Nature Immunology: doi: /ni.3694

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1 Supplementary Figure 1 Expression of Bhlhe41 and Bhlhe40 in B cell development and mature B cell subsets. (a) Scatter plot showing differential expression of genes between splenic B-1a cells and follicular (FO) B cells. RNA-seq data (Vilagos et al., J. Exp. Med. 209, ) are shown for known and predicted genes with regulatory functions. RPKM, reads per kilobase of exon per million mapped sequence reads. (b) Expression of Bhlhe41 (red) and Bhlhe40 (gray) across the Immgen Database. Several replicates are shown for each cell type. Fraction D (FrD), pre-b cells; fraction E (FrE), immature B cells. (c) Expression (RNA-seq) of Bhlhe41 and Bhlhe40 by the indicated precursor and B cell populations. TPM, transcripts per million. LMPP, lymphoid-primed multipotent progenitors; ALP, all-lymphoid progenitor; BLP, B-cell-biased lymphoid progenitor; pre-b(l), large pre-b cells; pre-b(s), small pre-b cells; PC, plasma cells; MZ B, marginal zone B cells; GC B, germinal center B cells; FL, fetal liver; PP, Peyer s patches; PerC, peritoneal cavity. (d,e) Cells from adult spleen (d), bone marrow (e) and peritoneal cavity (e) as well as neonatal liver (e, bottom row; day 1 after birth) of Bhlhe41-Cre-hCD2 transgenic and wild-type mice were stained with antibodies against human CD2 and markers defining the indicated cell populations. The flow cytometric definition of all cell types is described in the Online Methods. Histograms comparing hcd2 expression of the indicated cell types are shown. ΔMFI, difference in the median fluorescence intensity between Bhlhe41-Cre-hCD2 transgenic and wild-type cells of the indicated cell populations for the pair of wild-type and reporter mice shown in the plot. A representative result of two independent experiments is shown. (f) Expression (RNA-seq) of Bhlhe41 and Bhlhe40 in FO B cells stimulated with LPS for the indicated time (Wöhner et al., J. Exp. Med. 213, ). (g) hcd2 follicular B cells were sorted by flow cytometry from spleens of Bhlhe41-Cre-hCD2 transgenic and wild-type mice and then cultured in the presence of LPS, anti-igm plus IL-4 or anti-cd40 plus IL-4 for 2 or 4 days. One representative result of two independent experiments is shown.

2 Supplementary Figure 2 Bhlhe41 and Bhlhe40 are dispensable for B cell development. (a) Flow cytometric analysis showing CD19 and IgM expression (top) and Kit and CD2 expression (bottom) on CD19 + IgM cells from E18.5 fetal livers of wild-type (WT) and DKO embryos. One representative result of two independent experiments is shown. (b) Flow cytometric analysis showing IgM and IgD expression on CD19 + cells (top) and Kit and CD2 expression on CD19 + IgM IgD cells (bottom) from the bone marrow of mice with the indicated genotypes. One representative result of two independent experiments is shown. (c) Absolute numbers of the pro-b, pre-b, and immature B cells analyzed in (b). (d) Absolute numbers of peritoneal B-1b and B-2 cells (left) and splenic FO and MZ B cells (right) from adult mice of the indicated genotypes. The gating was preformed as shown in Fig. 1b,c. Horizontal bars indicate mean value, and error bars represent s.d. NS P > 0.05, * P < 0.05, ** P < 0.01 as determined by the Student s t-test. Five age-matched mice were analyzed per genotype. (e) Expression of CD5 and CD23 on peritoneal CD19 + B cells from wild-type and Bhlhe40 / mice. One representative result of two independent experiments is shown. (f) T, NK and IgM + B cell-depleted bone marrow cells from wild-type (WT) and DKO mice were mixed at a 1:1 ratio and injected into lethally irradiated Rag2 / recipients. Chimeras were analyzed weeks later, as described in the legend of Fig. 1f,g and Online Methods. Five chimeric mice were analyzed. Horizontal bars indicate mean value, and error bars represent s.d. NS P > 0.05, ** P < 0.01, *** P < as determined by the Student s t-test. One representative result of two independent experiments is shown.

3 Supplementary Figure 3 Analysis of B-1a cells and their progenitors in early ontogeny and adult mice. (a) RNA-seq datasets of the indicated wild-type (FO B, MZ B, and B-1a cells) and DKO B-1a cell populations from adult mice were examined by principal component analysis based on the 500 most differentially expressed genes among the wild-type B cell subsets. (b) Identification of B220 lo/ B-1-specified progenitors as described by Montecino-Rodriguez et al. (Nat. Immunol. 7, ). The gating strategy for identifying the Lin IgM CD93 + CD19 + B220 lo/ progenitors is shown for adult bone marrow and fetal liver (FL) cells (E15.5). Expression of the Bhlhe41-Cre-hCD2 reporter gene was analyzed in Lin IgM CD93 + CD19 + B220 lo/ progenitors of the E15.5 fetal liver (bottom row). (c) Cells with the B-1a phenotype and PtC-binding specificity emerge in the spleen at postnatal day 9. Cells from the indicated organs of wild-type neonatal and adult mice were stained with antibodies against CD19, B220, CD5 and V H12 as well as with PtC liposomes prior to flow cytometry.

4 Supplementary Figure 4 Analysis of B-1a cells from V H12- and V H12V 4-transgenic mice. (a) Peritoneal cells from mice of the indicated genotypes were stained with antibodies against V H12, IgM, CD5, CD23, CD19, and B220 and were analyzed by flow cytometry. All plots are gated on CD19 + cells. One representative result of four (V H12 transgenic) or at least seven (non-transgenic) independent experiments is shown. (b) Principal component analysis of RNA-seq data of the indicated wildtype populations (FO B, MZ B and polyclonal B-1a cells) and V H12/V 4 transgenic (Tg) B-1a cells based on the 500 most differentially expressed genes for the wild-type B cell subsets. (c) Bone marrow cells from mice of the indicated genotypes were stained with anti- IgM and anti-v H12 antibodies and analyzed by flow cytometry. One representative result of two independent experiments is shown.

5 Supplementary Figure 5 Generation and characterization of Bhlhe41 Tag/Tag mice. (a) Schematic domain structure (not drawn to scale) of the tagged Bhlhe41 protein that is expressed from the Bhlhe41 Tag allele. The tag sequences added at the last codon of Bhlhe41 contained epitopes for Flag and V5 antibodies, two cleavage sites for the TEV protease and a biotin acceptor sequence for biotinylation by the Escherichia coli biotin ligase BirA. (b) Frequency of peritoneal and splenic B-1a cells in wild-type and Bhlhe41 Tag/Tag mice. Three mice of each genotype were analyzed. Horizontal bars indicate mean value, and error bars represent s.d. (c) Gene set enrichment analysis (GSEA). The Bhlhe41/Bhlhe40-repressed genes identified in V H12/V 4 transgenic B-1a cells were compared to the ranked log2-fold gene expression changes between polyclonal DKO and wildtype B-1a cells (left). Likewise, the Bhlhe41/Bhlhe40-repressed genes identified in polyclonal B-1a cells were compared to the ranked log2-fold gene expression changes between V H12/V 4 transgenic DKO and wild-type B-1a cells (right). (d) The average density of open chromatin (determined by ATAC-seq) at all genomic Bhlhe41 peaks (left) or at the 117 Bhlhe41 peaks present at repressed target genes was assessed for a region extending from -1 kb to +1 kb relative to the Bhlhe41 peak summit.

6 Supplementary Figure 6 BCR signaling in V H12V 4-transgenic B-1a cells. (a) Intracellular Ca 2+ flux in V H12/V 4 transgenic wild-type and DKO splenic B-1a cells, as measured by the change in fluorescence emission of a Ca 2+ sensor dye after addition (arrow) of a goat anti-mouse IgM F(ab ) 2 fragment. (b) Scatter plot of gene expression differences between ex vivo sorted V H12/V 4 transgenic wild-type and DKO peritoneal B-1a cells. Genes encoding components of the BCR signaling pathway (GO: B cell receptor signaling pathway plus manually added genes) are highlighted in the left panel, while genes implicated in B-1a cell development and homeostasis as well as genes encoding the - and -chains of the IL-5, IL-3 and GM-CSF receptor family are indicated in the right panel. Genes that are discussed in the text are marked in red. RPM, reads per million mapped sequence reads. (c) Expression of the immunoglobulin -heavy chain (Ighm) mrna (RNA-seq; left) and the cellsurface IgM protein (right) is shown for peritoneal B-1a cells from V H12/V 4 transgenic wild-type and DKO mice. The Ighm mrna data were determined by two independent RNA-seq experiments per cell type, and error bars indicate s.e.m. * P < 0.05 as determined by the Student s t-test. TPM, transcripts per million. (d) Peritoneal cells from V H12/V 4 transgenic DKO and wild-type mice as well as peritoneal B-2 cells from wild-type mice were stained on ice for CD19, B220 and CD5, fixed, permeabilized and stained intracellularly (ic) for IgM and phosphorylated epitopes of the indicated signal transducers. Overlay contour plots displaying IgM expression and the phosphorylation status of the indicated BCR signaling components are shown for polyclonal B-2 cells (CD19 + CD5 B220 hi ) as well as for V H12/V 4 transgenic wild-type and DKO CD19 + B cells. One representative result of three independent experiments is shown. (e) Frequency of V H12 and V H12 + PtC liposome-binding cells (gated as shown in Fig. 6e) of peritoneal wild-type and Cd19 / IgM + CD5 + B-1a cells. Horizontal bars indicate mean value, and error bars represent s.d. NS P > 0.05, * P < 0.05 as determined by the Student s t-test. The data of eight wild-type and seven Cd19 / mice analyzed in three independent experiments are shown.

7 Supplementary Figure 7 Bhlhe41 represses genes encoding cell-cycle regulators. (a) Expression of genes encoding the indicated cell cycle regulators in V H12/V 4 transgenic and polyclonal wild-type and DKO B-1a cells, as determined by RNA-seq. The data are from two independent RNA-seq experiments per cell type, and error bars indicate s.e.m. TPM, transcripts per million. (b) Bhlhe41 binding (ChIP-seq) at the genes indicated in (a). RPM, reads per million mapped sequence reads.

8 Supplementary Figure 8 Bhlhe41 and Bhlhe40 regulate expression of the receptors for IL-5 and IL-3. (a) Expression of the Tnfrsf13b (TACI) and Tnfrsf13c (BAFF-R) genes in V H12/V 4 transgenic (top) and polyclonal (bottom) wild-type and DKO B-1a cells, as determined by two independent RNA-seq experiments per cell type. Error bars indicate s.e.m. TPM, transcripts per million. (b) Surface IL-5R and IL-3R expression on polyclonal B-1a cells of the indicated genotypes (top) as well as surface IL- 5R expression on V H12/V 4 transgenic B-1a cells (bottom left) and B-1a cells from mixed fetal liver (FL) chimeras (bottom right). One representative result of at least two independent experiments is shown.