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Supplemental Materials and Methods Supplemental Material for: Transcriptional silencing of γ-globin by BCL11A involves long-range interactions and cooperation with SOX6 Jian Xu, Vijay G. Sankaran, Min Ni, Tobias F. Menne, Rishi V. Puram, Woojin Kim, Stuart H. Orkin* *To whom correspondence should be addressed. E-mail: [email protected] Supplemental Materials and Methods Flow cytometry Cells at various stages of differentiation were analyzed by flow cytometry using FACSCalibur (BD Biosciences, San Jose, CA). Live cells were identified and gated by exclusion of 7-amino-actinomycin D (7-AAD; BD Pharmingen). The cells were analyzed for expression of cell surface receptors with antibodies specific for CD34, CD45, CD71, CD235, and CD36 conjugated to phycoerythrin (PE), fluorescein isothiocyanate (FITC), or allophycocyanin (APC; BD Pharmingen). Data were analyzed using FlowJo software (Ashland, OR). Cytology Cytocentrifuge preparations were stained with May-Grunwald-Giemsa as previously described (Sankaran et al. 2008). Real-time RT-PCR Real-time quantitative RT-PCR was performed using the iQ SYBR Green Supermix (Bio- Rad). The following primers were used for real-time RT-PCR: human and mouse BCL11A-XL (forward, 5’-ATGCGAGCTGTGCAACTATG-3’; reverse, 5’- GTAAACGTCCTTCCCCACCT-3’), human and mouse BCL11A-L (forward, 5’- CAGCTCAAAAGAGGGCAGAC-3’; reverse, 5’-GAGCTTCCATCCGAAAACTG-3’), and human BCL11A exon 1 and 2 (common between all known isoforms; forward, 5’- AACCCCAGCACTTAAGCAAA-3’; reverse, 5’-GGAGGTCATGATCCCCTTCT-3’). Supplemental Figure Legends Supplemental Figure 1. Expression of BCL11A isoforms in human and mouse erythroid cells. (A) Schematic diagram of human BCL11A isoforms (Liu et al. 2006). The antibodies used for ChIP experiments and their corresponding epitopes are indicated. Locations of primers used for RT-PCR analysis of all BCL11A isoforms (forward and reverse primers indicated by arrowheads), XL and L isoforms are indicated. (B) Relative mRNA expression of human BCL11A XL and L isoforms in adult human proerythroblasts (Pro-E) were quantified by real-time RT-PCR. Transcript levels were normalized against human GAPDH transcript levels. (C) Relative mRNA expression of mouse Bcl11a XL and L isoforms in mouse erythroleukemia (MEL) cells and FACS-sorted Ter119+CD71+ E14 fetal liver erythroid cells were quantified by real-time RT-PCR. Transcript levels were normalized against mouse Gapd transcript levels. Supplemental Figure 2. Lack of genome-wide cooccupancy between BCL11A, H3K4me3, and H3K27me3. (A) De novo BCL11A motif search was performed in SeqPos using MDscan algorithm (Liu et al. 2002) (B) Venn diagram of genome-wide colocalization of BCL11A, H3K4me3, and H3K27me3 ChIP-chip sites in adult human erythroid cells. (C) Genome-wide analysis of BCL11A, H3K4me3, H3K27me3 cooccupancy. Scatter plots displays mutual enrichment between indicated ChIP-chip datasets relative to the input. Correlation values are shown. Supplemental Figure 3. Expression of human β-like globin genes in β-YAC transgenic mice. Relative mRNA expression of human ε-, γ- and β-globin ain E14.5 fetal liver erythroid cells from wild-type (YAC+; Bcl11a+/+) and mutant (YAC+; Bcl11a-/-) mice were measured by qRT-PCR. Transcript levels were normalized against mouse Gapd transcript levels and calculated as percentage of total human β-like globin expression. The same cells were used for chromatin conformation capture (3C) experiments as described in Figure 2. All results are means ± SEM. Supplemental Figure 4. Expression of cell surface markers during ex vivo erythroid differentiation. CD34+ human hemotopoietic progenitors at day 3 in expansion phase (shaded histogram) and ex vivo differentiated erythroid precursors at day 5 in differentiation phase (open histogram) were analyzed by flow cytometry for expression of CD34, CD45, CD71 (transferrin receptor), CD235 (glycophorin A), and CD36 antigens. Supplemental Figure 5. Expression of human β-like globin genes during ex vivo erythroid differentiation. Relative mRNA expression of human ε-, γ- and β- globin at various stages during ex vivo maturation was quantified by real-time RT-PCR. The percentage of β-globin (β/ε+γ+β) is indicated at the top of the diagram. Supplemental Figure 6. Expression of human BCL11A and SOX6 during erythroid differentiation. Human BCL11A and SOX6 mRNA levels were measured by real-time RT-PCR. Transcript levels were normalized against human GAPDH transcript levels. All results are means ± s.d. of at least three independent experiments. Supplemental Figure 7. Physical interaction between BCL11A and SOX6. FLAG- tagged SOX6 cDNA was coexpressed in COS7 cells with V5-tagged Vector, BCL11A, GATA1, and MTA2 cDNA, respectively. Nuclear extracts were immunoprecipitated using anti-FLAG antibody, and copurified proteins were analyzed by Western blot with anti-V5 antibody. Inputs (10%) are shown. Supplemental Figure 8. Chromatin occupancy of BCL11A, SOX6, and GATA1 at the human β-globin cluster. In vivo chromatin occupancy of BCL11A, SOX6, GATA1, and RNA polymerase II (Pol II) was examined by ChIP-qPCR in human erythroid progenitors. Normal rabbit IgG was used as a negative control. Precipitated DNA samples were quantified using primers designed to amplify discrete regions across the human β-globin locus. The ChIP signals are shown as a percentage of the input DNA signal and are representative of three independent experiments. The human β-globin locus is depicted at the top containing five β-like globin genes (ε, Gγ, Aγ, δ, and β), flanked by the upstream locus control regions (LCR) and the downstream hypersensitive site (3’HS1). Supplemental Figure 9. Morphology of ex vivo differentiated erythroid precursors. At day 5 of differentiation, the cells appear to be morphologically indistinguishable shown by May-Grunwald-Giemsa staining of cytospins. This is also the case at other stages of differentiation. Supplemental Figure 10. SOX6 contributes to silencing of HbF expression. HPLC analysis of hemolysates shows the presence of mature HbF. The HbF peaks are labeled with an arrow in each chromatogram, with the first peak corresponding to acetylated HbF (Garlick et al. 1981) and the second unmodified HbF. References Garlick, R.L., Shaeffer, J.R., Chapman, P.B., Kingston, R.E., Mazer, J.S., and Bunn, H.F. 1981. Synthesis of acetylated human fetal hemoglobin. J Biol Chem 256(4): 1727-1731. Liu, H., Ippolito, G.C., Wall, J.K., Niu, T., Probst, L., Lee, B.S., Pulford, K., Banham, A.H., Stockwin, L., Shaffer, A.L., Staudt, L.M., Das, C., Dyer, M.J., and Tucker, P.W. 2006. Functional studies of BCL11A: characterization of the conserved BCL11A-XL splice variant and its interaction with BCL6 in nuclear paraspeckles of germinal center B cells. Mol Cancer 5: 18. Liu, X.S., Brutlag, D.L., and Liu, J.S. 2002. An algorithm for finding protein-DNA binding sites with applications to chromatin-immunoprecipitation microarray experiments. Nat Biotechnol 20(8): 835-839. Sankaran, V.G., Menne, T.F., Xu, J., Akie, T.E., Lettre, G., Van Handel, B., Mikkola, H.K., Hirschhorn, J.N., Cantor, A.B., and Orkin, S.H. 2008. Human Fetal Hemoglobin Expression Is Regulated by the Developmental Stage-Specific Repressor BCL11A. Science 322(5909): 1839-1842. Supplemental Figure 1 A Exon 5 Exon 1 Exon 2 Exon XS Exon 3 Exon 4 33 aa (S) 18 aa 110 aa 14 aa 34 aa 673 aa 29 aa (L) 5’ UTR 1 2 3 4 5 6 Antibodies BCL11A.ab1 BCL11A.ab2 BCL11A.ab3 used for ChIP 1-171 aa 172-434 aa 775-835 aa Primers used XL all for RT-PCR L XL 5.9 kb, 835 aa 1 2 3 4 5 6 L 4.0 kb, 773 aa 1 2 3 S 2.4 kb, 243 aa 1 XS 1.5 kb, 142 aa ORF NuRD Interacting Domain C2H2 ZnF Proline-Rich C2HC ZnF NLS (631-637 aa) B C 1.2 4.0 BCL11A (XL) Bcl11a (XL) 1.0 BCL11A (L) Bcl11a (L) 3.0 0.8 0.6 2.0 0.4 Relative mRNA Relative mRNA 1.0 0.2 0 0 Human Pro-E MEL E14 FL Pro-E Supplemental Figure 2 A B BCL11A 108 sites 98 6 4 457 39 612 H3K27me3 H3K4me3 502 sites 655 sites C 2.0 r = 0.28 2.0 r = 0.48 2.0 r = 0.05 1.0 1.0 1.0 0.0 0.0 0.0 -1.0 -1.0 -1.0 H3K4me3 ChIP-chip signal H3K27me3 ChIP-chip signal H3K27me3 ChIP-chip signal -0.8 -0.4 0.0 0.4 -0.8 -0.4 0.0 0.4 -1.0 0.0 1.0 2.0 BCL11A.ab1 ChIP-chip signal BCL11A.ab1 ChIP-chip signal H3K4me3 ChIP-chip signal Supplemental Figure 3 100 + +/+ YAC ; Bcl11a -/- -like 80 YAC + ; Bcl11a β 60 40 20 globin expression % of total human 0 ε γ β Supplemental Figure 4 100 100 80 80 60 60 40 40 20 20 0 Counts Counts 0 0 1 2 3 4 10 10 10 10 10 100 101 102 103 104 CD34 CD45 100 100 80 80 60 60 40 40 20 20 0 Counts 0 Counts 0 1 2 3 4 10 10 10 10 10 100 101 102 103 104 CD71 CD235 100 80 60 40 20 Counts 0 100 101 102 103 104 CD36 Supplemental Figure 5 94.5 97.0 95.9 93.7 94.4 92.4 100 80 60 β /ε +γ +β γ /ε +γ +β 40 20 % of mRNA expression 0 0 3 5 7 9 12 Days in Differentiation Supplemental Figure 6 1 BCL11A 0.8 0.6 0.4 Relative mRNA 0.2 0 0 3 5 7 9 12 Days in Differentiation 0.1 SOX6 0.08 0.06 0.04 Relative mRNA 0.02 0 0 3 5 7 9 12 Days in Differentiation Supplemental Figure 7 Vector BCL11A GATA1 MTA2 WB 100 kDa α IP: FLAG 75 -V5 50 100 V5 Input 75 α-V5 50 FLAG Input α-FLAG (SOX6) Supplemental Figure 8 LCR 5 4 3 2 1 3’HS1 ε Gγ Aγ δ β 0.2 0.15 0.1 BCL11A 0.05 0 0.5 0.4 0.3 0.2 SOX6 0.1 0 0.3 0.2 GATA1 0.1 0 1 0.8 0.6 0.4 Pol II 0.2 Relative enrichment (% of input) 0 0.2 0.15 0.1 IgG 0.05 0 -25 0 +25 +50 +75 Position (Kb) Supplemental Figure 9 Control BCL11A shRNA SOX6 shRNA BCL11A+SOX6 Supplemental Figure 10 Control shRNA BCL11A shRNA SOX6 shRNA BCL11A+SOX6 9.2% HbF 35.6% HbF 19.6% HbF 53.8% HbF.
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