Sox 6 expression during erythroid maturation. (A) Erythroid liquid cultures from CD34⁺ cells purified from HCB. Day 0: purified CD34⁺ cells; Day 8: beginning of the erythroid differentiation phase. Day 14: end of the culture, when 90% of cells are GpA⁺. (B) Erythroid liquid cultures from CD34⁺ cells purified from HPB. Day 0: purified CD34⁺ cells; Day 7: beginning of erythroid differentiation; Day 14: end of erythroid differentiation (97% GpA⁺cells) (C) Semi-quantitative RT–PCR on cDNA from K562 cells, treated (+) or not (−) with hemin (50 µM for 4 days). PCR amplification cycles are indicated below the lanes. (D) Mouse Bone Marrow cells sorted according to their erythroid maturation, from more immature kit⁺CD71⁻ to progressively more mature stages CD71^highTer119^low, CD71^highTer119^high, CD71^low Ter119^high. Semi-quantitative (left) and RealTime (right) PCRs were performed on cDNA from cells indicated in the figures. The Sox6 expression level is normalized on GAPDH (human cells) and HPRT (mouse cells), respectively.

Fine mapping of the double Sox6 binding site on the human Sox6 promoter. (A) Schematic representation of the 5′-region of the human Sox6 locus on chromosome 11. The thick arrows indicate the position of the double Sox6 binding site, located within the region immediately upstream the main transcript start site. Note that the transcription orientation is from right to left. (B) UCSC genome browser (http://genome.ucsc.edu/) graphical map of the human Sox6 promoter region. The thick arrows indicate the same Sox6 binding sites as in A. The −1116 nt promoter region studied in transfection experiments is indicated with a dotted line under the panel. (C) Nucleotide conservation of the region (−775/−759) containing the two Sox6 binding sites (black arrows). The few substitutions in the spacer region between the two single sites are underlined. (D) The −1116 nt conserved region behaves as a promoter in K562 cells. The constructs used are schematically represented on the left of the figure. Luciferase activity is given in arbitrary units, standard deviations are represented on the top of each column.

Sox6 represses its own promoter in cotransfection experiments in K562 cells. (A) The −1116/−1 region, either WT or mutated in the Sox6 consensus was cloned upstream to the Luciferase reporter gene. The mutations in the Sox6 consensus are the same as in Figure 4A, shown to abolish Sox6 binding in EMSA. (B) The two constructs were transfected either in K562 cells (left panel) with increasing doses of a Sox6 expressing plasmid, or in P19 cells (right panel) induced or not with RA. RA treatment induces Sox6 in P19 cells as shown by RT–PCR (right panel). In both cell lines, Sox6 induces a repression of the WT construct when compared to the mutated one. Statistical analyses are indicated above the chart. (C) Primary erythroblasts obtained from CD34⁺ cells purified from either Peripheral Blood (left panels) or Cord Blood (right panels) were nucleofected with the 1116-S6wt and 1116-S6mut constructs at Days 8 and 12 of the cultures, in the presence of a Renilla-expressing plasmid to normalize for relative transfection efficiencies. Upper panels: RealTime PCR assessing the expression level of the endogenous Sox6 (relative to GAPDH). Middle panels: Luciferase activity driven by the 1116-S6wt element; Lower panels: Luciferase activity driven by the 1116-S6mut element at the same days (no statistical significant differences were found). Luciferase activity is given in arbitrary units. (D) The activity of the 1116-S6mut is higher than that of the 1116-S6wt construct at late phases of erythroid differentiation (Day 12) when data from Peripheral and Cord Blood cells are plotted together (normalized on Renilla expression). (E) ChIP demonstrates that at Day 12 SOX6 is indeed bound to its promoter. IgG antibodies were used as a control. (F) A 234-nt fragment (either the WT or the mutated Sox6 double site) was cloned upstream to a highly active Gata-1-derived erythroid cassette (pe construct). (G) All constructs were cotransfected in K562 cells together with increasing amounts (from 0.2 to 1.6 µg) of a Sox6 expressing plasmid. The pe+WT construct is repressed in a dose dependent manner by the addition of the cotransfected Sox6 expressing plasmid (black bars, columns 7, 11, 15), while the corresponding mutated element, pe+MUT, is not (columns 8, 12, 16). A Sox4 expressing plasmid (at the highest concentration of 1.6 µg used for Sox6) fails to repress pe+WT, suggesting a Sox6 highly specific effect (lane 19). Sox6-FLAG and Sox4-FLAG expressing plasmids produce comparable amounts of protein, as demonstrated by using the anti-FLAG antibody in the western blot on the right (1.6 µg of transfected plasmids, extracts from 5 × 10⁵ transfected cells per lane).

Sox6 binds the murine homologous Sox6 consensus region in bone marrow erythroid cells. (A) ChIP experiments. The anti-Sox6, rabbit IgG, and the unrelated anti-Brn3 antibodies were used to immunoprecipitate chromatin from mouse total bone marrow. Upper gel: double Sox6 consensus region. Lower gel: region within the Sox6 exon 16, used as a negative control. (B) FACS analysis and sorting. Two cells populations were sorted from murine bone marrow: erythroid CD71⁺Ter119⁺ (R2) and CD71⁻Ter119⁻ (R1). (C) Chromatins from R1 and R2 cells populations were immunoprecipitated using rabbit IgG, anti-Sox6 and anti-HDAC1 antibodies. (D) The same chromatins were immunoprecipitated using rabbit IgG and anti-H3K4me1 (left) and anti-H3K27me3 (right) antibodies.

Schematic representation of the bioinformatic strategy used to identify new direct Sox6 targets. (A) Sequence of the double Sox6 binding site located within the mouse εy-globin promoter. Nucleotides positions are indicated on top of the sequence. Search criteria set in TFBScluster search are indicated. (B) TFBScluster search output. The identified potential Sox6 binding sites are clustered on the basis of their relative position with respect to known genes. The absolute number—and the corresponding percentages—of sites for each group is reported in the pie-chart. (C) The merge of the list of the 56 potential Sox6 binding sites located within 10Kb upstream to known genes with the list of genes differentially expressed during mouse erythropoiesis identified seven erythroid genes potentially regulated by Sox6 (listed in D). For each potential target site (including the one within the Sox6 locus itself) the distance from the transcriptional start site, the exact Sox consensus sequence, and the evolutionary conservation are indicated.

Sox6 binds with high affinity to the −775/−759 double consensus on human Sox6 promoter. (A) Nucleotide sequence of the four probes used in EMSA experiments. The two Sox6 sites are underlined and the mutations destroying the Sox6 consensus are marked by asterisks. (B) EMSA experiments: probes in panel A were ³²P labeled and incubated with either nuclear (lanes 1–6 and 10–16) or total K562 cell extracts (lanes 7–9 and 16–18). Extracts from K562 cells transfected with the pCMV-Sox6FLAG expressing vector: black bars over lane numbers; increasing amounts of antibodies are indicated as + and ++, respectively. ³²P labelled probes are indicated below the panel. The retarded band produced by Sox6 binding is indicated by the grey arrow: its specificity is demonstrated by the use of a specific anti-FLAG antibody (lanes 3 and 4). The MUT probe, mutated in both Sox6 consensus sites, fails to give any retarded band when tested in the same conditions as for the wild-type (WT) probe (lane 10–18). (C) Unlabeled competitor oligonucleotides (sequences in panel A) were added at three increasing concentrations and are indicated on top of the panel. WT: lane 2 (at the intermediate concentration). Col2a1-derived Sox6 consensus site (ref. 4): lanes 3–5. Mut1: lanes 6–8. Mut2: lanes 9–11 MUT: lanes 12–14.). Right panel: direct binding of the WT and Mutated probes.

Overexpression of exogenous Sox6 represses the endogenous Sox6 transcription. (A) Semiquantitative RT–PCR on cDNA from K562 and HCB-derived progenitors cells transduced with either the Sox6 overexpressing vector or the corresponding empty vector. GAPDH was used to normalize for cDNAs loading. The number of amplification cycles used for each set of primers is indicated on the right of the figure. In Real Time quantification, the endogenous Sox6 transcript—in K562 and HCB cells overexpressing the Sox6FLAG protein—was scored as undetectable (not shown). Right panel: primers combinations used to discriminate between endogenous versus exogenous Sox6 transcript. (B) Western blot analysis. Nuclear extracts from 7 × 10⁵ cells were loaded in each lane. Right panel: antibodies used to discriminate between endogenous versus exogenous Sox6 protein: the anti-FLAG antibody detects exogenous SOX6, while the endogenous protein is revealed by anti-SOX6 antibody raised again the C-terminal portion of the protein, absent in the exogenous protein. The anti c1/c2 hnRNP antibody was used to normalize for protein loading. (C) ChIP experiments. The anti-FLAG or rabbit IgG antibodies were used to immunoprecipitate chromatin from either K562 (transduced with the empty vector) or K562 overexpressing Sox6FLAG (K562-S6) cells. Lanes 1 and 2: input chromatins. Lanes 3 and 4: normal rabbit IgG. Lane 5 and 6: anti-FLAG antibody. Lane 7: water. Upper gel: Sox6 promoter region. Lower gel: GAPDH genomic locus was used as a negative control. Lower panel: Real Time quantification of the Sox6 promoter enrichment in chromatin immunoprecipitated with the anti-FLAG antibody. GAPDH and SOX4: negative control loci, cyclin D1: positive control. Real Time analysis was performed twice in triplicate. (D) Restriction enzyme accessibility assay. Schematic representation (not in scale) of the position of the PstI endonuclease restriction site relative to the Sox6 binding sites within the Sox6 promoter. Grey arrows indicate primers used to amplify the uncut copies of this region. In presence of low Sox6 level, PstI accesses the Sox6 promoter region (30% of cut copies, with P-value <0.0005) while in the presence of high Sox6 level it does not. A PstI recognition site within the human GATA1 promoter is used as a positive control.