Figure 1.
TCL1A interacts with KDM1A and enhances its demethylase activity. (A) Experimental setting of MS of TCL1A coimmunoprecipitations (co-IPs) in human CLL B cells (nonmalignant B cells from tonsils as biological controls). Immunoglobulin G (IgG) co-IPs in CLL B cells served as technical controls. CLL sample strata: immunoglobulin heavy chain variable (IGHV) mutated (M-CLL, N = 6) vs IGHV unmutated (U-CLL, N = 5). Proteins that showed a significant enrichment between the IgG control and CLL samples (459 proteins) or tonsils (889 proteins) were considered TCL1A interacting partners (Welch test, FDR q-value ≤ 0.05, FCh ≥ 2.0). Four hundred nine and 407 TCL1A-interacting proteins were identified in M-CLL and U-CLL cells, respectively (324 proteins overlapped between both subsets). (B) Left: selected pathways identified by overrepresentation analysis (ORA; supplemental Table 6), bars illustrate the percentage of involved proteins from the TCL1A interactome within each pathway. Right: heatmap of chromatin-modifying enzymes that interact with TCL1A protein. KDM1A (∗) tended to interact with TCL1A at a higher abundance in U-CLL than M-CLL (P = .052, Mann Whitney test). (C) Immunoblots of co-IPs using IgG or TCL1A antibodies in primary CLL B cells cocultured with differentiated THP-1 monocytic cells supplemented with CpG and IL-15 for 36 hours to induce proliferation (Ki67+ 38.9%-54.1%). Each lane represents an individual CLL sample. (D) Immunoblots of TCL1A co-IPs from Eμ-TCL1A splenic leukemic cells (2 mice) treated with DNA double strand break–inducing etoposide (10 μM, at 0, 1, and 3 hours). (E) Histone demethylase activity assay: higher demethylase activity of KDM1A in DoHH2 B cells transfected with TCL1A (N = 3, each experiment in duplicates; P = .008, Mann Whitney test, mean ± standard error of the mean [SEM]). (F) Quantitative MS analysis of histone posttranslational modifications (PTMs) in DoHH2±TCL1A B cells (N = 3 independent experiments). Left: principal component analysis (PCA) of MS data showing distinct groups by the presence of TCL1A (blue DoHH2, red DoHH2TCL1A). Each data point represents 1 independent experiment. Middle: heatmap showing differential histone single marks in TCL1A positive and negative DoHH2 cells. Right: lower levels of H3K9me2 (P = .013) and H3K9me3 (P < .001) in TCL1A-positive DoHH2 cells (Student t test, mean ± SEM). (G) Immunoblots showing lower levels of H3K9me3 in TCL1A-positive DoHH2 cells. (H) Top: immunoblots showing KDM1A levels in 14 CLL (lanes = case numbers). Bottom: higher demethylase activity of KDM1A in cases with higher KDM1A levels as per KDM1A activity assay (N = 4 CLL per group, P = .028, Mann Whitney test, mean ± SEM; KDM1A levels shown in top panel). Supplemental Figure 1A shows no correlation between KDM1A and H3K9me2/3 protein signals (nonexclusive impact of KDM1A on H3K9me2/3 levels). (I) Immunoblots of co-IPs using IgG or TCL1A antibodies illustrating higher abundance of TCL1A-KDM1A interaction in primary CLL B cells vs T-PLL T cells. Each lane represents an individual B-CLL or T-PLL sample. RFU, relative fluorescence units.

TCL1A interacts with KDM1A and enhances its demethylase activity. (A) Experimental setting of MS of TCL1A coimmunoprecipitations (co-IPs) in human CLL B cells (nonmalignant B cells from tonsils as biological controls). Immunoglobulin G (IgG) co-IPs in CLL B cells served as technical controls. CLL sample strata: immunoglobulin heavy chain variable (IGHV) mutated (M-CLL, N = 6) vs IGHV unmutated (U-CLL, N = 5). Proteins that showed a significant enrichment between the IgG control and CLL samples (459 proteins) or tonsils (889 proteins) were considered TCL1A interacting partners (Welch test, FDR q-value ≤ 0.05, FCh ≥ 2.0). Four hundred nine and 407 TCL1A-interacting proteins were identified in M-CLL and U-CLL cells, respectively (324 proteins overlapped between both subsets). (B) Left: selected pathways identified by overrepresentation analysis (ORA; supplemental Table 6), bars illustrate the percentage of involved proteins from the TCL1A interactome within each pathway. Right: heatmap of chromatin-modifying enzymes that interact with TCL1A protein. KDM1A (∗) tended to interact with TCL1A at a higher abundance in U-CLL than M-CLL (P = .052, Mann Whitney test). (C) Immunoblots of co-IPs using IgG or TCL1A antibodies in primary CLL B cells cocultured with differentiated THP-1 monocytic cells supplemented with CpG and IL-15 for 36 hours to induce proliferation (Ki67+ 38.9%-54.1%). Each lane represents an individual CLL sample. (D) Immunoblots of TCL1A co-IPs from Eμ-TCL1A splenic leukemic cells (2 mice) treated with DNA double strand break–inducing etoposide (10 μM, at 0, 1, and 3 hours). (E) Histone demethylase activity assay: higher demethylase activity of KDM1A in DoHH2 B cells transfected with TCL1A (N = 3, each experiment in duplicates; P = .008, Mann Whitney test, mean ± standard error of the mean [SEM]). (F) Quantitative MS analysis of histone posttranslational modifications (PTMs) in DoHH2±TCL1A B cells (N = 3 independent experiments). Left: principal component analysis (PCA) of MS data showing distinct groups by the presence of TCL1A (blue DoHH2, red DoHH2TCL1A). Each data point represents 1 independent experiment. Middle: heatmap showing differential histone single marks in TCL1A positive and negative DoHH2 cells. Right: lower levels of H3K9me2 (P = .013) and H3K9me3 (P < .001) in TCL1A-positive DoHH2 cells (Student t test, mean ± SEM). (G) Immunoblots showing lower levels of H3K9me3 in TCL1A-positive DoHH2 cells. (H) Top: immunoblots showing KDM1A levels in 14 CLL (lanes = case numbers). Bottom: higher demethylase activity of KDM1A in cases with higher KDM1A levels as per KDM1A activity assay (N = 4 CLL per group, P = .028, Mann Whitney test, mean ± SEM; KDM1A levels shown in top panel). Supplemental Figure 1A shows no correlation between KDM1A and H3K9me2/3 protein signals (nonexclusive impact of KDM1A on H3K9me2/3 levels). (I) Immunoblots of co-IPs using IgG or TCL1A antibodies illustrating higher abundance of TCL1A-KDM1A interaction in primary CLL B cells vs T-PLL T cells. Each lane represents an individual B-CLL or T-PLL sample. RFU, relative fluorescence units.

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