Recruitment of MLL1 complex is essential for SETBP1 to induce myeloid transformation

Abstract

Abnormal activation of SETBP1 due to overexpression or missense mutations occurs frequently in various myeloid neoplasms and associates with poor prognosis. Direct activation of Hoxa9/Hoxa10/Myb transcription by SETBP1 and its missense mutants is essential for their transforming capability; however, the underlying epigenetic mechanisms remain elusive. We found that both SETBP1 and its missense mutant SETBP1(D/N) directly interact with histone methyltransferase MLL1. Using a combination of ChIP-seq and RNA-seq analysis in primary hematopoietic stem and progenitor cells, we uncovered extensive overlap in their genomic occupancy and their cooperation in activating many oncogenic transcription factor genes including Hoxa9/Hoxa10/Myb and a large group of ribosomal protein genes. Genetic ablation of Mll1 as well as treatment with an inhibitor of the MLL1 complex OICR-9429 abrogated Setbp1/Setbp1(D/N)-induced transcriptional activation and transformation. Thus, the MLL1 complex plays a critical role in Setbp1-induced transcriptional activation and transformation and represents a promising target for treating myeloid neoplasms with SETBP1 activation.

Keywords: Biological sciences; Cancer; Cell biology; Molecular biology.

Graphical abstract

Figure 1

MLL1 is a critical partner for SETBP1/SETBP1(D/N) to activate Hoxa9/Hoxa10/Myb transcription (A) Chromatin immunoprecipitation (ChIP) analysis of indicated regions of Hoxa9 promoter relative to transcriptional start site using an antibody specific for H3K4me3 in mouse myeloid progenitor cells immortalized by transduction with pMYs-3xFLAG-Setbp1-IRES-GFP virus at 72 h after transduction with a lentiviral shRNA targeting GFP (GFP-sh) and a non-targeting shRNA (NC-sh). Data are represented as mean ± SD (n = 3). ∗, p <0.05; ∗∗, p <0.01; ∗∗∗, p <0.001 (two-tailed Student's t test). (B) ChIP analysis of indicated regions of Hoxa9 promoter in same cells as in (A) using an H4K16ac-specific antibody. Data are represented as mean ± SD (n = 3). ∗, p <0.05; ∗∗∗, p <0.001 (two-tailed Student's t test). (C) ChIP analysis of Hoxa9 promoter regions in same cells as in (A) using antibodies specific for MLL1-N and MLL1-C fragments (left panel) and anti-FLAG M2 (right panel). Data are represented as mean ± SD (n = 3). (D) Left panel, real-time RT-PCR analysis of relative Hoxa9/Hoxa10/Myb mRNA levels in Setbp1-immortalized cells at 72 h after transduction with Mll1-specific shRNAs (Mll1-sh1 and -sh2) or a non-targeting control shRNA (NC-sh). Data are represented as mean ± SD (n = 3). ∗, p <0.05; ∗∗, p <0.01 (two-tailed Student's t test). Right panel, western blotting analysis of the same transduced cells using indicated antibodies. (E) ChIP analysis of Hoxa9 promoter regions in mouse myeloid progenitor cells immortalized by transduction with pMYs-3xFLAG-Setbp1(D/N)-IRES-GFP virus using antibodies specific for MLL1-N and MLL1-C fragments (left panel) and anti-FLAG M2 (right panel). Data are represented as mean ± SD (n = 3). (F) Left panel, real-time RT-PCR analysis of relative Hoxa9/Hoxa10/Myb mRNA levels in Setbp1(D/N)-immortalized cells at 72 h after transduction with the same lentiviral shRNAs as in (D). Data are represented as mean ± SD (n = 3). ∗, p <0.05; ∗∗, p <0.01 (two-tailed Student's t test). Right panel, western blotting analysis of the same transduced cells using indicated antibodies. (G) Left panel, real-time RT-PCR analysis of relative HOXA9, HOXA10, and MYB mRNA levels in primary human sAML cells with a SETBP1 missense mutation (p.D868Y) at 72 h after transduction with indicated negative control or MLL1-specific shRNAs. Data are represented as mean ± SD (n = 3). ∗∗, p <0.01; ∗∗∗, p <0.001 (two-tailed Student's t test). Right panel, Western blotting analysis of same transduced cells using indicated antibodies.

Figure 2

MLL1 physically associates with SETBP1 and SETBP1(D/N) (A) Western blotting analysis of M2 immunoprecipitates prepared from nuclear extract of HEK293T cells transiently transfected with empty pMYs vector or pMYs construct expressing 3xFLAG-tagged Setbp1 or Setbp1(D/N) using indicated specific antibodies. (B) Western blotting analysis of immunoprecipitates prepared with an MLL1-N-specific antibody from the same nuclear extracts as in (A) using the indicated specific antibodies. (C) Western blotting analysis of M2 immunoprecipitates similarly prepared as in (A) using additional specific antibodies. (D) Western blotting analysis of M2 and IgG immunoprecipitates prepared from myeloid progenitors immortalized by 3xFLAG-tagged Setbp1 or Setbp1(D/N) using indicated specific antibodies. (E) Immunofluorescence staining of Setbp1-immortalized myeloid progenitors using a SETBP1-specific antibody together with either an MLL1-N-specific or MLL1-C-specific antibody. Nuclei were counterstained with DAPI. Scale bar, 10 μm. (F) Same immunofluorescence staining as in (D) for Setbp1(D/N)-immortalized myeloid progenitors. Scale bar, 10 μm.

Figure 3

Both MLL1-N1 and MLL1-C directly interact with SETBP1 and SETBP1(D/N) (A) Schematic representation of domain structure of full-length MLL1 protein and the different 3xHA-tagged mouse MLL1 fragments used for co-immunoprecipitation studies to test possible direct interactions with 3xFLAG-tagged SETBP1 and SETBP1(D/N). Results of the co-immunoprecipitation studies shown in (B) and (C) are also summarized, with “+” indicating interaction detected with both SETBP1 and SETBP1(D/N) and “-” indicating no interaction detected. (B) Co-immunoprecipitation experiments were performed using either anti-FLAG M2 antibody (left panels) or anti-HA antibody (right panels) on mixtures of SETBP1 with different MLL1 fragments. Inputs and immunoprecipitates were subsequently analyzed by western blotting analysis using anti-SETBP1, M2, or anti-HA antibody. SETBP1 protein and all MLL1 fragments were synthesized by in vitro transcription and translation in wheat germ extract. (C) Same co-immunoprecipitation experiments as in (B) to detect possible direct interaction between SETBP1(D/N) and MLL1 fragments.

Figure 4

Genome-wide transcriptional regulation by SETBP1 and SETBP1(D/N) in mouse LSK cells (A) Upper panels, mapping of top 5,000 peaks (by q-value) from SETBP1 and SETBP1(D/N) ChIP-seq data. Lower panels, Venn diagrams showing overlaps of SETBP1-bound or SETBP1(D/N)-bound peaks in indicated genomic regions. p Values for overlaps were calculated by hypergeometric test. (B) Pie charts showing the numbers of differentially expressed genes induced by Setbp1 and Setbp1(D/N) expression in mouse LSK cells. (C) Top GO gene sets with positive enrichment from GSEA analysis of differentially expressed genes in mouse LSK cells induced by ectopic Setbp1 and Setbp1(D/N) expression. (D) GSEA analysis of differentially expressed genes in mouse LSK cells induced by Setbp1 expression using indicated gene sets. (E) Genome browser tracks representing SETBP1 and SETBP1(D/N) shared occupancy at indicated loci of oncogenic transcription factor genes and ribosomal protein genes.

Figure 5

Co-localization of SETBP1 and SETBP1(D/N) with MLL1 at oncogenic transcription factor genes and ribosome protein genes (A) Mapping of MLL1-N ChIP-seq peaks in mouse LSK cells overexpressing Setbp1 or Setbp1(D/N). (B) Venn diagrams showing overlaps of top 5,000 SETBP1-bound or SETBP1(D/N)-bound peaks with top 5,000 MLL1-bound peaks in LSK cells overexpressing Setbp1 or Setbp1(D/N). p Values were calculated using hypergeometric test. (C) Average binding profiles of H3K4me3 at promoter regions of SETBP1-bound, MLL1-bound, and SETBP1/MLL1 co-bound targets. (D) Genome browser tracks showing co-localization of SETBP1, MLL1, H3K4me3, and H4K16ac at indicated loci of oncogenic transcription factor gene targets and ribosomal protein gene targets. (E) Genome browser tracks showing co-localization of SETBP1(D/N), MLL1, H3K4me3, and H4K16ac at the same target loci as in (D).

Figure 6

Mll1 is essential for transformation induced by Setbp1 and Setbp1(D/N) (A) Schematic diagram showing the experimental design for the generation of Mll1 conditional immortalized and AML cells. (B) Relative colony formation by Setbp1- and Setbp1(D/N)-immortalized Mll1^F/F;Cre⁺ and Mll1^+/+;Cre⁺ myeloid progenitors at 48 h after treatment with 4-OHT or control ethanol (EtOH). Data are represented as mean ± SD (n = 3). ∗∗∗, p <0.001 (two-tailed Student's t test). (C) Cytospin analysis of Setbp1- and Setbp1(D/N)-immortalized Mll1^F/F;Cre⁺ and Mll1^+/+;Cre⁺ myeloid progenitors at 72 h after treatment with 4-OHT. Scale bar, 10 μm. (D) Real-time RT-PCR analysis of indicated SETBP1/MLL1 co-bound targets in Setbp1-immortalized Mll1^F/F;Cre⁺ and Mll1^+/+;Cre⁺ cells at 48 h after treatment with 4-OHT or EtOH. Data are represented as mean ± SD (n = 3). (E) Real-time RT-PCR analysis of indicated SETBP1(D/N)/MLL1 co-bound targets in Setbp1-immortalized Mll1^F/F;Cre⁺ and Mll1^+/+;Cre⁺ cells at 48 h after treatment with 4-OHT or EtOH. Data are represented as mean ± SD (n = 3). (F) Survival curves of mice receiving 5 × 10⁵Setbp1-induced (left panel) or Setbp1(D/N)-induced (right panel) Mll1^F/F;Cre⁺ AML cells treated with Tamoxifen or control corn oil. Tamoxifen or corn oil treatments are indicated by dotted lines. p Values were calculated using the log rank test. ∗∗∗, p <0.001.

Figure 7

Setbp1- and Setbp1(D/N)-induced AML cells are sensitive to WDR5 inhibition (A) Real-time RT-PCR analysis of indicated SETBP1/MLL1 co-bound targets in Setbp1-induced AML cells at 72 h after treatment with OICR-9429 at indicated concentration or control DMSO. Data are represented as mean ± SD (n = 3). (B) Real-time RT-PCR analysis of same genes in Setbp1(D/N)-induced AML cells at 72 h after treatment with OICR-9429 or control DMSO. Data are represented as mean ± SD (n = 3). (C) Relative colony formation by Setbp1-induced AML cells upon treatment with WDR5 inhibitor OICR-9429 at indicated concentration or control DMSO. Data are represented as mean ± SD (n = 3). (D) Relative colony formation by Setbp1(D/N)-induced AML cells upon treatment with WDR5 inhibitor OICR-9429 at indicated concentration or control DMSO. Data are represented as mean ± SD (n = 3). (E) Left panel, real-time RT-PCR analysis of relative indicated SETBP1/MLL1 co-bound targets in Setbp1-induced AML cells after transduction with Wdr5-specific shRNAs (Wdr5-sh1 and -sh2) or a non-targeting control shRNA (NC-sh). Data are represented as mean ± SD (n = 3). Right panel, western blotting analysis of the same transduced cells using indicated antibodies. (F) Same analyses as in (E) for Setbp1(D/N)-induced AML cells.