentiation of LCs, suggesting that CHAF1B has a major role in maintaining leukemic stem cells. In fact, heterozygous deletion of Chaf1b or overexpression of a dominant negative allele was sufficient to substantially reduce colony formation, as well as completely prevent leukemia in vivo .

Transcriptome analysis of Chaf1b- deficient LCs revealed an increase in expression of over 1500 genes, including many of those associated with differentiation of myeloid leukemia cells. In comparison, we only identified 255 significantly downregulated genes, suggesting that CHAF1B primarily functions as a transcriptional repressor. By ChIP-seq, we determined that CHAF1B, but not the DNA replication factor PCNA, co-occupied the promoter and enhancer regions of many of these upregulated genes. Surprisingly, by ATAC-seq we found a widespread decrease in accessibility at CHAF1B binding sites following its knockdown, suggesting that other mechanisms are likely the key contributors to the differentiation phenotype. Motif analysis of CHAF1B-bound peaks in MLL-AF9 cells revealed specific enrichment of differentiation-driving transcription factor binding sites, including CEBPA, FLI1, and RUNX2. Therefore, we hypothesized that CHAF1B competes with transcription factors for occupancy at promoters of differentiation genes, preventing their expression and maintaining a leukemia stem cell state. We tested our hypothesis by comparing the occupancy of CEBA, FLI1, and RUNX2 at CHAF1B bound sites by ChIP-seq in cells replete or deficient for CHAF1B. We found that occupancy of these factors was significantly enhanced at CHAF1B-bound regions in LCs following Chaf1b deletion, and that this was associated with concomitant increases in expression of the differentiation target genes. We conclude that CHAF1B maintains the leukemic stem cell state by preventing differentiation through direct competition for chromatin occupancy with transcription factors at regulatory regions of genes that promote terminal maturation.