Supplemental Materials

Three-step transcriptional priming that drives the commitment of B cell progenitors toward B cells.

Tomohiro Miyai, Junichiro Takano, Takaho A. Endo, Eiryo Kawakami, Yasutoshi Agata, Yasutaka Motomura, Masato Kubo, Yukie Kashima, Yutaka Suzuki, Hiroshi Kawamoto, and Tomokatsu Ikawa*

*Correspondence author. E-mail: [email protected]

1 Supplemental Materials & Methods

Antibodies, growth factors and flow cytometry. The following antibodies were purchased from BD Pharmingen or BD Horizon: c- (2B8), Sca-1 (D7), Flk2

(A2F10.1), CD34 (RAM34), IL-7Ra (SB/199), CD24 (M1/69), BP-1 (6C3), CD43 (S7), erythroid lineage cells (TER119), Mac-1 (M1/70), Gr-1 (RB6-8C5), CD11c (HL3),

B220 (RA3-6B2), CD8 (53-6.7), CD4 (H129.19), NK1.1 (PK136), CD3ε (145-2C11), and CD19 (1D3). IgM (II/41), IgD (11-26C) and human CD25 (M-A251). TER119,

Mac-1, Gr-1, B220, CD19, NK1.1, CD3ε, CD4, and CD8 were used as Lin markers.

For single cell RNA-seq analysis, CD11c, CD25, IgM, IgD antibodies were also added to Lin markers whereas CD4 was excluded. Recombinant murine IL-3, IL-7, stem cell factor (SCF), Flt-3 ligand (Flt-3L), thrombopoietin (Tpo) were purchased from R&D.

6-10 wk old mice were used for all experiments. Mice were sacrificed and single cell suspensions of BM, spleen and thymus were prepared. Cell number was counted, then

~1 × 107 cells were stained with combination of antibodies in FACS buffer (1× PBS

with 2% FCS and 0.05% NaN3). Cellularity is assessed with FACSAria II or

FACSCanto II (Beckton Dickinson).

Isolation of hematopoietic progenitors and transduction of retrovirus. Single cell suspensions of FL cells from 14 dpc or BM cells of B6.CD45.1 mice were prepared as described previously(Ikawa et al. 2004). Cells were then incubated with purified monoclonal antibodies specific for lineage markers (Lin: TER119, Mac-1, Gr-1, B220

2 and Thy1.2) for 20 min on ice. Lin+ cells were depleted with Dynabeads Sheep anti-Rat

IgG (Invitrogen) according to the manufacturer’s protocol. LSK cells or HSCs from FL and adult BM were isolated by FACS Aria II. Retroviral vector pMCs-IRES-GFP containing human ID3 cDNA fused with modified human estrogen ERT2

(pMCs-hId3-ERT2-IG) were generated as previously described. shRNA against various targets were prepared by Open Biosystems LMP system (MSCV-LTRmiR30-PIG,

Thermo Scientific). The vector of the coding sequence of GFP was replaced by human

CD25 (a gift from Dr. C. Murre). Vector construction of specific targets was performed according to the manufacturer’s protocols, and target sequences are shown in

Table S1. Retrovirus was generated by transfection of pMCs-hId3-ERT2-IG or shRNA construct plasmid together with pantropic envelope VSV-G plasmid into Plat E packaging cell line using FuGENE 6 Transfection Reagent (Promega). Viral supernatant was collected at day 2 and 3 after transfection.

PCR analysis for genomic rearrangement of IgH . Genomic DNA is extracted by DNeasy Blood and Tissue Kit (Qiagen). PCR is performed with KAPATaq Extra

HotStart ReadyMix with dye (KAPA Biosystems). Primer sequences are shown in

Supplementary Table 2.

ChIP and ChIP-seq. Detailed ChIP protocol was described elsewhere(Ikawa et al.

2006). Cells were fixed with 1% formaldehyde solution for 10 (for methylated histone) or 20 (for TFs) minutes at 25°C. Soluble was immunoprecipitated with

3 normal rabbit IgG (as negative control, Santa Cruz Biotechnology, #sc-2027), anti-H3K4me3 (Millipore, #07-473), anti-H3K27me3 (Millipore, #07-449), anti-H3K9me3 (Medical & Biological Laboratories, #MABI0308), anti-E2A (Santa

Cruz Biotechnology, #sc-349X). For quantification, precipitated and input DNA were purified with MinElute PCR Purification Kit (Qiagen) and performed quantitative PCR with SYBR Premix Ex Taq (TaKaRa). Primer sequences are shown in Table S2. For sequencing, purified DNA were fragmented with DNA shearing system M220 (Covaris) then libraries were prepared with NEBNext Ultra DNA Library Prep Kit for Illumina

(New England Biolabs). Procedures for quality check of libraries and sequencing were corresponded to RNA-seq.

Data analysis. Sequences obtained in RNA-seq experiments were aligned on mouse genome (NCBI build 37, mm9) using tophat2 (version 2.1.0)(Kim et al. 2013) and assigned to mouse using cufflinks (version 2.1.1)(Trapnell et al. 2010) with gene annotation provided by iGenomes. Fold changes and p-values of in B cell progenitors of Eµ-Nfil3 and WT mice was performed using cuffdiff (version

2.1.1)(Trapnell et al. 2013). Expression similarity among experimental replicates was estimated using Pearson correlation of RPKM values of all genes. In the PCA plot, we used 4290 representative genes that had different expression (difference of maximum and minimum FPKM values is more than twice) among time points with less than 0.01 q-value using ANOVA. To extract the TF (E2A, EBF1 and PAX5) target genes, previously deposited ChIP-seq records (Accession numbers of Gene Expression

4 Omnibus are as follows; GSM546523 for E2A (Lin et al. 2010), SRX018714 for EBF1

(Treiber et al. 2010) and SRX148793 for PAX5 (Revilla-i-Domingo et al. 2012)) were used. Alignment on mouse genome (mm9) was performed with bowtie2-2.2.2

(Langmead and Salzberg 2012). Peaks were called with MACS2 (Zhang et al. 2008) following peak annotation with ChIPseeker package (Yu et al. 2015). Based on the obtained gene sets of respective TF targets, temporal expression signatures of TF target or non-target genes were calculated among highly variable genes during time-course.

ChIP-Seq reads were aligned on mouse genome (mm9) using bowtie 2.1.0 and compared with gene locations Mapping to reference genome is performed with

Integrative Genomics Viewer (https://www.broadinstitute.org/igv). To see epigenetic changes of differently expressed genes before and after differentiation, we evaluated expression changes between 0 hr and 144 hr samples using DESeq2 package(Love et al.

2014) and collected significant genes (q-value < 0.01) expressed more than 2 times (410 genes) or less than 0.5 times (304 genes). RPKM around TSS (-5kb to +2kb, 100bp window size) was subtracted between two time points and represented.

PCA, heat maps, correlation plots, k-means clustering, GO analysis, ChIP-seq subtraction diagrams were analyzed using in-house computer programs developed by

Takaho A Endo, RIKEN IMS.

Statistics. All experiments were performed at least 2 times. The two-tailed Student’s t-test was used to analyze differences between two groups.

5 References for Supplemental Materials & Methods

Ikawa T, Kawamoto H, Goldrath AW, Murre C. 2006. E and Notch signaling

cooperate to promote T cell lineage specification and commitment. J Exp Med 203:

1329–1342.

Ikawa T, Kawamoto H, Wright LYT, Murre C. 2004. Long-Term Cultured

E2A-Deficient Hematopoietic Progenitor Cells Are Pluripotent. Immunity 20: 349–

360.

Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. 2013. TopHat2:

accurate alignment of transcriptomes in the presence of insertions, deletions and

gene fusions. Genome Biol 14: R36.

Langmead B, Salzberg SL. 2012. Fast gapped-read alignment with Bowtie 2. Nat

Methods 9: 357–359.

Lin YC, Jhunjhunwala S, Benner C, Heinz S, Welinder E, Mansson R, Sigvardsson M,

Hagman J, Espinoza CA, Dutkowski J, et al. 2010. A global network of

transcription factors, involving E2A, EBF1 and Foxo1, that orchestrates B cell fate.

Nat Immunol 11: 635–643.

Love MI, Huber W, Anders S. 2014. Moderated estimation of fold change and

dispersion for RNA-seq data with DESeq2. Genome Biol 15: 550.

Revilla-i-Domingo R, Bilic I, Vilagos B, Tagoh H, Ebert A, Tamir IM, Smeenk L,

Trupke J, Sommer A, Jaritz M, et al. 2012. The B-cell identity factor Pax5

regulates distinct transcriptional programmes in early and late B lymphopoiesis.

6 EMBO J 31: 3130–3146.

Trapnell C, Hendrickson DG, Sauvageau M, Goff L, Rinn JL, Pachter L. 2013.

Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat

Biotechnol 31: 46–53.

Trapnell C, Williams B a, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL,

Wold BJ, Pachter L. 2010. Transcript assembly and quantification by RNA-Seq

reveals unannotated transcripts and isoform switching during cell differentiation.

Nat Biotechnol 28: 511–515.

Treiber T, Mandel EM, Pott S, Györy I, Firner S, Liu ET, Grosschedl R. 2010. Early B

cell factor 1 regulates B cell gene networks by activation, repression, and

transcription- independent poising of chromatin. Immunity 32: 714–725.

Yu G, Wang LG, He QY. 2015. ChIP seeker: An R/Bioconductor package for ChIP

peak annotation, comparison and visualization. Bioinformatics 31: 2382–2383.

Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE, Nussbaum C,

Myers RM, Brown M, Li W, et al. 2008. Model-based Analysis of ChIP-Seq

(MACS). Genome Biol 9: R137.

7 Supplemental Figures� A� B�

Expansion of iLS cells� ExpansionExpansion of HSCs of HSCs� Expansion of iLS cells 1.55 47.9 34.3 58 51.6 19.6 4-OHT�

Cytokines: � � IL-7, SCF, SCF, Flt3L, Flt3-L,Tpo Tpo � Cytokines: � IL-7,IL-7, SCF, SCF, Flt-3 ligandFlt3-L� c-Kit c-Kit CD34

SortSorting GFP+ GFP+ cellscells� 2.8 47.8 5.25 2.54 21.2 7.57 LSK cells TSt-4TSt-4 stromal stromal cells cells� LSK cells� RetroviralRetroviral transfection transduction of of pMCS-hId3-ER IRES-GFP Sca-1� Flt-3� IL-7Rα� pMCS-hId3-ERT2 IRES-GFP� C� D� E� F� 14 � ) 100 6 � 12 IL-3IL-3� Remove 4-OHT 4-OHT� IL-7IL-7� Remove 4-OHT 4-OHT� 80 10

60 8 系列Ctrl�1 iLSiLS cellscells� TSt-4� iLSiLS cellscells � TSt-4/DLL1� 6 40 系列Id3-ER2 T2� 4 2.73 0.0217 41 2.26 1.01 0.0208 21.5 3.51 � � 20 2 Foldchange Lin-of cells10 (x % of Lin-%of cells in totalcells CD19 0 0 NK1.1 0 10 20 30 1 96.8 0.401 1.47 55.3 98.7 0.229 60.4 14.5 Days� Mac-1� CD3e�

3

G� L� M� � )

Day 0 / Day 6 5 i.v.� 2 �

SSC 1 Cellnumber (x10 FSC� H� I� 0 � � � � CD4 CD19 NK1.1 NK1.1

GFP� CD3e� Mac-1� GFP� CD8a� CD3e� J� K� � � � � IgD CD19 CD4 CD19

� GFP� IgM� IgM� CD8a� GFP� Mac-1

Figure S1. Establishment and characterization of iLS cells. (A) Scheme for establishment of iLS cells. (B) Flow cytometric profiles of Id3-ERT2 transduced cells. (C) Proportion of lineage negative (Lin-: CD19-Mac-1-NK1.1-) populations of LSK(control) or Id3-ERT2 transduced cells. Values represents mean ± SD in a representative of two independent experiments. (Continue to next page) (D) Number of Lin- cells after 1 month culture in LSK or Id3-ERT2 transduced cells. Values represents mean ± SD in a representative of two independent experiments. (E) Myeloid lineage potential of iLS cells in vitro. iLS cells were cultured in the presence of 10 ng/ml IL-3 on TSt-4 stromal cells for 10 days (upper). FACS profiles before (lower, left) and after (lower, right) the culture are shown. (F) T and NK lineage potential of iLS cells in vitro. iLS cells were cultured in the presence of 5 ng/ml IL-7 on TSt-4/DLL1 stromal cells for 3 wks (upper). FACS profiles of before (lower, left) and after (lower, right) the culture are shown. (G-K) Hematopoietic reconstitution potential of iLS cells. iLS cells were transferred into γ-ray-irradiated NOG mice, then primary and secondary lymphoid organs (BM: H and K, thymus: I and spleen: J) were analyzed at 4 (H,I) or 6 (J,K) wks after transfer. (L) Forward Scatter (FSC) vs Side Scatter (SSC) profiles of iLS cells before (day 0) and after (day 6) the culture for B cell induction. (M) Comparison of cell proliferation between iLS cells and iLS-derived B cells. 1 x 104 of iLS cells and iLS derived-B cells (at day 6 of the culture) were harvested and further cultured for 3 days. The numbers of GFP+ cells were analyzed by FACS. � A Fold enrichment� B 0 1 2 3 4 5 6 7 8 � � 0 Ebf1 p<0.001 indicated loci. Values represent means in ±representative SD ofthree independent experiments. **p<0.01, *** means in ±representative SD oftwoindependent experiments. ( Figure S2. *** mb-1 Relative mRNA expression� 0.6 0.2 0.4 0.8 1.2 1.4 0.5 1.5 2.5 � 2 � 0 1 2 3 0 1 ), Igll1 0 1 2 3 4 5 6 7 8

0 0 (C) rearrangementVDJ in Cd19 Flt3 Induction of 1 1 0 Pax5 (l5) forBlineage-associated genes, and *** 2 2 2 � 4 4 � 6 0 1 2 3 4 5 6 iLS 0 0.2 0.3 0.4 0.5 0.1 Igll1 (days) 0.05 0.15 0.25 0.35 0.45 ** 0.1 0.2 0.3 0.4 0.5 0 cells into Blineage cells. 0 � 2 � 0 0 � Rag1 Ikzf1 12 10 1 2 4 6 8 0 1 Igh 2 Vpreb1 2 0 locus during Bcell differentiation. Acta1 asa loading isshown control. *** 4 4 2 6 6 � � 20 40 60 0 iLS 0.1 0.2 0.3 0.4 0.6 0.2 0.4 0.8 1.2 1.4 0 0 1 0 IgHie time course (days) time course *** Flt3

0 0 (A) (A) 2 � Tcf3 Igll1 1 1 � and qRT 2 2 V C V V H -PCR analysis-PCR of ( TFs Cd34 4 4 H H (B) H558-DJ 7183-DJ � Q52-DJ V 6 6 κ Acta1 ChIP-qPCR D forstem cell-associated genes. Values represent -J H κ -J 0.6 0.5 1.5 2.5 0.2 0.4 0.8 1.2 2 H H � H H 0 1 2 3 0 1 � � � � � 0 0

Ladder� Vpreb1 0 Ebf1 1 1 analysis forE2A occupancy at the � � iLS 1 2 2 � time course (days) time course 2 4 4 Tcf3 � 3 6 6 � , Ebf1 4 � 0.6 0.1 0.2 0.3 0.4 0.5 5 0 � , Pax5 6 � 0 7 Pax5 � 1 ), 10 � Cd79a 2 � +� BM CD19 4 6 NTC�

- DJ2 Ladder - VJ1 - DJ3 - DJ1 - VJ2 - VDJ3 - VDJ2 - VDJ1 - VDJ3 - VDJ2 - VDJ1 - VDJ3 - VDJ2 - VDJ1 � � � � � � � � � � � � � � � Cluster # GO term ClusterRepresentative # genesGO term -log (p-value) -log (p-value) 0 10 15 0 10 15

B cell receptor signaling pathway Flt3 cell adhesion I B cell homeostasis Cd34VI cell migration intracellular signal transduction Spi1 negative regulation of protein phosphorylation leukocyte cell-cell adhesion Dnmt3a apoptotic process chromatin modification hippo signaling integrin-mediated signaling pathway Runx2 collagen fibril organization regulation of catalytic activity positive regulation of peptidase activity negative regulation of endocytosis peptidyl-tyrosine phosphorylation

cholesterol biosynthetic process Jmjd1c response to virus II sterol biosynthetic process Nr4a1VII innate immune response steroid metabolic process Jun defense response to virus cholesterol metabolic process Nfil3 cellular response to interferon-beta steroid biosynthetic process Egr1 negative regulation of cell proliferation positive regulation of apoptotic process positive regulation of fat cell differentiation transcription from RNA polymerase II positive regulation of epithelial cell proliferation regulation of transcription, DNA-templated cell cycle transforming growth factor beta receptor Tgfb2VIII III signaling pathway cell division negative regulation of cell proliferation Nr1d1 mitotic nuclear division negative regulation of transcription from RNA polymerase II promoter Cebpb segregation protein phosphorylation Srf positive regulation of cell cycle cell cycle inflammatory response Smad3IX Fosl2 cellular response to DNA damage stimulus actin cytoskeleton organization DNA repair positive regulation of smooth muscle contraction cell division DNA replication positive regulation of cell migration Wnt5a Hmga2 Foxd1 chromatin modification IV angiogenesis Wnt10b Ctgf Tead1 B cell proliferation focal adhesion assembly

regulation of Rho protein signal transduction Irs1 Cd9 E2f7 ClusterCluster # # GO GOterm term ClusterVCluster # # GO GOterm term Fzd2 Gli3cellular response to DNA damage stimulus -log (p-value) positive regulation of B cell proliferation X Cdk1 cell cycle 10-log10(p-value) -log10-log(p-value)10(p-value) 0 0 15 15 0 0 15 15 DNA repair B cell receptorB cell receptor signaling signaling pathway pathway Igf1r somatic hypermutation of I cell adhesioncell adhesion immunoglobulin genes I B cell homeostasisB cell homeostasis VI VI cell migrationcell migration Bdnf intracellularintracellular signal transduction signal transduction negativenegative regulation regulation of protein of phosphorylationprotein phosphorylation Twist1 leukocyteleukocyte cell-cell cell-celladhesion adhesion apoptoticapoptotic process process Sema3a chromatinchromatin modification modification hippo signalinghippo signaling integrin-mediatedintegrin-mediated signaling signaling pathway pathway collagencollagen fibril organization fibril organization Ddit3 regulationregulation of catalytic of catalytic activity activity positive positiveregulation regulation of peptidase of peptidase activity activity Cdk14 negativenegative regulation regulation of of endocytosisendocytosis peptidyl-tyrosinepeptidyl-tyrosine phosphorylation phosphorylation cholesterolcholesterol biosynthetic biosynthetic process process responseresponse to virus to virus Bmp4 II II sterol biosyntheticsterol biosynthetic process process VIIVII innate immuneinnate immune response response Fgfr2 steroid metabolicsteroid metabolic process process defensedefense response response to virus to virus Fzd1 cholesterolcholesterol metabolic metabolic process process cellular responsecellular response to interferon-beta to interferon-beta Igf1 steroid biosyntheticsteroid biosynthetic process process negativenegative regulation regulation of cell proliferation of cell proliferation positive positiveregulation regulation of apoptotic of apoptotic process process positive positiveregulation regulation of of Vdr fat cell differentiation fat cell differentiation transcriptiontranscription from RNA from polymerase RNA polymerase II promoter II promoter positive positiveregulation regulation of of Id3 epithelial epithelial cell proliferation cell proliferation regulationregulation of transcription, of transcription, DNA-templated DNA-templated cell cyclecell cycle Cd24a Foxo1 transformingtransforming growth factorgrowth beta factor receptor beta receptor VIIIVIII Ly6a Pcgf2 III III signaling signaling pathway pathway cell divisioncell division negativenegative regulation regulation of cell proliferation of cell proliferation mitotic nuclearmitotic nucleardivision division Ccnf Ccna2 negativenegative regulation regulation of transcription of transcription from from RNA polymerase RNA polymerase II promoter II promoter chromosomechromosome segregation segregation protein phosphorylationprotein phosphorylation positive positiveregulation regulation of cell cycle of cell cycle cell cyclecell cycle Igll1, Cdca3 inflammatory response inflammatory response IX IXcellular responsecellular response to DNA damageto DNA damage stimulus stimulus Vpreb1 Cdca8 actin cytoskeleton organization actin cytoskeleton organization DNA repairDNA repair Ebf1 positive positiveregulation regulation of of smooth smoothmuscle contractionmuscle contraction cell divisioncell division Bach2 DNA replicationDNA replication positive positiveregulation regulation of cell migration of cell migration chromatinchromatin modification modification Dnmt1 IV IV angiogenesisangiogenesis B cell proliferationB cell proliferation Uhrf1 focal adhesionfocal adhesion assembly assembly regulationregulation of Rho protein of Rho signalprotein transduction signal transduction Notch1 Hhex V V cellular responsecellular response to DNA damageto DNA damage stimulus stimulus positive positiveregulation regulation of B cell of proliferation B cell proliferation X X cell cyclecell cycle Id2 Sox4 DNA repairDNA repair Kit Zfpm1 somatic somatichypermutation hypermutation of of immunoglobulin immunoglobulin genes genes Myb

Figure S3. Characteristic gene ontologies of gene clusters in Figure 1F. Statistical significances of gene enrichment are indicated as the bar graphs of their p-values.� A� D� Downregulated genes Upregulated genes

H3K4me3 H3K27me3 H3K4me3 H3K27me3 0 0.5 1 2 4 8 10 12 36 48 72 96 144

0 0.5 1 2 4 8 10 12 36 48 72 96 144

0.8 1.0 Correlation -5 0 2 -5 0 2 B� C� Distance to TSS (kb)

-5 0 2 -5 0 2 -1 0 1 RPKM change Distance to TSS (kb)

Figure S4. Histone modification during B cell lineage commitment. (A-C) ChIP-sequencing analysis using H3K4me3 ( blue), H3K9me3 (dark blue) and H3K27me3 (green) –specific antibodies of iLS cells differentiating into B cells. Loci of B cell-associated genes: Ebf1, Pax5, Igll1 and Vpreb1 (A), stem cell-associated genes: Flt3 and CD34 (B) and other lineage-associated genes: Csf2ra and Epor (C) are shown, respectively. (D) Alterations of H3K4me3 and H3K27me3 occupancy around TSS of down-regulated and up-regulated (over 2-fold difference with high significance (q < 0.01) between day 0 and day 6) gene loci in iLS cells differentiating into B cells. � A� Time �

B� Suz12� E2A�

Jun� Klf4�

Tead1� Gata2� Egr1� Nfil3� Aiolos� Irf4� Pax5�

Bach2� Ebf1� Ezh2�

Figure S5. Direct connection of TFs in the TF network during B cell commitment. (A) Gene expression profiles of 115 TFs (33 constant, 27 early, 23 mid and 32 late) which comprise the transcriptional network. Time phases are parallel to Figure 3C. Each line represents the expression pattern of the factor shown in the node of network (B). Details are shown in Supplementary Table S4. (B) Inter-regulation between time phase-specific TFs. Frequently associated interactions (detected in over 70% of ChIP-seq datasets) are represented as an edge. Regulatory connections of individual genes are listed in Supplementary Table S5. A� B� C� Single cells Single cells (d0) (d8) � 8 days RNA-seq induction tSNE2 iLS cells B cell progenitors (multipotent) (committed)

tSNE1� D�

E� Ebf1� Vpreb1� Cd79b� Igll1� � tSNE2

tSNE1� tSNE1� tSNE1� tSNE1�

F Cd34� Mycn� Sfpi1� Nfil3� � tSNE2

tSNE1� tSNE1� tSNE1� tSNE1�

Figure S6. Digital single cell RNA-sequencing analysis during B cell commitment. (A) Scheme for single cell RNA-seq analysis using iLS cells. (B) Distribution of number of UMI counts (left) and genes (right) detected per about 3000 iLS cells. (C) t-SNE projection of multipotent (d0) and B-committed (d8) progenitors isolated from iLS cells. (D) Hierarchical clustering of gene expression data. The representative genes enriched in each cluster are displayed in the heat map,

showing normalized gene expression on a log2 scale from white to red (low to high). (E-F) tSNE projection of iLS cells, with each cell colored based on their normalized expression of Ebf1, Vpreb1, Cd79a, Igll1, Cd34, Mycn, Sfpi1 and Nfil3. � A� B�

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batch labels

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● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ●● ● ● ● ● ● ●● ● ● ●● ●● ●● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●●● ● ● ● ● ● ●●● ●● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●●● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ●● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ID ● ● ● ● ● ● ● ●● ● ● ● ● ● ● 19.5 ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ●● ●● ● ●● ● ● ●● ● ● ● ● ● 89.5 ● ● ● ● ● ● ● ● ●● ● ● ● ● ●●● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 1 ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●●● ●● ●● ● ● ● ● ● ● 0 ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● 2 ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ●● ● ● ●● ●● ●● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● TSNE.2 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 3 ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ●● ● ● ●● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ●●● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●●● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● 99.5 ● ● ● ● ● ●● ● ●● ● ●● ●● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ●● ●● ● ●● ● ●● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● −20 ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● 34.9 ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ●● ● ●● ● ● ●● ● ● ●● ● ● proB ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● LMPP ●●● ● ● ●●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● (1123 cells)� ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● (599 cells) ● ● � ● ● ● ●

−20 0 20 40 TSNE.1 Single cell RNA-seq

Figure S7. Single cell RNA sequencing analysis using BM-derived B cell progenitors. (A) Gating strategies for isolation of LMPP, CLP and pro-B cells. Lineage antibody cocktail contains anti-CD3e, CD8, CD11b, CD11c, CD25, IgM, IgD, TER119, NK1.1 and Gr-1 antibodies. (B) Distribution of number of UMI counts (left) and genes (right) in each cell types. (C) t-SNE projection of LMPP, CLP and pro-B cells.� withdrawal of:� 4-OHT A� B� 0.8 Nfil3� EtOH 0.4 Junb� Time (hr) Klf4� � 0.6 0.3 0.1 0 0.5 1 2 4 6 8 10 12 24 48 72 96 120 144 168 0.4 0.2 Mxd1 Klf11 expression Relative mRNA RelativemRNA 0.2 0.1 Nfil3 Mnt 0 0 0 Csrnp1 0 0.5 1 2 4 0 0.5 1 2 4 0 0.5 1 2 4 Time (h) Jund Bach1 Junb Nr4a1 Klf4 Egr1 C� Junb CLP2 Nr4a1 Klf4 Egr1 Klf7 17.8� 2.6� 13.9� 44.9� Rybp CLP1 14.0� 1.8� 12.6� 20 pro-B1val 20 val 20 val 20 val Rel 1.00 0.2� 1.00 1.5� 1.00 0.3� 1.00 43.3� 5.9� Zfp281 0.75 0.75 0.75 0.75 0 0 0 0 Mafk 0.50 0.50 0.50 0.50 TSNE.2 TSNE.2 TSNE.2 TSNE.2 0.25 0.25 0.25 0.25 Egr2 TSNE.2 −20 −20 −20 −20 Atf3 0.00 0.00 0.00 0.00 pro-B2 20.4� 1.7� 21.1� Nr4a2 LMPP 0.4� 3.4� 2.6� Nr4a1 54.3� −20 0 17.420 40� −20 0 20 40 −20 0 20 40 −20 0 20 40 TSNE.1 TSNE.1 TSNE.1 TSNE.1 Egr1 TSNE.1 Klf4 D� � 1.2 1.2 1.2 Klf6 * 1 Junb 1 1 Klf2 0.8 * 0.8 0.8 * Jun 0.6 0.6 0.6 0.4 mRNA expression mRNA 0.4 0.4

Min Max 0.2 0.2 0.2 0 0 0 Control shNr4a2 Control shKlf4 Control shEgr1 Control shNr4a2 shKlf4 shEgr1 E� F� 6 ) 91 76 93 63 5 5

4

hCD25 3 *

54 26 18 18 2 ** ** B cellB number (x10

CD19 1

0 Mac-1 Control Nr4a2 Klf4 Egr1 )

5 1.2 G� Control shEgr1 shKlf4 shPax5 H� 1 32 13 11 10 0.8 ** * 45 68 60 60 0.6 ***

CD19 0.4 0.2

Mac-1 cellB number (x10 0 Control Egr1 Klf4 Pax5

Figure S8. Early responding TFs are essential for B cell differentiation. (A) Gene expression profiles of transcription factors among cluster II (1st wave) genes in Figure 1F. (B) qRT-PCR analysis of indicated genes in Id3-transduced progenitors after withdrawal of 4-OHT or EtOH (as vehicle control) at an early time point. Values represent mean ± SD in representative of two independent experiments. (C) tSNE projection of LMPP, CLP and pro-B cells in BM, with each cell colored based on their normalized expression of Junb, Nr4a1, Egr1 and Klf4. The numbers indicate the frequency of expressing cells in each cluster. (D-H) shRNA-mediated KD of indicated genes using iLS cells (D-F) or BM LSK cells (G, H). KD efficiency (D), flow cytometric profiles (E, G) and B cell numbers (F, H) are shown. shRNA-transduced cells (hCD25+) are gated for Mac-1 vs CD19 profiles. Values represent mean ± SD in three independent experiments. * p<0.05, ** p<0.01, *** p<0.001. compared to control. � (Ter119, Mac-1, Gr-1, Thy1, B220 depleted)� Lineage-� Sca-1+c-Kit+� � � � � LMPP� Flk2 c-Kit c-Kit SSC-A � HSC� MPP FSC-A� Lineage� Sca-1� CD34� Lineage- Sca-1+� � � � � CLP c-Kit Sca-1 FSC-W

FSC-H� Lineage� IL-7Rα� - + Lineage B220 � - +� Fr. C’ IgM � pre-B� CD43 � �

� Fr. C � � � AA4.1+� BP-1 B220 B220 FSC-A SSC-W � Fr. A� Fr. B SSC-H� CD3ε, Mac-1, Gr-1, CD43� AA4.1� CD24 (HSA)� CD11c, Ter119, IgM�

Figure S9. Gating strategies for isolation of hematopoietic populations in BM. Cell debris and doublets were excluded by width/height plot of forward and side scatter. The lineage cocktail contains CD3ε, CD11b, TER-119, B220, Gr-1, CD8α and NK1.1.� A� B� Nfil3� pLG-E4BP4 6.0kbp

� 1.9kbp 1.4kbp 1.4kbp

3 FLAG

IgH chain enhancer IgH chain promoter E4BP4 FPKM mean FPKM

time point (h)� 1.2 CD43+ E� 1 C� IgM-� AA4.1+ -� 0.8 1.40±0.12� Lin 0.129±0.006� 3.87±0.13� *** 0.126±0.011� 0.6 1.53±0.03� 0.4 � 1.52±0.11� WT 0.2 0 Relative mRNA expression RelativemRNA 0.25±0.018� Control shNfil3 0.049±0.003� 2.00±0.16� F� 4.26±0.75� 0.080±0.01� Control shNfil3 0.109±0.032� 1.50±0.16� � 32 63 Nfil3-/- 2.00±0.21

� � � 45 25 BP-1 B220 B220 �

0.25±0.072 CD19 0.068±0.007� IgM� CD43� HSA�

Mac-1� D� Fr. A� Fr. B� Fr. C� Fr. C’� pre-B� Immature B� 4 � 4 � 4 � 4 � 6 � 5 � (×10 ) � (×10 ) n.s.� (×10 ) � (×10 ) n.s.� (×10 ) n.s.� (×10 ) � 3 * 16 4 * 6 2 10 * 14 3.5 5 8 � 12 3 2 4 10 2.5 6 8 2 3 1 4 1 6 1.5 2 Cellnumber 4 1 1 2 2 0.5 0 0 0 0 0 0 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3

Figure S10. Critical role of Nfil3 in B lineage commitment. (A) Expression pattern of Nfil3 in iLS cells differentiating into B cells by RNA-seq analysis. (B) Schematics of the Eµ-Nfil3 vector. (C, D) FACS profiles (C) and actual cell number (D) of B cell progenitor populations in wild type (WT) and Nfil3-/- mice. Values represent mean ± SD in a representative of three independent experiments. * p<0.05. (E, F) shRNA-mediated KD of Nfil3 in BM LSK cells differentiating into B cells. Cells were cultured on TSt-4 stromal cells for 6 days after shRNA induction. KD efficiency (E) and FACS profiles (F) are shown. *** p<0.001. Supplemental Tables�

Table S1. Sequences of shRNA oligonucleotides. Nucleotides shown in magenta are gene- specific shRNA sequences.

Target Sequence TGCTGTTGACAGTGAGCGAAGACACTCTATGCTTTGCAAGTAGTGAAGCCACAG Cbx2 ATGTACTTGCAAAGCATAGAGTGTCTCTGCCTACTGCCTCGGA TGCTGTTGACAGTGAGCGCGGCCACACACTCTTCGATTATTAGTGAAGCCACAG Uhrf1 ATGTAATAATCGAAGAGTGTGTGGCCATGCCTACTGCCTCGGA TGCTGTTGACAGTGAGCGAATAGATAATGTGAGAAGGAAGTAGTGAAGCCACAG Mysm1 ATGTACTTCCTTCTCACATTATCTATGTGCCTACTGCCTCGGA TGCTGTTGACAGTGAGCGAGCAGAGCGAGTCTGTGACAATTAGTGAAGCCACAG Pax5 ATGTAATTGTCACAGACTCGCTCTGCCTGCCTACTGCCTCGGA TGCTGTTGACAGTGAGCGACCTGTCACTCTTCTCCTTTAATAGTGAAGCCACAGA Nr4a2 TGTATTAAAGGAGAAGAGTGACAGGCTGCCTACTGCCTCGGA TGCTGTTGACAGTGAGCGCTCTGCCTTGCTGATTGTCTATTAGTGAAGCCACAGA Klf4 TGTAATAGACAATCAGCAAGGCAGATTGCCTACTGCCTCGGA TGCTGTTGACAGTGAGCGCATTCTTGATGTGAAGATAATTTAGTGAAGCCACAGA Egr1 TGTAAATTATCTTCACATCAAGAATATGCCTACTGCCTCGGA TGCTGTTGACAGTGAGCGACTGCTCTCCCTGAAATTAAAGTAGTGAAGCCACAG Nfil3 ATGTACTTTAATTTCAGGGAGAGCAGCTGCCTACTGCCTCGGA Table S2. Primer sequences for qRT-PCR, ChIP-qPCR and rearrangement PCR.

Primer sequences for qRT-PCR

�� Forward Reverse Flt3 AGTTCACCAAAATGTTCACG ACTTCTTCCAGGTCCAAGAG Ikzf1 GAGCTGGACTTGTGTCTTTG TTTCACACCTTGGTGAACTC Tcf3 CAGAACTGGAAACAAAGCAG TGGTGGCTCTGAAGTAGAAG Ebf1 TGGGTTACAGGTCATATTCG GAACTGCTTGGACTTGTACG Pax5 CATTCGGACAAAAGTACAGC GATGCCACTGATGGAGTATG Cd19 CAGTGATGGGACTAGCAGAC GTAGTGTTGCCAGAAACTCG Rag1 GAAGCTTCTGGCTCAGTCTACATCT ACCCTCATAGCGCTGCAGGTT Igll1 GTTCTAATGGGATGCTAGGC AGCGTCCTTCTCTTATCAGG Vpreb1 GAGTGGGAAGGAGAAAAGTC CCTTCCCATACCAGACTAGC Nr4a2 ATGCTGGTGCTAGTGTCTGC TGGATCCCCAGCTTTCTCAAG Klf4 GCACACCTGCGAACTCACAC GTTTGCGGTAGTGCCTGGTC Egr1 ACGACAGCAGTCCCATCTACTCGG GGACTCGACAGGGCAAGCATATGG Nfil3 GAACTCTGCCTTAGCTGAGGT ATTCCCGTTTTCTCCGACACG Ikzf3 GACACGTGCCCTATGACAACAGCAG GCATGCGTAGTTGCAGAGGTGACAC Bach2 TGGATGGTTGACTTTGTTTC AGGGTTTCACTTTTGCCTAC Irf4 TGGGTAAACACTCCTGACAC AGTGTTCCTCAGCCTACCTC Jun TTGCCCCAACAGATCCCGGT TGCGGTTCCTCATGCGCTTC Cbx2 CAAACACAACAGCTGGGAGC GGTCTCTTGCCTCTCTTCCG Mysm1 ATAGGACAGTCAGCCTCCTC AAAAGCTAGGTTGAGGGCTA Uhrf1 TGAAGTACTGGCCAGAGAGA CAGGGTACTGCATAGTGAGC Dnmt1 ATGGTGTTGTCTACCGACTG CAGAATACTTGCGGTAGTGC �� �� �� Primer sequences for ChIP-qPCR �� Forward Reverse Ebf1 CCACGCTCCTTCCAGTTTAG ATCCTTCCGCCTTATCACAG Pax5 CCTGTACTAGCACAATTGTCAACC CTGAATGGGACCTAGATACTGTAGTG Igll1 GGGTTAAGACAGGCAGCTGTGAG CAAACCCCAGGCTGTCTCTAGTT Vpreb1 TGCCAAGCTGGCCATGTGAACAC GATGTTCCTCTACCATATGTGAG IgHie GGTGGGGCTGGACAGAGTGTT TCAGAACCAGAACACCTGCAG �� �� �� Primer sequences for rearrangement PCR DHL MTTTTTGTSAAGGGATCTACTACTGTG �� VH7183 GAASAMCCTGTWCCTGCAAATGASC �� VHQ52 ACTGARCATCASCAAGGACAAYTCC �� VHJ558 CARCACAGCCTWCATGCARCTCARC �� JH3 CTCACAAGAGTCCGATAGACCCTGG �� mVkdeg-F1 GSTTCAGTGGCAGTGGRTCWGGRAC �� mJk2-R1 TTTCCAGCTTGGTCCCCCCTCCGAA �� Acta1-F GGCATCGTGTTGGATTCTG �� Acta1-R CACGAAGGAATAGCCACGC ��