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 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 ),

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 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 . 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 -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 Figure7 IP: FLAG FLAG Input V5 Input 100 kDa 100 50 75 50 75

Vector BCL11A GATA1 MTA2 α α α -V5 -V5 -FLAG (SOX6) WB 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