Sequences within and upstream of the mouse Ets1 gene drive high level expression in B cells, but are not sufficient for consistent expression in T cells
The levels of transcription factor Ets1 are high in resting B and T cells, but are downregulated by signaling through antigen receptors and Toll-like receptors (TLRs). Loss of Ets1 in mice leads to excessive immune cell activation and development of an autoimmune syndrome and reduced Ets1 expression has been observed in human PBMCs in the context of autoimmune diseases. In B cells, Ets1 serves to prevent premature activation and differentiation to antibody-secreting cells. Given these important roles for Ets1 in the immune response, stringent control of Ets1 gene expression levels is required for homeostasis. However, the genetic regulatory elements that control expression of the Ets1 gene remain relatively unknown. Here we identify a topologically-associating domain (TAD) in the chromatin of B cells that includes the mouse Ets1 gene locus and describe an interaction hub that extends over 100 kb upstream and into the gene body. Additionally, we compile epigenetic datasets to find several putative regulatory elements within the interaction hub by identifying regions of high DNA accessibility and enrichment of active enhancer histone marks. Using reporter constructs, we determine that DNA sequences within this interaction hub are sufficient to direct reporter gene expression in lymphoid tissues of transgenic mice. Further analysis indicates that the reporter construct drives faithful expression of the reporter gene in mouse B cells, but variegated expression in T cells, suggesting the existence of T cell regulatory elements outside this region. To investigate how the downregulation of Ets1 transcription is associated with alterations in the epigenetic landscape of stimulated B cells, we performed ATAC-seq in resting and BCR-stimulated primary B cells and identified four regions within and upstream of the Ets1 locus that undergo changes in chromatin accessibility that correlate to Ets1 gene expression. Interestingly, functional analysis of several putative Ets1 regulatory elements using luciferase constructs suggested a high level of functional redundancy. Taken together our studies reveal a complex network of regulatory elements and transcription factors that coordinate the B cell-specific expression of  Ets1. Overall design: B cells were isolated from two wild type C57BL/6 mouse spleens (numbers 1060 and 1062) using the EasySep Mouse B Cell Isolation Kit (Stem Cell Technologies) and cultured in RPMI-1640 media with 10% fetal calf serum, penicillin/streptomycin, glutamax and 50 micromolar beta-mercaptoethanol at 37°C in a tissue culture incubator. For each mouse number, cells were either left unstimulated (no treatment, NT) or stimulated (stim) via the BCR with goat F(ab')2 fragment of goat anti-mouse IgM, µ chain specific antibody (Jackson ImmunoResearch) for two hours. 100,000 cells from each sample were pelleted and stored frozen prior to Assay for transposase accessible chromatin followed by sequencing (ATAC-seq). Reads were aligned to the mm10 version of the mouse genome.