PMC

Classification of the regulated genes. (A) MA plots generated from 8 biological samples were M = log2R − log2G and A = 1/2 × (log2R + log2G). (B) Venn diagram indicating the number of genes significantly regulated in each microarray study (knockdown with si323 and si2992) and the overlap between the 2 sets of regulated genes after c-Myb knockdown in K562 cells. (C) Pie chart representing major functional protein groups found among the encoding genes regulated by c-Myb knockdown.

Chromatin immunoprecipitation (ChIP) assays. The K562-hcM cell line, which stably expresses HA- and FLAG-tagged c-Myb, was used to detect direct binding of c-Myb to the promoters of LMO2, MYADM, GATA2, STAT5A, and IKZF1. An unrelated DNA region (UDR) was used as negative control. Anti-FLAG antibody was used to immunoprecipitate tagged cMyb, while IgG control antibody was used as negative control.18 qPCR reactions were run in triplicates. The results are given as percentage recovery ± standard deviation compared to input material and are representative of 3 independent experiments. ChIP primer positions and sequences are given in and and Supplementary Table S2, respectively.

Description of the siRNAs. (A) Graphic representation of the localization and sequence of the c-Myb siRNAs used in this study. (B) Western analysis of protein levels 24 hours after siRNA-mediated knockdown of c-Myb. Protein extracts (50 µg) were subjected to Western analysis with H141 (anti–c-Myb antibody). The blot was also probed with α-tubulin antiserum for loading control. (C) MYB mRNA expression in K562 cells 24 hours after transfection with si323 and si2992. The mRNA expression was measured by qRT-PCR, as described in Materials and Methods, and represented as percentage of expression in control K562 cells (transfected with siLuc). (D) MYBL2 mRNA expression was not affected by the transfection of K562 cells with c-Myb siRNAs si323 and si2992. The expression of MYBL2 was measured by qRT-PCR 24 hours after transfection of K562 cells and represented relative to the levels of expression in K562 cells transfected with siLuc (C). (E) Log2 of the fold change in the expression of the 3 members of the MYB family in the microarray analysis.

Analysis of target genes using the K562-hcM cell line. (A) Left panel: qRT-PCR–mediated relative quantification of the mRNA levels for the indicated genes in parental K562 cells (K562-p) or in the K562 cell line that stably expresses exogenous c-Myb (K562-hcM). Total RNA was extracted and cDNA synthesized, as described in Materials and Methods. Four independent experiments were performed, and at least 2 different dilutions for each cDNA were analyzed by qPCR. The expression levels were normalized versus the expression levels of the housekeeping gene POLR2A and represented as percentage of expression in untransfected parental K562 cells (K562-p). The bar graph represented the average of expression from at least 3 independent transfections ± standard error. Right panel: Corresponding Western analysis of the expression levels of exogenous (exo) and endogenous (endo) c-Myb in K562-p and K562-hcM cells. (B) Left panel: qRT-PCR–mediated relative quantification of the mRNA levels for the indicated genes 24 hours after si2992 transfection of the K562-p and K562-hcM cell lines. Total RNA was extracted and cDNA synthesized, as described in Materials and Methods. Four independent experiments were performed, and at least 2 different dilutions for each cDNA were analyzed by qPCR. The expression levels were normalized versus the expression levels of the housekeeping gene POLR2A and represented as percentage expression relative to the K562-hcM cells. The bar graph represented the average of expression from at least 3 independent transfections ± standard error. Right panel: Corresponding Western analysis of the expression levels of exogenous (exo) and endogenous (endo) c-Myb in K562-hcM and K562-p cells 24 hours after transfection with si2992 or siLuc (ctrl).

3C analysis of spatial interactions between c-Myb target genes. The upper panel of the figure shows a simplified map of the locus under study. Arrows on the map depict genes with exons given as black lines. The anchor restriction fragment harboring the LMO2 promoter is shown in more detail with the positions of unique restriction sites indicated (H = HindIII; E = EcoRI; N = NlaIII). Black arrowheads show primers used for the 3C analysis, while the white arrowheads represent the TaqMan probe (for primer sequences, see Suppl. Table S4). The graphs in the lower panel demonstrate relative cross-linking frequencies between the LMO2 anchor fragment (dark gray shadowing) and other fragments (light gray shadowing). The fragments’ relative chromosomal positions are shown on the x-axis (0 point: start of chromosome 11 genomic contig sequence NW001838022.2). The results from both parental K562 cells (black curves) and K562 cells after transfection with si323 (gray curves) are included. The interaction frequency is normalized to the cross-linking frequency observed between the anchor and the adjacent fragment, which is set to 100. Error bars represent standard error for 3 independent experiments.

Validation of the most regulated genes. (A) qRT-PCR–mediated validation of the microarray indicated down-regulation of MYADM and LMO2 after c-Myb knockdown. mRNA expression was measured 24 hours after transfection of K562 cells with si323 or si2992. Total RNA was extracted and cDNA synthesized, as described in Materials and Methods. Two different dilutions for each cDNA were analyzed by qPCR. The expression levels were normalized versus the expression levels of the housekeeping gene POLR2A and represented as percentage of expression in siLuc-transfected K562 cells (ctrl). The bar graph represented the average of expression from at least 3 independent transfections ± standard deviation. (B) Promoter maps. Schematic presentation of the promoter area (−2,000 to +700) of MYADM and LMO2, aligned relative to their transcription start site (TSS) (position +1) depicted by an arrow. The GenBank accession numbers are given to the right of the TSS. Putative MREs following the consensus YAAC[NG/GN] are shown as gray boxes. The solid black lines specify the promoter regions that were amplified and cloned into the luciferase reporter plasmid pGL3 basic. Arrowheads indicate the position of the primers used in the ChIP. (C) Reporter assay–based study of the responsiveness of MYADM and LMO2 promoter regions to c-Myb–2KR. CV-1 cells were transfected with a reporter construct, where the luciferase gene expression was driven by either the MYADM or LMO2 promoter, and increasing concentration of c-Myb expression plasmid (0-300 ng). A reporter plasmid containing 3 MYB recognition elements (MREs) upstream of a core promoter from human MYC driving the luciferase reporter was used as a positive control (3×MRE). The empty reporter vector was used as negative control. Results are represented as average luciferase units from 3 independent transfections ± standard error. The values in brackets refer to the slope of activation (RLU/ng), based on a linear regression analysis of the reporter assay data (Suppl. Fig. S1).

Validation of the regulation of transcriptional regulators. (A) qRT-PCR–mediated validation of the microarray indicated down-regulation of IKZF1, STAT5A, and GATA2 after c-Myb knockdown. The mRNA expression of the 3 genes was measured 24 hours after transfection of K562 cells with si323 or si2992. Total RNA was extracted and cDNA synthesized, as described in Materials and Methods. Two different dilutions for each cDNA were analyzed by qPCR. The expression levels were normalized versus the expression levels of the housekeeping gene POLR2A and represented as percentage of expression in siLuc-transfected K562 cells (ctrl). The bar graph represented the average of expression from at least 3 independent transfections ± standard deviation. (B) Promoter maps. Schematic presentation of the promoter area (−2,000 to +700) of IKZF1, STAT5A, and GATA2, aligned relative to their transcription start site (TSS) (position +1) depicted by an arrow. The GenBank accession numbers are given to the right of the TSS. Putative MREs following the consensus YAAC[NG/GN]53 are shown as gray boxes. The solid black lines specify the promoter regions that were amplified and cloned into the luciferase reporter plasmid pGL3 basic. Arrowheads indicate the position of the primers used in the ChIP. (C) Reporter assay–based study of the responsiveness of respective promoter regions to c-Myb–2KR. CV-1 cells were transfected with a reporter construct, where the luciferase gene expression was driven by either the IKZF1, the STAT5A, or the GATA2 promoter, and increasing concentration of c-Myb expressing plasmid (0-300 ng). A reporter plasmid containing 3 MYB recognition elements (MREs) upstream of a core promoter from human MYC driving the luciferase reporter was used as positive control (3×MRE). The empty reporter vector was used as negative control. Results are represented as average luciferase units from 3 independent transfections ± standard error. The values in brackets refer to the slope of activation (RLU/ng), based on a linear regression analysis of the reporter assay data (Suppl. Fig. S1).