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J. Ropa et al.
associates with RNA polymerase II (RNAPII) and both positively and negatively regulates gene transcription.7–11 In AML, the PAF1c is critical for the regulation of a pro- leukemic HOXA gene program in AML cells through the recruitment of MLL and MLL-fusion proteins to the HOXA locus via direct physical interactions.12–15 Hoxa9 and its co-factor MEIS1 are upregulated in about 50% of AML and associated with a poor patient prognosis.16 Given our recent data linking H3K9 methyltransferases with Hoxa9 and Meis1 repression along with altered H3K9me3 in AML patients compared to CD34+ cells,17 it is important to understand the epigenetic and biological impact of H3K9 methyltransferases on AML.
SETDB1 is a H3K9 mono/di/tri-methyltransferase involved in heterochromatin regulation and euchromatic gene silencing.18 SETDB1 was shown to bind gene loci associated with development in mouse embryonic stem (ES) cells, such as the Hoxd cluster of genes.19 SETDB1 has been implicated as an oncogene in melanoma, breast can- cer, liver cancer, and lung cancer.4 Importantly, Ceol et al. reported amplification of SETDB1 in melanoma results in aberrant binding and regulation of the HOXA locus.20 In contrast to these oncogenic roles for SETDB1, Avgustinova and colleagues report that depletion of the H3K9 methyltransferase G9a in squamous tumors leads to a delayed, but more aggressive phenotype due to expand- ed cancer progenitor pools with increased genomic insta- bility.21 In the hematopoietic system, the methyltrans- ferase activity of G9a is required for leukemogenesis due to a physical interaction with HOXA9.22 Importantly, loss of G9a has no effect on hematopoietic stem cells.22,23 SETDB1, however, is required for both hematopoietic stem and progenitor cell (HSPC) maintenance and leukemic stem cells.23 Further, Cuellar and colleagues show that SETDB1 mediated silencing of endogenous retroviral elements is required for the growth of AML cell lines.24 Together, these studies suggest that therapeutic tar- geting of SETDB1 may benefit AML patients. However, we recently demonstrated that SETDB1 negatively regu- lates the expression of the pro-leukemic Hoxa9 and Meis1 genes in MLL-AF9 transformed AML cells through associ- ation with the PAF1c, which localizes to Hoxa and Meis1 loci. The PAF1c-SETDB1 interaction mediates promoter H3K9me3 and repression of Hoxa9 and Meis1 expression.5 Further, SETDB1 expression is inversely correlated with HOXA9 and MEIS1 expression in AML patient samples.5 These data imply a more complex role for H3K9 methyla- tion in AML similar to skin tumors whereby H3K9 methyltransferases display both oncogenic and suppres- sive roles.20,21 Thus, further investigation into the role of H3K9 methyltransferases in AML is required.
Here we show that AML patients with higher expres- sion of SETDB1 display a better prognosis, consistent with repression of HOXA9 and MEIS1 expression. SETDB1 overexpression induces cellular differentiation and delays disease onset in a mouse model of AML, recapitulating AML patient survival. We also investigated the utility of inhibiting H3K9 methyltransferases in AML cells and HSPC, demonstrating that inhibition of H3K9 methyla- tion in HSPC leads to retention of self-renewal capacity in HSPC and more efficient transformation by the MLL-AF9 fusion protein. Finally, we show that SETDB1 regulates gene expression by inducing changes in the epigenetic landscape and chromatin accessibility at gene targets crit- ical to leukemogenesis.
Methods
Patient sample data
Data for patient gene expression relative to normal hematopoi- etic cells were mined from BloodPool on BloodSpot database.25 Bloodspot assigns AML patient samples to a closest normal hematopoietic counterpart using transcriptomic profiles.26 AML patient RNA sequencing (RNA-seq) and survival data were mined from The Cancer Genome Atlas.2
Cell line generation
Cell lines were generated from C57Bl/6 (Taconic Farms) mouse bone marrow or from SETDB1floxed27 mice. Platinum-E viral packaging cells were transfected with the indicated constructs: MSCVneo-FLAG-MLL-AF9 (MA9), MSCVneo-FLAG-E2A-HLF (EHF), MSCVhygro-FLAG-EHMT2 (G9a) (Ge lab; Addgene #41721), MSCVpuro-HA-SETDB1 or empty vector (EV) controls. Cells were spinfected with viral supernatants and 5 ug/mL poly- brene (Millipore), selected with 1 mg/mL G418 (Invitrogen) and 1 ug/mL puromycin (Invitrogen) or 200 ug/mL hygromycin (Invitrogen) and cultured in IMDM with 15% stem cell fetal bovine serum (Millipore), 1% penicillin/ streptomycin (Invitrogen), 10 ng/mL interleukin-3 (IL-3) and 100 ng/mL stem cell factor (R&D).
Mouse modelling
Primary MLL-AF9 mouse leukemia cells were retrovirally trans- duced with MSCVpuro-HA-SETDB1 or EV and selected in 2 ug/mL puromycin for 4 days. 100,000 cells were injected intra- venously into lethally irradiated (950 rads) C57Bl/6 mice. Mice were monitored for survival, moribund mice were euthanized, and bone marrow, spleen, and liver were harvested. Animal stud- ies were approved by the University of Michigan’s Committee on Use and Care of Animals and Unit for Laboratory Medicine.
Quantitative PCR (qPCR)
RNA was harvested using the Qiagen RNeasy mini plus kit. cDNA synthesis was performed using the SuperScript III kit (Invitrogen). qPCR was performed using fast SYBR-green master- mix (Thermo Fisher). Primers sequences are listed in the Online Supplementary Table S2.
Sequencing libraries preparation
MA9+ empty vector (EV) and MA9+SETDB1 cells were harvest- ed for next generation sequencing library preparation for RNA- seq, ChIP-seq, and ATAC-seq. ChIP-seq antibodies were validated using Epicypher histone peptide arrays (Online Supplementary Figure S6). Library preparation details are in the Online Supplementary Materials and Methods.
Data availability
Sequencing data is available via the Gene Expression Omnibus, accession GSE136850.
Results
SETDB1 expression is correlated with AML patient prognosis
Given our data linking H3K9 methyltransferases with HOXA9 and MEIS1 expression, we investigated their expression in AML patient sample data. Interestingly, SETDB1, SUV39H1, and SUV39H2 exhibit lower expres- sion in AML patient samples when compared with normal hematopoietic cells, with median expression levels that
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haematologica | 2020; 105(9)