HMG20B stabilizes association of LSD1 with GFI1 on chromatin to confer transcription repression and leukaemia cell differentiation block

Pharmacologic inhibition of LSD1 induces molecular and morphologic differentiation of blast cells in acute myeloid leukemia (AML) patients harboring MLL gene translocations. In addition to its demethylase activity, LSD1 has a critical scaffolding function at genomic sites occupied by the SNAG domain transcription repressor GFI1. Importantly, inhibitors block both enzymatic and scaffolding activities, in the latter case by disrupting the protein:protein interaction of GFI1 with LSD1. To explore the wider consequences of LSD1 inhibition on the LSD1 protein complex we applied mass spectrometry technologies. We discovered that the interaction of the HMG-box protein HMG20B with LSD1 was also disrupted by LSD1 inhibition. Downstream investigations revealed that HMG20B is co-located on chromatin with GFI1 and LSD1 genome-wide; the strongest HMG20B binding co-locates with the strongest GFI1 and LSD1 binding. Functional assays demonstrated that HMG20B depletion induces leukemia cell differentiation and further revealed that HMG20B is required for the transcription repressor activity of GFI1 through stabilizing LSD1 on chromatin at GFI1 binding sites. Interaction of HMG20B with LSD1 is through its coiled-coil domain. Thus, HMG20B is a critical component of the GFI1:LSD1 transcription repressor complex which contributes to leukemia cell differentiation block.

. Depletion of HMG20B in primary patient MLL leukaemia cells.
Primary patient MLL-rearranged AML cells were infected with lentiviral vectors targeting HMG20B for knockdown (KD), or a non-targeting control (NTC), with puromycin drug resistance as the selectable marker.     in at least one condition, excluding proteins likely to be contaminants -www.crapome.org).
Proteins previously identified as part of an LSD1 complex are in bold red [1]. BMbone marrow. PBblood.

Reagents and antibodies
Antibodies used for western blotting were as follows: Goat (NI02, Millipore). penicillin/streptomycin supplemented with 20ng/ml GCSF, 20ng/ml IL3 and 20ng/ml TPO (Peprotech, London, UK). The same growth factor combination was used for primary cell semisolid culture.

Viral particle manufacture
Lentiviral supernatants were prepared, and cells were infected with viral particles as previously described [2]. Briefly, 293FT cells were plated in 10cm dishes at a density of 4.5x10 6 cells per dish in 9ml DMEM with 10% FBS. Next day cells were transfected with 21ug polyethylenimine (PEI), 4ug of the lentiviral construct, 2ug of pCMVD8.91 and 1ug of pMDG.2 in 1ml serum-free DMEM. Follow 30 minutes of room temperature incubation to allow the formation of DNA-PEI complexes the mixture was added dropwise to 293FT cells. Next day the medium was replaced with 10ml fresh DMEM+10%FBS. All viral supernatants were collected after 24 and 48 hours and filtered through a 0.45um filter prior to use. Lentiviral supernatants were either used immediately or stored long-term at -80°C. For cell transduction 8ug/ul of polybrene was added to lentiviral supernatants and cells were centrifuged for 30 minutes at 900g and 37°C followed by an overnight incubation. Next day cells were transferred to normal media, and antibiotic selection was started 24 hours following spinoculation.

Isolation and culture of CD117 + murine cells
Bone marrow (BM) cells were harvested from the long bones of four-to eight-week-old mice by crushing them with a mortar and pestle. Next CD117 + BM stem and progenitor cells were recovered using immunomagnetic beads and an AutoMACS Pro Device (Milteny Biotec, Bergisch Gladbach, Germany). Cells were incubated overnight in RPMI with 20% fetal bovine serum (R20) with 20ng/ml SCF, 10ng/ml IL6, 10ng/ml GM-CSF and 10ng/ml IL3 (Peprotech, London, UK) to promote cell cycle entry. Next day cells were spinoculated with lentiviral supernatant in the presence of cytokines and 8ug/ul polybrene (Millipore) for 30 min at 900xg and 37ºC. Following spinoculation, cells were incubated overnight with R20 with growth factors to allow for expression of antibiotic resistance in transduced cells prior to the addition of puromycin (3ug/ml). Cells were either (i) harvested for RNA 48 hrs later to confirm knockdown efficiency or (ii) transferred to semisolid culture for clonogenic assays at a starting culture density of 10 3 /ml. Colonies were enumerated 7 days later and cells were analyzed by cytospin staining and flow cytometry. The PCR product was sub-cloned into pGEM-T and after sequence verification cDNAs were excised using EcoRI and XbaI and sub-cloned into pLentiGS-minCMV-TET-blasticidin [2].

Generation of expression and knockdown constructs
To generate a GFI1 ZNF HMG20B fusion construct, fragments coding for FLAG-GFI1 ZNF and full length HMG20B were amplified from existing plasmids [3] [9]. DESeq2 was used to perform differential gene expression analysis and calculate FPKM (fragments per kilobase of transcript per million mapped reads) values for each gene, counting only reads that mapped to exonic regions [10]. All sequencing data files are available at the Gene Expression Omnibus: GSE192975

Gene set enrichment analysis
Pre-ranked gene set enrichment analysis was performed with GSEA v2.0.14 software from www.broadinstitute.org/gsea [11]. Expressed protein coding genes were rank ordered according to log2 fold change in expression (Table S5). Gene sets were from [32].

Cytospin analyses
2-5x10 4 cells were resuspended in 150μl PBS and, through centrifugation at 60xg for 5 min, were spun onto a microscope glass slide and left to air dry. Cells were fixed by incubation in methanol for 10 minutes followed by May-Grunwald (Sigma;

SILAC and nuclear immunoprecipitation-mass spectrometry and analysis
The stable isotope labeling with amino acid in cell culture (SILAC) of THP1 cells was done as previously reported [12]. Briefly, cells were grown in heavy methionine and arginine or normal/light labeled media for 15 days (7 passages), and complete isotopic labeling was confirmed by mass spectrometry. Heavy-labeled cells were exposed to 250nM of OG86 and the (StatsDirect, Altrincham, UK). All data met the assumptions of the statistical tests used and variance was similar between groups.