An Erg-driven transcriptional program controls B cell lymphopoiesis

B lymphoid development is initiated by the differentiation of hematopoietic stem cells into lineage committed progenitors, ultimately generating mature B cells. This highly regulated process generates clonal immunological diversity via recombination of immunoglobulin V, D and J gene segments. While several transcription factors that control B cell development and V(D)J recombination have been defined, how these processes are initiated and coordinated into a precise regulatory network remains poorly understood. Here, we show that the transcription factor ETS Related Gene (Erg) is essential for early B lymphoid differentiation. Erg initiates a transcriptional network involving the B cell lineage defining genes, Ebf1 and Pax5, which directly promotes expression of key genes involved in V(D)J recombination and formation of the B cell receptor. Complementation of Erg deficiency with a productively rearranged immunoglobulin gene rescued B lineage development, demonstrating that Erg is an essential and stage-specific regulator of the gene regulatory network controlling B lymphopoiesis.


Supplementary
Mature recirculating B (Hardy Fraction F) Blood was collected into EDTA and differential cell counts performed using an ADVIA 120 Hematology System. RBC, red blood cells; WBC, white blood cells. * Padj < 10 -11 by Student's two-tailed unpaired t-test corrected for multiple testing by Benjamini-Hochberg procedure to control for false discovery rate.

Supplementary Table 3. Primers and PCR reactions
Primer 1 Primer 2 Expected sizes See Figure 3. Chromatin Immunoprecipitation (ChIP). Chromatin immunoprecipitation was performed on 2x10 7 cultured proB cells for Erg binding using the ab133264 antibody. Cells were cross-linked with 1% formaldehyde for 15 min at room temperature, terminated by the addition of 0.125M glycine.
Lysates were sonicated in a Covaris ultrasonicator to achieve a mean DNA fragment size of 500 bp.
Immunoprecipitation was performed using 10μg of antibodies for a minimum of 12h at 4°C in modified RIPA buffer (1% Triton X-100, 0.1% deoxycholate, 90mM NaCl, 10mM Tris-HCl, pH8.0 and protease inhibitors). An equal volume of protein A and G magnetic beads (Life Technologies) were used to bind the antibody and associated chromatin. Reverse crosslinking of DNA was performed at 65 o C overnight with RNaseA digestion followed by DNA purification using QIAquick PCR purification kits (Qiagen). Immunoprecipitated DNA was analysed on an Applied Biosystems StepOnePlus System with SYBR green reagents for iEμ μA and intergenic negative control regions using specific primers as detailed in Supplementary Table 3. Relative ChIP PCR enrichment of the iEμ μA containing region in proB cells compared to input was performed and normalized to the intergenic negative control region using the 2 -ΔΔCT method 43 .

ChIP-seq.
For sequencing analysis of immunoprecipitated DNA, DNA was quantified using the Qubit dsDNA HS Assay (Life Technologies). Library preparations were performed using the standard ThruPLEXTM-FD Prep Kit protocol (Rubicon Genomics) and size selected for 200-400bp fragments using Pippen Prep (Sage Science Inc.). Fragment sizes were confirmed using either the High Sensitivity DNA assay or the DNA 1000 kit and 2100 bioanalyzer (Agilent Technologies). Libraries were quantified with qPCR, normalized and pooled to 2nM before sequencing with single-end 75bp reads using standard protocols on the NextSeq (Illumina). DNA reads were adapter trimmed using Trimmomatic 49 and aligned to the GRCm38/mm10 build of the Mus musculus genome using the BWA aligner 50 . Peaks were called using MACS2 51 with default parameters to identify peaks using C17 antibody for Erg binding with Rag1Cre T/+ ;Erg Δ/Δ thymocytes as a negative control to filter peaks not due to Erg binding or ab133264 antibody in Rag1Cre T/+ ;Erg Δ/Δ pre-proB cells and uA Δ/Δ proB cells and wild-type proB cell controls, and were annotated to closest (peak start within 10kb from TSS) and overlapping genes using the R/Bioconductor package ChIPpeakAnno 52