Fig 2: Regulation of SMAD5 by L3MBTL1 Occurs in Erythroid Cells(A) KD of L3MBTL1 induces early expression of the erythroid-specific marker GlyA in iPSC-derived CD34+ cells. Representative flow cytometry analysis of iPSCs after 2 days in Epo-induced culture.(B) Expression levels of EKLF, NF-E2, LMO2, EPOR, and fetal globin gene were assessed in iPSC-derived erythroid progeny by qRT-PCR. Data were normalized by GAPDH expression. The data represent the mean ± SD of the three independent experiments. ∗∗p < 0.01 by Student’s t test.(C) K562 erythroleukemia cells were retrovirally transduced to express L3MBTL1-HA (blue bars) or empty MIGR1 control (gray bars), crosslinked with 1% formaldehyde, and immunoprecipitated with an anti-SMAD5 antibody or IgG antibody. Primers covering the SMAD-binding motifs (Adelman et al., 2002) across the upstream enhancer, the proximal promoter, and the intronic enhancer of the EKLF were utilized. Data were normalized by 10% input. The data represent the mean ± SD of the three independent experiments.(D) EKLF, NF-E2, GATA-1, LMO2, EPOR, and BCL11A expression levels were evaluated by qRT-PCR in L3MBTL1-KD CB CD34+ cells after 7 days of erythroid-supporting culture (100 ng/ml SCF and 6 U/ml EPO) compared to controls. GAPDH served as a housekeeping gene control. The data represent the mean ± SD of the three independent experiments. ∗∗p < 0.01 by Student’s t test.(E) Fetal globin levels were evaluated in L3MBTL1-KD CB CD34+ cells by western blot. Tubulin served as the loading control.(F) Overexpression of L3MBTL1 decreases globin gene mRNA levels in cord blood CD34+ cells, as shown by qRT-PCR. Cells were cultured for 3 days in liquid culture that supports erythroid differentiation. GAPDH served as the housekeeping gene. The data represent the mean ± SD of the three independent experiments. ∗∗p < 0.01 by Student’s t test.(G) Overexpression of L3MBTL1 dramatically decreases protein expression levels of gamma globin in K562 cells, as shown by western blot assay. Tubulin served as the loading control. See also Figure S4.

Fig 3: L3MBTL1 Transcriptionally Represses SMAD5(A) GEP of undifferentiated L3MBTL1-KD iPSCs, compared to controls, based on microarray analysis. We utilized independent clones of iPSCs (generated from cord blood CD34+ cells), which we independently infected with lentiviral vectors expressing shRNAs against L3MBTL1.(B) mRNA expression levels of several upregulated and downregulated genes, identified by microarray analysis, was confirmed by qPCR. Data were normalized by GAPDH expression and shown as shRNA versus control. The data represent the mean ± SD of the three independent experiments.(C) GSEA for the combined set of SMAD and hematopoietic stem cells genes compared with the differentially expressed genes in L3MBTL1-KD iPSCs.(D) Expression levels of phospho SMAD1/5, total SMAD5, phospho SMAD2, total SMAD2, and HHEX were evaluated in L3MBTL1-KD and control iPSCs by western blot. Tubulin served as the loading control.(E) iPSCs were crosslinked with 1% formaldehyde and immunoprecipitated with anti-L3MBTL1 or IgG antibody (as a nonspecific control). Plotted values are relative enrichments (y axis) to 10% input and measured for sites in the SMAD5 promoter and actin (x axis). The data represent the mean ± SD of the three independent experiments. ∗p < 0.05 by Student’s t test.(F) L3MBTL1-KD and control iPSCs were crosslinked with 1% formaldehyde and immunoprecipitated with an anti-H3K27 tri-methyl antibody or an IgG antibody (as a nonspecific control). Plotted values are relative enrichments (y axis) to 10% input and measured for sites in the SMAD5 promoter (x axis). The data represent the mean ± SD of the three independent experiments. ∗p < 0.05 by Student’s t test. See also Figure S3.

Fig 4: Genetic and enzymatic reduction of HS reduces hepcidin expression and SMAD5 phosphorylation in human hepatocytes. A, a model for an HS chain using the symbol nomenclature for glycans (42). B, primary human hepatocytes were treated for 30 min with a combination of heparin lyases I, II, and III (gray bars) to decrease cell surface and extracellular matrix HS and then stimulated for 6 h with BMP6 (50 ng/ml). HAMP mRNA was measured by qPCR in triplicate. The average values obtained were normalized to GAPDH mRNA and then expressed as -fold change over untreated cells (n = 2). C, EXT1 and EXT2 mRNA expression was silenced in HepG2 cells by transfection of the corresponding siRNAs. As a control, some cells were transfected with siRNA directed to GFP (n = 3). D, HAMP mRNA expression was measured 48 h after silencing of EXT1 and EXT2 (EXT1/2) with or without stimulation by BMP6 (10 ng/ml) during the last 6 h (n = 3). An siRNA targeting GFP served as a negative control. E and F, Western blotting of pSMAD5 and SMAD5 after silencing EXT1/2 or GFP with or without stimulation with 10 ng/ml of BMP6. The bands were quantitated by ImageJ software and the values were normalized to SMAD5 as shown in (F). G, the expression of ID1 mRNA was evaluated 48 h after silencing of EXT1/2 or GFP with or without stimulation by 10 ng/ml BMP6 (n = 3). In C, D, and G, the average values for mRNA expression were normalized to HPRT1 and scaled to the average value obtained from untreated cells.

Fig 5: Transgenic mice overexpressing Heparanase showed increased BMP6/SMAD signaling.(A) evaluation of BMP6 mRNA in the liver of WT and TG-HPA mice (B) evaluation of Id1 mRNA, in relation to Hprt1. (C) Western blot of pSMAD5, of total SMAD5 and of GAPDH as housekeeping for normalization in densitometry quantification. The values are expressed as fold change of wild type mice.