BackgroundThe transcription factor Ets1 is highly expressed in B lymphocytes. Loss of Ets1 leads to premature B cell differentiation into antibody-secreting cells (ASCs), secretion of autoantibodies, and development of autoimmune disease. Despite the importance of Ets1 in B cell biology, few Ets1 target genes are known in these cells.ResultsTo obtain a more complete picture of the function of Ets1 in regulating B cell differentiation, we performed Ets1 ChIP-seq in primary mouse B cells to identify >10,000-binding sites, many of which were localized near genes that play important roles in B cell activation and differentiation. Although Ets1 bound to many sites in the genome, it was required for regulation of less than 5% of them as evidenced by gene expression changes in B cells lacking Ets1. The cohort of genes whose expression was altered included numerous genes that have been associated with autoimmune disease susceptibility. We focused our attention on four such Ets1 target genes Ptpn22, Stat4, Egr1, and Prdm1 to assess how they might contribute to Ets1 function in limiting ASC formation. We found that dysregulation of these particular targets cannot explain altered ASC differentiation in the absence of Ets1.ConclusionWe have identified genome-wide binding targets for Ets1 in B cells and determined that a relatively small number of these putative target genes require Ets1 for their normal expression. Interestingly, a cohort of genes associated with autoimmune disease susceptibility is among those that are regulated by Ets1. Identification of the target genes of Ets1 in B cells will help provide a clearer picture of how Ets1 regulates B cell responses and how its loss promotes autoantibody secretion.

Tight control of B cell differentiation into plasma cells (PCs) is critical for proper immune responses and the prevention of autoimmunity. The Ets1 transcription factor acts in B cells to prevent PC differentiation. Ets1−/− mice accumulate PCs and produce autoantibodies (autoAbs). Ets1 expression is downregulated upon B cell activation through the BCR and TLRs and is maintained by the inhibitory signaling pathway mediated by Lyn, CD22 and SiglecG, and SHP-1. In the absence of these inhibitory components, Ets1 levels are reduced in B cells in a Btk-dependent manner. This leads to increased PCs, autoAbs, and an autoimmune phenotype similar to that of Ets1−/− mice. Defects in inhibitory signaling molecules, including Lyn and Ets1, are associated with human lupus, although the effects are more subtle than the complete deficiency that occurs in knockout mice. Here, we explore the effect of partial disruption of the Lyn/Ets1 pathway on B cell tolerance and find that Lyn+/−Ets1+/− mice demonstrate greater and earlier production of IgM, but not IgG, autoAbs compared to Lyn+/− or Ets1+/− mice. We also show that Btk-dependent downregulation of Ets1 is important for normal PC homeostasis when inhibitory signaling is intact. Ets1-deficiency restores the decrease in steady state PCs and Ab levels observed in Btk−/− mice. Thus, depending on the balance of activating and inhibitory signals to Ets1, there is a continuum of effects on autoAb production and PC maintenance. This ranges from full-blown autoimmunity with complete loss of Ets1-maintaining signals to reduced PC and Ab levels with impaired Ets1 downregulation.

The transcription factor (TF) Ets1 is a critical regulator of B cell quiescence. Its expression must be maintained at a high level in resting B cells to prevent inappropriate differentiation and autoimmune responses, and its downregulation in response to antigen receptor stimulation is vital for proper B cell responses. How Ets1 expression is controlled remains unstudied. Quantification of newly-transcribed Ets1 pre-mRNA shows that Ets1 gene transcription is reduced upon BCR stimulation. Inhibition of new protein synthesis did not prevent repression of Ets1 transcription in response to stimulation, suggesting that the phenomenon depends on modulation of existing TF binding to Ets1 regulatory elements (REs) rather than de novo synthesis of a repressor protein. Epigenetic analyses identified numerous putative REs in the mouse Ets1 locus. A 217 kb BAC transgenic construct containing potential RE sequences drives eGFP reporter expression in a manner that recapitulates that of endogenous Ets1. To identify REs that may be important for regulating Ets1 expression and stimulation-induced repression, we performed ATAC-seq in untreated and BCR-stimulated B cells, and observed potentially meaningful changes to chromatin accessibility. Motifs for highly relevant TFs are enriched in accessible regions in the Ets1 locus, including members of the Smad, ETS, AP-1, and NFκB families. Following up on NFκB as a potential regulator of Ets1 expression, downregulation of Ets1 transcription was found to be dependent upon IKK2 activity but did not require RelA. These analyses suggest a potentially complex regulatory network behind the expression of Ets1 as it safeguards the resting B cell state. Funded by NIH R01 AI122720 and Lupus Research Alliance.