Transcriptomic and epigenomic profiling of SETDB1 mediated repression in Acute Myeloid Leukemia

Epigenetic regulators play a critical role in normal and malignant hematopoiesis. We recently showed that the Histone 3 Lysine 9 (H3K9) methyltransferase SETDB1 negatively regulates the expression of the pro-leukemic genes HoxA9 and its cofactor Meis1 through deposition of promoter H3K9 trimethylation (H3K9me3) in MLL-AF9 AML cells. Here, we investigated the microbiological impact of altered SETDB1 expression in AML cells. We explored changes in transcription using RNA-seq, promoter associated histone modifications using ChIP-seq, and chromatin accessibility using ATAC-seq. Next generation sequencing of AML cells with or without overexpression of SETDB1 shows that high expression of SETDB1 induces repressive changes to the promoter epigenome and downregulation of genes linked with AML, including Dock1 and the MLL-AF9 target genes Hoxa9, Six1, and others. These data reveal novel targets of SETDB1 in AML that point to a role for SETDB1 in negatively regulating pro-leukemic target genes and suppressing AML. MLL-AF9 (MA9) acute myeloid leukemia cell lines were generated from mouse bone marrow by retroviral transduction of the MLL-AF9 oncogene. These cells were co-transduced with SETDB1 overexpression or empty vector (EV) control. RNA was harvested from three biological replicates of MA9+SETDB1 and MA9+EV and poly-A selected RNA-seq was performed. Two biological replicates each were obtained for each ChIP experiment. Cells were crosslinked using 1% paraformaldehyde and harvested to perform immunoprecipitation using antibodies targeting H3K9me3, H3K79me2, and H3K9ac. 10% input was used as a control. Two biological replicates for MA9+SETDB1 or MA9+EV were harvested for ATAC-seq. These cells were pretreated in culture with DNase I to eliminate cell-free DNA. They were then harvested, frozen, and sent to Active Motif for ATAC-seq processing, library prep, and sequencing. This protocol was performed as described in Buenrostro, et al Curr Protoc Mol Biol. 2015.