Figure 1.
SMARCD1, enriched in hematopoietic progenitors, impedes myeloid differentiation genes by maintaining a repressive chromatin state. (A) Tags per million (TPM) count of SMARCD1 expression from cap analysis gene expression (CAGE)-sequencing data of CD34+ HSPCs and mature myeloid cells with indicated surface markers. (B) Relative log2 fold change in expression of SMARCD1 normalized to ACTB expression in CD34+ HSPCs isolated from cord blood and differentiated to monocytes/macrophages using macrophage-colony stimulating factor (M-CSF) for 10 days. Log2 fold changes were plotted from CD34+ cells isolated from 3 biological replicates. (C) Relative log2 fold change in expression of SMARCD isoforms in uninduced HL-60 cells compared with that after 48 hours of vitamin D3 (50 nM)–induced cells from 3 biological replicates. (D) Relative log2 fold change of SMARCD isoforms in CD34+ AML cells compared with that in the CD34− population. (E) SMARCD1 expression profile across FAB-classified patients with AML from the TCGA database. (F-L) HL-60 cells were transduced with lentiviruses expressing either empty vector (shcontrol) or short hairpin RNA targeting SMARCD1 (shSMARCD1), and all experiments were conducted 96 hours after selection in puromycin-containing media. (F) Total protein cell lysate (50 µg) from wild type, shcontrol, and shSMARCD1 was used for immunoblotting with SMARCD1 antibody. Representative immune blot images for HL-60 cells; ACTB was used as the loading control. (G-J) Flow cytometry analysis of shcontrol or shSMARCD1 HL-60 cells for myeloid differentiation markers CD11b and CD14. (G-J) Representative contour plots with grids, showing percent negative and positive population for single- and double-stained CD14-allophycocyanin-H7 (APC-H7) and CD11b-FITC (fluorescein) populations. (I) Mean fluorescent intensity (MFI) plots of shcontrol and shSMARCD1 HL-60. (H-J) Same analysis as panels G-I after induction with 10 nM vitamin D3 for 48 hours. (K-L) Representative May Grünwald Giemsa–stained images of shcontrol and shSMARCD1 HL-60 in uninduced cells (K) and after 48 hours of vitamin D3 induction (L) using a Zeiss Apotome 2 (63×, NA 1.4). All statistical parameters used in this figure are for n = 3 independent experiments; error bars indicate means ± standard deviation. *P = .05; **P = .005; ***P < .001; ****P < .0001: 2-tailed Student t test. (M-R) Transcriptomic analysis of SMARCD1 knockdown in HL-60 cells. (M) K-means clustering heat map of RNA-sequencing representing 1175 significantly differentially expressed genes (log2 fold change ≥ 2 and FDR ≤ 0.05; log2 fold change ≤ 2 and FDR ≤ 0.05) annexed from pairwise comparison of shcontrol vs shSMARCD1 under uninduced and induced (vitamin D3, 10 nM) conditions. (N) CellRadar analysis of differentially expressed genes after SMARCd1 knockdown in HL60 cells (https://karlssong.github.io/cellradar/). (O) Heat map representing delta cycle threshold values of indicated genes with respect to ACTB in shcontrol (mean of 3) and shSMARCD1 (n = 3). (P-S). Epigenetic regulation of SWI/SNF complex bound myeloid differentiation genes. (P) Co-immunoprepitation in HL-60 cells using SMARCA4 and SMARCD1 antibodies. Immunoglobulin G (IgG) was used as the negative control. Single representative blot from 2 independent experiment confirming BRG1 and SMARCD1 interaction using pull down and reverse pull down experiments. (Q) Chromatin immunoprecipitation (ChIP)-qPCR for SMARCA4 enrichment and control IgG in the promoter regions for indicated genes in HL-60 cells. (R-S) ChIP-qPCR for H3K4me3 (R) and H3K27me3 (S) marks normalized to the respective input control. IgG pull down was used as the control. Enrichment is plotted as percent input. P values (Student t test) for individual genes are shown in adjacent tables. All ChIP data are from 3 independent experiments. (T) Gene regulatory model for SMARCD1.

SMARCD1, enriched in hematopoietic progenitors, impedes myeloid differentiation genes by maintaining a repressive chromatin state. (A) Tags per million (TPM) count of SMARCD1 expression from cap analysis gene expression (CAGE)-sequencing data of CD34+ HSPCs and mature myeloid cells with indicated surface markers. (B) Relative log2 fold change in expression of SMARCD1 normalized to ACTB expression in CD34+ HSPCs isolated from cord blood and differentiated to monocytes/macrophages using macrophage-colony stimulating factor (M-CSF) for 10 days. Log2 fold changes were plotted from CD34+ cells isolated from 3 biological replicates. (C) Relative log2 fold change in expression of SMARCD isoforms in uninduced HL-60 cells compared with that after 48 hours of vitamin D3 (50 nM)–induced cells from 3 biological replicates. (D) Relative log2 fold change of SMARCD isoforms in CD34+ AML cells compared with that in the CD34− population. (E) SMARCD1 expression profile across FAB-classified patients with AML from the TCGA database. (F-L) HL-60 cells were transduced with lentiviruses expressing either empty vector (shcontrol) or short hairpin RNA targeting SMARCD1 (shSMARCD1), and all experiments were conducted 96 hours after selection in puromycin-containing media. (F) Total protein cell lysate (50 µg) from wild type, shcontrol, and shSMARCD1 was used for immunoblotting with SMARCD1 antibody. Representative immune blot images for HL-60 cells; ACTB was used as the loading control. (G-J) Flow cytometry analysis of shcontrol or shSMARCD1 HL-60 cells for myeloid differentiation markers CD11b and CD14. (G-J) Representative contour plots with grids, showing percent negative and positive population for single- and double-stained CD14-allophycocyanin-H7 (APC-H7) and CD11b-FITC (fluorescein) populations. (I) Mean fluorescent intensity (MFI) plots of shcontrol and shSMARCD1 HL-60. (H-J) Same analysis as panels G-I after induction with 10 nM vitamin D3 for 48 hours. (K-L) Representative May Grünwald Giemsa–stained images of shcontrol and shSMARCD1 HL-60 in uninduced cells (K) and after 48 hours of vitamin D3 induction (L) using a Zeiss Apotome 2 (63×, NA 1.4). All statistical parameters used in this figure are for n = 3 independent experiments; error bars indicate means ± standard deviation. *P = .05; **P = .005; ***P < .001; ****P < .0001: 2-tailed Student t test. (M-R) Transcriptomic analysis of SMARCD1 knockdown in HL-60 cells. (M) K-means clustering heat map of RNA-sequencing representing 1175 significantly differentially expressed genes (log2 fold change ≥ 2 and FDR ≤ 0.05; log2 fold change ≤ 2 and FDR ≤ 0.05) annexed from pairwise comparison of shcontrol vs shSMARCD1 under uninduced and induced (vitamin D3, 10 nM) conditions. (N) CellRadar analysis of differentially expressed genes after SMARCd1 knockdown in HL60 cells (https://karlssong.github.io/cellradar/). (O) Heat map representing delta cycle threshold values of indicated genes with respect to ACTB in shcontrol (mean of 3) and shSMARCD1 (n = 3). (P-S). Epigenetic regulation of SWI/SNF complex bound myeloid differentiation genes. (P) Co-immunoprepitation in HL-60 cells using SMARCA4 and SMARCD1 antibodies. Immunoglobulin G (IgG) was used as the negative control. Single representative blot from 2 independent experiment confirming BRG1 and SMARCD1 interaction using pull down and reverse pull down experiments. (Q) Chromatin immunoprecipitation (ChIP)-qPCR for SMARCA4 enrichment and control IgG in the promoter regions for indicated genes in HL-60 cells. (R-S) ChIP-qPCR for H3K4me3 (R) and H3K27me3 (S) marks normalized to the respective input control. IgG pull down was used as the control. Enrichment is plotted as percent input. P values (Student t test) for individual genes are shown in adjacent tables. All ChIP data are from 3 independent experiments. (T) Gene regulatory model for SMARCD1.

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