advances.sciencemag.org/cgi/content/full/5/8/eaaw0706/DC1

Supplementary Materials for

Bcl11b prevents catastrophic autoimmunity by controlling multiple aspects of a

regulatory T cell gene expression program

Syed Nurul Hasan, Amit Sharma, Sayantani Ghosh, Sung-Wook Hong, Sinchita Roy-Chowdhuri, Sin-Hyeog Im, Keunsoo Kang, Dipayan Rudra*

*Corresponding author. Email: rudrad@ibs.re.kr

Published 7 August 2019, Sci. Adv. 5, eaaw0706 (2019)

DOI: 10.1126/sciadv.aaw0706

The PDF file includes:

Fig. S1. Treg-specific deletion of Bcl11b results in systemic autoimmunity comparable to that observed in mouse lacking functional Foxp3 expression. Fig. S2. Marked immune activation in KO mice. Fig. S3. Functional annotation and GSEA of Bcl11b-dependent up-regulated (Up) or down-regulated (Down) genes. Fig. S4. Motif analysis to identify potential transcription factor binding sites. Fig. S5. Characterization of genes harboring Lost or Gained Foxp3 peaks in Bcl11b-deficient Treg cells. Legends for tables S1 to S5

Other Supplementary Material for this manuscript includes the following: (available at advances.sciencemag.org/cgi/content/full/5/8/eaaw0706/DC1)

Table S1 (Microsoft Excel format). Data tables related to Fig. 3. Table S2 (Microsoft Excel format). Data tables related to Fig. 4 (A to E). Table S3 (Microsoft Excel format). Data tables related to Fig. 4F to I. Table S4 (Microsoft Excel format). Data tables related to Fig. 5. Table S5 (Microsoft Excel format). Data tables related to Fig. 6.

Fig. S1. Treg-specific deletion of Bcl11b results in systemic autoimmunity comparable to that

observed in mouse lacking functional Foxp3 expression. (A) Intracellular staining to confirm Treg

cell specific deletion of Bcl11b in Bcl11bf/f

Foxp3IRES-YFP-cre

mice. (B) Appearance of

Bcl11b+/+

Foxp3IRES-YFP-cre

(WT), Bcl11b in Bcl11bf/f

Foxp3IRES-YFP-cre

(KO) and Foxp3GFPKO

(harboring

GFP cDNA followed by stop codons knocked into Foxp3 locus) euthanized at ~1 month of age. (C)

Appearance of representative lymph nodes and spleens harvested from ~1 month old WT, KO and

Foxp3GFPKO

mice. (D) Representative hematoxylin and eosin (H+E) stained histological sections of

indicated organs of WT, KO and Foxp3GFPKO

mice. Original magnification ×100. Data is representative

of at least 3 independent experiments. (Photo Credits: Parts B and C, Dipayan Rudra, Academy of

Immunology and Microbiology, Institute for Basic Science; Part D, Sinchita Roy-Chowdhuri,

University of Texas MD Anderson Cancer Center).

Fig. S2. Marked immune activation in KO mice. (A) Quantification of expression of indicated

activation markers on CD4+Foxp3

- T-cells from lymph nodes and spleens of 3-5 weeks old WT (filled

red circles) and KO (filled black circles) mice. Each circle represents one animal. Data is from 2-3

independent experiments. (B) Representative FACS plots and quantification of CD44+CD62L

lo effector

Treg population in WT (red lines and filled circles) and KO (black lines and filled circles) mice. (C)

Quantification of expression of indicated activation markers on CD4+Foxp3

+ Treg cells from lymph

nodes and spleens of 3-5 weeks old WT and KO mice. Each circle represents one animal. Data is from

2-3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001 (Student’s t-test,

error bars, s.d.).

Fig. S3. Functional annotation and GSEA of Bcl11b-dependent up-regulated (Up) or down-

regulated (Down) genes. FACS plots demonstrating pre- and post-sort purity of Treg cells sorted from

WT and KO heterozygous female (het-KO and het-WT) mice, which were used to prepare libraries for

RNA-seq analysis. Data is representative of at least four identical experiments, for which 4-5 mice

were used per group in each sorting sessions. (B) Gene ontology (GO) term enrichment in the

“biological process” category of Bcl11b-dependent Up (right) or Down (left) genes. Top 12 GO terms

ranked according to the number of counts are plotted. Genes representing each category are listed in

Supplementary Table 1. (C, D) GSEA plots demonstrating negative correlation of Treg signature genes

with Bcl11b-dependent gene expression (genes activated in Treg cells compared to conventional T-

cells are Down (C), and genes repressed in Treg cells compared to conventional T-cells are Up (D) in

Bcl11b-deficient Treg cells compared to WT). Two independent GSEA data sets (left and right panels

that show similar results are shown. (E) GSEA analysis demonstrating “Foxp3-enriched” gene

expression clusters GAVIN_FOXP3_TARGET_CLUSTER_T4 and

GAVIN_FOXP3_TARGET_CLUSTER_P4 to be over-represented in Bcl11b-dependent Down genes.

Net enrichment scores (NES) and false discovery rate (FDR) q-values are shown for each plot.

Fig. S4. Motif analysis to identify potential transcription factor binding sites. Transcription factor

binding motif enrichment within 100 bp upstream or downstream of Bcl11b peaks corresponding to

genes exclusively harbouring Tn/Tr-common sites (A) or Tr-specific sites (B). P-values are indicated in

red. Sites with P value less than 1e-4 are not included.

Fig. S5. Characterization of genes harboring Lost or Gained Foxp3 peaks in Bcl11b-deficient Treg

cells. Additional examples of IGV tracks of Foxp3 ChIP-seq (blue) and corresponding ATAC-seq (red

and black) peaks of the indicated categories in WT and KO Treg cells. Grey arrows indicate ATAC-seq

peaks altered in KO Treg cells compared to WT. (B) Gene ontology analyses of “biological process”

category for genes which gained Foxp3 peaks, as well were 1.5 folds upregulated in Bcl11b-deficient

Treg cells. (C) Representative Foxp3 bound peaks and corresponding ATAC-seq peaks (grey arrows)

‘Gained’ in Bcl11b-deficient Treg cells, the corresponding genes for which are downregulated

compared to WT.

Supplementary table legends:

Table S1. Data tables related to Fig. 3.

Spreadsheet 1: List of Bcl11b-dependent downregulated (Down) genes determined by DGE

analysis of RNA-seq data comparing het-KO with het-WT.

Spreadsheet 2: List of Bcl11b-dependent upregulated (Up) genes determined by DGE analysis of

RNA-seq data comparing het-KO with het-WT.

Spreadsheet 3: List of genes corresponding to top 12 GEOTERMs of Gene Ontology analysis of

“Biological Process” category of Down genes, arranged according to number of counts. Activated

Treg signature genes (Tn/Tr 1.5 × down) or Foxp3-dependent genes (Tfn/Tr 1.5 × down) are

highlighted in blue.

Spreadsheet 4: List of genes corresponding to top 12 GEOTERMs of Gene Ontology analysis of

“Biological Process” category of Up genes, arranged according to number of counts. Repressed

Treg signature genes (Tn/Tr 1.5 × up) or Foxp3-dependent genes (Tfn/Tr 1.5 × up) are highlighted

in red.

Spreadsheet 5: Tabular data for cumulative distribution fraction (CDF) curve plotted in Fig. 3E.

Spreadsheet 6: Tabular data for CDF curve plotted in Fig. 3F.

Table S2. Data tables related to Fig. 4 (A to E).

Spreadsheet 1: List of Bcl11b-dependent Up or Down genes identified by DGE analysis that are

bound by Bcl11b within ±20kb of their transcription start sites (TSS).

Spreadsheet 2: List of Bcl11b associated peaks that are common in naïve T and Treg cells.

Spreadsheet 3: List of Bcl11b associated peaks that are specific for Treg cells.

Spreadsheet 4: Tabular data for CDF curve plotted in Fig. 4E.

Table S3. Data tables related to Fig. 4F to I.

Spreadsheet 1: List of Bcl11b and Foxp3 overlapping peaks in Treg cells.

Spreadsheet 2: List of Bcl11b-specific peaks in Treg cells.

Spreadsheet 3: Tabular data for CDF curve plotted in Fig. 4I.

Table S4. Data tables related to Fig. 5.

Spreadsheet 1: List of Bcl11b bound sites lost in Foxp3null

(Tfn) cells derived from Foxp3GFPKO

mice.

Spreadsheet 2: Tabular data for CDF curve plotted in Fig. 5D.

Table S5. Data tables related to Fig. 6.

Spreadsheet 1: List of Foxp3 peaks lost in Bcl11b-deficient Treg cells.

Spreadsheet 2: List of Foxp3 peaks gained in Bcl11b-deficient Treg cells.

Spreadsheet 3: Tabular data for CDF curves plotted in Fig. 6D and 6F.

Spreadsheet 4: Gene Ontology analysis of 1.5-folds upregulated genes associated with Foxp3-

bound peaks gained in Bcl11b-KO Treg cells. Genes corresponding to top 12 GEOTERMs of

“Biological Process” category are listed.

Spreadsheet 5: List of ATAC-seq peaks lost in Bcl11b-deficient Treg cells.

Spreadsheet 6: List of ATAC-seq peaks gained in Bcl11b-deficient Treg cells.