Global Crosstalk between Two Histone Modifications That Control Transcriptional Elongation in MLL Leukemia

The SET domain containing 2 (SETD2) gene encodes the methyltransferase which is responsible for histone 3 lysine 36 tri-methylation (H3K36me3). Previously, we identified SETD2 loss-of-function (LOF) mutations in about 22% of MLL -rearranged acute leukemia patients (MLL leukemia). Using an Mll-AF9 knock-in (MA9) mouse model, we found that knockdown (KD) of Setd2 could significantly accelerate disease development. All of these findings indicate a strong cooperation between SETD2 inactivation and MLL fusions; however, the detailed mechanism remains unclear. H3K36me3 reduction results from SETD2 LOF has been widely reported. In MLL leukemia, high expression levels of MLL fusion target genes (MLL targets), driven by aberrant high level of histone H3 lysine 79 dimethylation (H3K79me2), have been well-studied. Both H3K79me2 and H3K36me3 are enriched at transcriptionally active gene bodies and are associated with phosphorylated RNA Polymerase II. As two frequently dysregulated elongation marks in leukemia patients, we are interested in the potential crosstalk between H3K79me2 and H3K36me3. We aim to clarify whether H3K79me2 changes could affect H3K36me3 levels in MLL leukemia without SETD2 LOF; whether down-regulation of SETD2-H3K36me3 axis could further affect DOT1L-H3K79me2 axis in MLL leukemia with SETD2 LOF; and how this epigenetic crosstalk contribute to leukemia progression.

Firstly, to investigate whether H3K79me2 changes could affect H3K36me3 levels in MLL leukemia when there is no genetic alteration for SETD2, we purified c-Kit-positive (c-Kit⁺) hematopoiesis stem progenitor cells (HSPCs) from MA9 and C57/BL/6 wild-type (WT) mice. Increased H3K79me2 and H3K36me3 levels were observed in MA9 HSPCs using immuno-blotting. Through integrated ChIP-seq and RNA-seq analysis, we found that most of the genes showed increased H3K79me2 and/or H3K36me3 in MA9. Moreover, H3K79me2 targets significantly overlapped with H3K36me3 targets in MA9, suggesting these two marks were co-enriched in, and may also co-regulate, a large set of genes. Changes in H3K79me2 and H3K36me3 were positively correlated with differences in gene expression. However, at mRNA levels, significantly up-regulated genes were mainly MLL targets but not H3K79me2 and H3K36me3 co-enriched genes. Secondly, to explore whether the H3K36me3 reduction mediated by SETD2 LOF could further affect H3K79me2 levels in MLL leukemia, we mimicked the SETD2 LOF with MLL fusions in patients by performing KD of Setd2 in MA9 HSPCs. Interestingly, an increased H3K79me2 level was observed besides a dramatic reduction of H3K36me3 in Setd2 -KD-MA9 cells through immune-blotting and ChIP-seq analysis. At mRNA levels, there was no further activation of the MLL targets, whereas the previously H3K79me2 and H3K36me3 co-enriched genes were changed. Among the co-enriched genes, a set of AML related tumor suppressor genes (TSGs), such as Asxl1, Asxl2, Bcor were down-regulated; but oncogenes, such as Erg, Stat3, Fndc3a, Nampt, Igf1r, were up-regulated. Next, functional studies of TSG Asxl1 and oncogene Erg were performed. We generated a MA9/ Asxl1 -knockout mouse model (Asxl1^-/-/MA9). Enhanced cell proliferation were observed in Asxl1^-/-/MA9 cells compared to Asxl1^+/+/MA9 cells. Overexpression of ERG was observed in both mouse MA9 and human CD34-MA9 cells by Setd2 / SETD2 KD. Importantly, ERG KD partially rescued the proliferation and self-renewal capacities of SETD2 KD effects in mouse and human MA9 cells. These results support the key role of ASXL1 and ERG in MLL leukemia acceleration as SETD2 targets modulated by H3K79me2 and H3K36me3 changes.

In conclusion, in MLL leukemia, both H3K79me2 and H3K36me3 are aberrantly elevated and co-enriched in a group of genes. SETD2 inactivation leads to a global reduction of H3K36me3 and a further elevation of H3K79me2, but does not change the expression of known MLL fusion target genes. Instead, this pattern of histone changes is associated with transcriptional deregulation of a novel set of genes; downregulating TSGs (e.g. ASXL1) and upregulating oncogenes (e.g. ERG). Taken together, our findings reveal a global crosstalk between the oncogenic DOT1L-H3K79me2 axis and the tumor suppressive SETD2-H3K36me3 axis in gene regulation, provide molecular insights into how SETD2 mutations accelerate MLL leukemogenesis through differential regulation of additional TSGs and oncogenes.