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.


Abstract:
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.

Introduction:
Autoimmune diseases are caused by the immune system attacking self-tissues.Genetic factors influence the development of autoimmune diseases and genome-wide association studies (GWAS) have been used to identify disease-associated single-nucleotide polymorphisms (SNPs) for many autoimmune diseases.GWAS has established that the human ETS1 gene is a susceptibility locus for multiple autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), psoriasis and others (1).Ets1 mRNA levels are reduced in PBMCs isolated from patients with a variety of different autoimmune diseases (2)(3)(4)(5)(6)(7).Supporting a role for Ets1 in autoimmunity, mice lacking Ets1 develop a lupuslike autoimmune disease, with excessive B and T cell activation and secretion of IgM and IgG autoantibodies against self-antigens (8)(9)(10)(11).Cell-type-specific deletion of Ets1 has demonstrated cellintrinsic roles for Ets1 in blocking the aberrant activation of both B and T cells (10,11).
Ets1 expression is high in resting lymphocytes (12)(13)(14), but is downregulated by stimulation through antigen receptors or Toll-like receptors (15)(16)(17)(18)(19). Loss of Ets1 leads to increased B cell differentiation into antibody-secreting plasma cells (20) and forced expression of Ets1 blocks development of plasma cells (7,15,16,21).Since the level of Ets1 in B cells is crucial to regulating the differentiation program, expression of the Ets1 gene must be tightly controlled.Maintenance of Ets1 expression in resting B cells relies heavily on inhibitory signaling pathways that limit the transduction of activating signals.B cells that lack expression of inhibitory signaling components such as inhibitory receptors CD22 or SiglecG, proximal kinase Lyn, or downstream phosphatases SHP-1 or SHIP-1 have increased BCR signaling and express lower levels of Ets1 mRNA and protein (16).Our studies have shown that the low level of Ets1 in Lyn-/-B cells is not due to decreased stability of the Ets1 protein or mRNA (16).Instead, BCR and TLR signaling result in a decrease in transcription of the Ets1 gene and this does not require new protein synthesis, implicating pre-formed transcription factors (17).
Transcriptional regulation of gene expression is controlled by three-dimensional chromatin architecture in the nucleus as well as the binding of cell-type specific transcription factors.As part of this process, the genome is partitioned into an array of chromatin loops generated by the combined actions of cohesin and CTCF proteins that define topologically-associated domains (TADs) (22)(23)(24).TADs counteract the formation of transcriptionally-silent heterochromatin domains (25,26) and also function to insulate promoters of genes within them from the influence of response elements outside the TAD (27)(28)(29)(30).TADs are further sub-divided into sub-TAD regions that contain one or a few genes along with regulatory elements such as gene enhancers or silencers that bind specific transcription factors (26)(27)(28).Recently, the organization TAD structures in the Ets1 locus in mouse and human T cells have been published (31).This analysis also identified a super-enhancer region located approximately 250 kb downstream of the Ets1 gene, which is required for CD4+ T helper 1 (Th1) differentiation (31).In contrast to the situation in T cells, the genomic organization of the Ets1 locus and the regulatory elements and transcription factors that bind to them in B cells remain unknown.
To identify B cell TADs and sub-TADs around Ets1 and also regulatory elements that coordinate expression of Ets1, we examined chromatin interactions, epigenetic profiles and DNA accessibility in mouse B cells.To further validate the roles of the regulatory elements, we generated a bacterial artificial chromosome (BAC) reporter construct in which eGFP expression was inserted under the direction of sequences from -113 kb to +105 kb of the mouse Ets1 gene and observed reporter expression that recapitulated endogenous Ets1 in B cells, but did not fully recapitulate Ets1 expression in T cells.Four putative regulatory elements experience changes in accessibility upon BCR stimulation and motif analysis identified transcription factors that may bind to these regulatory elements.Transient transfection assays with numerous putative regulatory element sequences failed to detect sequences that individually activate or repress reporter gene transcription at an appreciable level .These results suggest the possibility that Ets1 gene transcription in B cells may be regulated by a network of multiple regulatory elements the coordinately stimulate gene transcription, rather than being reliant on one or a few dominant regulatory elements.

BAC recombineering and transgenic mouse generation:
The RP23-350A20 BAC (BACPAC Resources) was recombineered according to the protocol previous described (43) using a targeting construct that contained eGFP, an SV40 polyA sequence and a neomycin/kanamycin resistance cassette between homology arms to replace the first exon of the Ets1 gene.
Clones were selected and PCR-tested for eGFP insertion.FLP-FRT was induced to remove the neomycin/kanamycin resistance cassette.The final recombined BAC contains the eGFP cDNA and SV40 polyA sequence inserted in place of the first exon of the major isoform of Ets1 (mouse Ets1 isoform 1) and under the control of Ets1 gene regulatory elements.
The recombined BAC was microinjected into fertilized C57BL/6 mouse eggs by the Roswell Park Comprehensive Cancer Center (RPCCC) Gene Targeting and Transgenic Facility.BAC Transgenic (BACtg) mice were identified by PCR genotyping.All mice were house at RPCCC Lab Animal Shared Resource and experiments were performed in accordance with Institutional Animal Care and Use Committee protocol number UB1104M.Mice were euthanized by CO 2 inhalation followed by cervical dislocation.

BACtg flow cytometry:
Single-cell suspensions were made from the spleens of wild type and BACtg animals and stained with Ghost Dye Violet 510 (Tonbo Biosciences) and fluorescent dye-conjugated antibodies against B lineage surface markers, including B220, CD19, CD21, CD23, CD80, PDL2, Fas, PNA, CD138, and CD98, or T lineage surface markers, including CD3, CD4, and CD8.Cells were fixed and permeabilized for intracellular staining and stained with unconjugated rabbit monoclonal anti-Ets1 (D8O8A, Cell Signaling Technologies) or rabbit monoclonal IgG isotype control, followed by PE-conjugated anti-rabbit IgG.Data was collected using a LSR II flow cytometer and analyzed using FlowJo software.Samples were gated on singlets and live cells, and specific populations were identified based on marker expression.

Mouse B cell ATAC-seq and identification of DARs:
B cells were isolated from wild type mouse spleens and stimulated via the BCR for two hours.100,000 cells from each sample were pelleted and stored frozen prior to ATAC-seq (Active Motif).Illumina NextSeq500 sequencing was used to generate paired-end 42 bp sequencing reads that were mapped to the mm10 genome build using the BWA algorithm with default settings (44).Duplicate reads were removed and analysis was performed on reads that passed Illumina's purity filter, aligned with a maximum of 2 mismatches, and mapped uniquely.Data was normalized across samples by reducing read count to that of the smallest sample.Peaks were called using the MACS2 algorithm (45).DARs were identified by comparing the number of sequencing tags in each region and identifying regions with greater than 2-fold differences in tag count.Gene ontology analysis of genes within 10 kb of DARs was performed using DAVID (46), limited to Gene Ontology (GO)'s biological process annotation set.De novo motif analysis was performed using HOMER's findMotifsGenome.plprogram (47).The FIMO program within the MEME Suite of tools was used to scan the four DARs for transcription factor motifs (48).ATAC-seq data are available in the GEO database under the accession number GSE273513.

Luciferase construct design and luciferase assays:
A region of the mouse Ets1 proximal promoter of 469 bp (-451 to +17 bp) was cloned into the pGL3-Basic firefly luciferase vector.Sequences covering regions of Sites 1-6 (defined in the Results section below) were cloned upstream of this minimal promoter fragment.In addition, a shorter fragment of the minimal promoter of 193 bp (-114 to +59 bp) was cloned into pGL3-Basic along with shorter segments of putative regulatory regions.A20 B lymphoma cells were co-transfected with 2 μg of the firefly luciferase plasmids and 0.25 μg of pRL-Renilla luciferase plasmid using Nucleofection (Lonza) and analyzed at 24 hours after transfection.Firefly luciferase activity was normalized to Renilla luciferase activity.

Results: The 3-D chromatin organization of the mouse Ets1 gene in B cells
To define topologically-associated domains (TADs) around the mouse Ets1 gene, we examined Hi-C data from a previously-published study of resting mouse B cells (49).This analysis showed that the mouse Ets1 gene is contained within a CTCF-flanked TAD of ~900 kb that stretches from -445 kb to +452 kb from the major transcriptional start site (TSS) of Ets1 (boundaries of the TAD are shaded in aqua in Figure 1A) (34).In addition to the Ets1 gene, this TAD contains fellow ETS family member Fli1, as well as two potassium channel-encoding genes, Kcnj1 and Kcnj5.A closer look at the contact map reveals a CTCF-flanked sub-TAD stretching from -107 kb to +244 kb (indicated by the large aqua triangle in Figure 1B).This region contains only the Ets1 gene itself, with no other known genes.Within the Ets1 sub-TAD, there is a local hub of more intense interactions that stretches from -107 kb to the end of the Ets1 gene (approximately +62 kb from the TSS) (denoted by the smaller aqua triangle in Figure 1B).It is likely that regulatory elements crucial for B cell-specific expression of mouse Ets1 are present within the strong interaction hub.We suspect that the CTCF binding site located at -107 kb likely functions to insulate the Ets1 promoter from regulatory elements of the Fli1 gene, which is oriented in a head-to-head manner with Ets1 and separated by 154 kb (Figure 1B).Indeed, Hi-C detects little interaction between the Fli1 promoter and sequences in the Ets1 sub-TAD.

A region extending from -113 kb to +105 kb from the mouse Ets1 TSS is directs expression in B cells
Prior transgenic mouse studies have shown that sequences located from -5.3 kb to +9 kb from the TSS of Ets1 were not sufficient to drive expression of a transgene in B cells or other lymphoid lineages (50).To identify sequences that drive B cell-specific expression of Ets1, we generated BAC transgenic mice harboring a 219 kb piece of mouse DNA including sequences from -113 kb upstream of the Ets1 gene (stretching just beyond the CTCF-bound boundary) to +105 kb downstream of the transcriptional start site.
This BAC construct contains the entire local hub of robust interactions around the Ets1 gene (Figure 2A).
The first exon of the Ets1 gene was replaced with an eGFP cDNA, followed by a stop codon and an SV40 polyA signal, allowing eGFP expression from the Ets1 promoter under the direction of putative regulatory elements located within the BAC (Figure 2A).Analysis of both mRNA and protein showed high eGFP expression in lymphoid tissues, including the thymus, spleen, and lymph nodes, where B and T lymphocytes are abundant and where Ets1 is normally expressed at high levels (Figure 2B-C).Non-lymphoid tissues such as the brain and kidneys showed much lower expression as expected.
To compare the potential overlap in the Ets1 and eGFP expression in these BACtg mice at a cellular level, we performed intracellular flow cytometry using an antibody specific for Ets1.B220 + CD19 + B cells from both wild type mice and BACtg mice showed high levels of intracellular Ets1 expression compared to isotype control staining (Figure 3A, bottom).Mature B cell subsets within this B220 + CD19 + population, including CD23 hi CD21 lo follicular B cells, CD23 lo CD21 hi marginal zone B cells, and Fas + PNA + germinal center B cells, all showed strong positive staining for both Ets1 and eGFP (Figure 3B).Ets1 is downregulated upon B cell differentiation to plasma cells (12,15,51).In keeping with this, eGFP levels were lower in B220 lo CD138 + CD98 + plasma cells than in B220+CD19+ B cells of BACtg mice (Figure 3B).
However, in contrast to the B cells, eGFP expression did not fully reflect Ets1 expression in the T cell populations of BACtg mice.While both CD3 + CD4 + and CD3 + CD8 + T cells had uniformly high staining for intracellular Ets1, GFP expression showed a broad range in these populations (Figure 3B), indicating variegation of BACtg expression.
BCR stimulation induces the downregulation of Ets1 in B cells, mainly through a decrease in Ets1 gene transcription (16,17).To determine if the response elements required for this phenomenon are within the 219 kb of the BAC, B cells from the BACtg animals were isolated and stimulated ex vivo, and expression of Ets1 and eGFP were assessed.As shown in Supplemental Figure S1, stimulation by the BCR resulted in a decrease in Ets1 pre-mRNA and mRNA in B cells.Because the pre-mRNA is a short-lived product in the cells, its measurement serves as a surrogate for the transcriptional level of the Ets1 gene.In the BACtg B cells, eGFP mRNA was similarly downregulated in response to BCR stimulation, although not quite as dramatically.We surmise that the attenuated downregulation of eGFP compared to Ets1 is due to a difference in mRNA stability of the two transcripts (16,52).Overall, eGFP expression in the mature B cell populations of these BACtg mice recapitulates that of endogenous Ets1 under both basal conditions and upon BCR stimulation.

Multiple putative regulatory elements have the potential to regulate Ets1
To identify candidate regulatory elements that might control Ets1 expression in mouse B cells, we examined epigenetic characteristics of regions included in the BAC transgene in more detail.We identified regions that had open chromatin, as defined by peaks of ATAC-seq accessibility in mouse B cells (highlighted in aqua in Figure 4A).H3K4me3 histone methylation, a mark of active promoters, was enriched at the TSS.H3K4me1 histone methylation and H3K27ac histone acetylation, both marks of active regulatory regions (53), were enriched at numerous DNA segments upstream and within the Ets1 gene.On the other hand, regions downstream of the Ets1 gene did not harbor any regions that showed enrichment for H3K27Ac or H3K4me1 in B cells.These observations, combined with the Hi-C data shown in Figure 1 and the BAC transgenic data shown in Figures 2-3, suggest that in mouse B cells Ets1 is likely regulated by DNA sequences upstream and within the gene itself, rather than sequences located downstream of the gene.
The BAC transgenic mice have variegated expression of eGFP in T cell populations, suggesting that the BAC lacks all the elements required for reproducible expression of Ets1 in T cells.In contrast to B cells which lack putative regulatory elements downstream of the Ets1 gene, ATAC-seq data from mouse T cell populations shows that T cells have accessible sites downstream of the Ets1 gene (highlighted in aqua in Supplemental Figure S2).These sites are located beyond the downstream regions included in the BAC (highlighted in yellow in Supplemental Figure S2) and could represent T cell-specific enhancers required for optimal and sustained expression.Since some of the T cells in BACtg mice express high levels of GFP, the 219 kb region of the BAC must encompass regulatory elements that can induce expression in T cells.
However, these elements appear to be insufficient to prevent spreading of heterochromatin into the Ets1 locus in T cells thus resulting in variegated expression of the transgene.Recently a region located ~250 kb downstream of Ets1 was shown to function as a multi-enhancer for mouse Th1 cells (31).
While peaks of H3K27Ac and H3K4me1 histone marks were discrete in regions 5' to the Ets1 gene, there was broad enrichment of these marks across the body of the Ets1 gene (Figure 4A).To better define potentially important regulatory regions within the Ets1 gene itself, we examined histone marks in a human B cell line GM12878 that expresses Ets1.As shown in Figure 4B, GM12878 cells have a more discrete pattern of enrichment of H3K27Ac at seven specific regions within and around the human ETS1 gene.These regions of H3K27Ac enrichment are characterized by the presence of DNAse I hypersensitivity and were labeled Site 1-7 (Figure 4B).Site 4 corresponds to the Ets1 proximal promoter and beginning of intron 1, sequences previously tested in transgenic mice and shown to not mediate lymphoid-specific expression (50).Thus, this region was not further analyzed.Site 1 encompasses the CTCF binding peaks located at -104 kb upstream of the mouse Ets1 promoter.Sites 1-7 contained regions of DNA sequence conservation between mouse and human, supporting the idea that they may be functionally important.In summary, there are multiple potential regulatory elements found within and upstream of the mouse Ets1 gene that may be crucial for B cell-specific expression.

Chromatin patterns in resting B cells versus plasma cells
Analysis of previously-published RNA-seq data from sorted purified mouse bone marrow progenitors (37) shows that Ets1 is expressed at low levels in hematopoietic stem cells (HSC), multi-potent progenitors (MPP) and common lymphoid progenitors (CLP), but is elevated once cells commit to the B cell lineage and again reduced in fully-differentiated plasma cells (PC) (Figure 5A).Open chromatin patterns assessed by ATAC-seq in the same sorted populations showed that there were differences in the regions of open chromatin between resting follicular (FO) B cells, where Ets1 levels are high, and PC, where Ets1 levels are low (Figure 5B).The ATAC-seq peaks located near the sub-TAD boundary where CTCF binds (-100 to -106 kb region) and the ATAC-seq peaks located in the proximal promoter of Ets1 were equally present in both FO B cells and PC.When examining the other ATAC-seq peaks, we found that peaks located at approximately -87 kb, -44 kb, -16 kb, -10 kb, +39 kb and +44 kb were strong in B cells, but reduced in PC (Figure 5B, blue highlighting).The differences in chromatin accessibility at these regions may indicate the presence of regulatory elements bound by activating transcription factors promoting Ets1 expression in B cells.

Putative regulatory elements within and upstream of the Ets1 gene change in accessibility upon BCR stimulation
As mouse Ets1 gene transcription is decreased in response to BCR stimulation and this effect is mediated by response elements within the BAC, we explored changes in chromatin accessibility in B cells in response to BCR stimulation.The downregulation of Ets1 gene transcription occurs early after activation, within 2 hours of BCR engagement (16).We therefore generated ATAC-seq datasets for resting mouse B cells and B cells stimulated for 2 hours with a BCR crosslinking antibody.Regions that experienced a twofold or greater change in accessibility between resting and stimulated B cells were deemed as differentially accessible regions (DARs) (Figure 6A).Upon stimulation, changes in chromatin accessibility were induced genome-wide, with 2259 DARs increasing and 598 DARs decreasing in accessibility.The disproportionate amount of DARs increasing in accessibility versus those decreasing is in line with previous studies of immune cell ex vivo stimulation (54,55).Genes within 10 kb of DARs that gained or lost accessibility were found to be functionally associated with immune responses and signal transduction (Figure 6B).Motif enrichment analysis revealed that DARs that gained accessibility frequently contained binding sites for AP-1 and NF-κB proteins, which are downstream effectors of pathways activated by BCR signaling, while those that lost accessibility were most highly enriched for the Ets1 motif (Figure 6C).The loss of Ets1 binding in BCR-stimulated B cells is consistent with the decreased transcription of the Ets1 gene upon BCR stimulation.However, it is also likely that BCR-induced phosphorylation of the Ets1protein, which has been shown to decrease its DNA binding activity and in T cells leads to its nuclear export (56,57), contributes to loss of Ets1 binding in stimulated B cells.
Four ATAC-Seq peaks in the region contained within the BACtg were identified as DARs, with 2 gaining and 2 losing accessibility after 2 hours of BCR stimulation (Figure 7A, DARs highlighted in blue and red, respectively).These sites were located at -85 kb (included within Site 2 defined in Figure 4B), at +36 and +39 kb (included within Site 6) and at +49 kb (included within Site 7) from the TSS.The changes in DNA accessibility could reflect stimulation-induced alterations in transcription factor binding.A decrease in accessibility upon stimulation could reflect the loss of binding of a transcriptional activator, while an increase in accessibility may be caused by increased binding of a transcriptional repressor.Both scenarios could lead to downregulation of Ets1 gene transcription.
DNA sequences in the +39 kb and +49 kb DARs showed conservation with corresponding sequences in human ETS1 gene (Supplemental Figure S3).On the other hand, the other two mouse DAR sequences at -85 kb and +36 kb didn't show strong DNA sequence conservation, although these DARs were located within Sites 2 and 6 that did have regions of conserved sequence.To identify transcription factors that may bind to the four DAR sites, we used the FIMO program to scan the sequences for DNA-binding motifs.The five strongest motif matches for each site are mapped in Figure 7B.Transcription factors sites found within the DARs that lose accessibility after BCR stimulation include those for NF-κB proteins, SpiC, Smad proteins and Irf5, among others.Both sites that gained accessibility contained motifs for AP-1 family members and for Srebf2, a sterol RE-binding protein (Figure 7B).ChIP-seq data from the CH12 mouse B cell lymphoma line indicates that both c-Jun and JunD can bind at the +49 kb DAR site that gains accessibility (Figure 7C) (36).With the exception of Hoxd3 and Hnf4g, all of the transcription factors shown in Figure 7B are expressed in B cells (37) and could potentially be involved in regulating Ets1 transcription.These changes in chromatin accessibility that occur in response to BCR stimulation could highlight the response elements important for regulation of Ets1 expression and implicate AP-1, in particular, in its downregulation.

A selection of regulatory elements tested in transient transfections are insufficient to activate the Ets1 promoter
To determine if the potential regulatory elements identified above govern Ets1 expression, we next generated a series of luciferase reporter constructs with putative regulatory elements cloned upstream of the Ets1 minimal promoter (Figure 8).We generated two sets of constructs.In the first set, a 469 bp fragment of the Ets1 minimal promoter (451 bp to +18 bp from the TSS) was cloned along with putative regulatory element fragments containing regions of Sites 1-3 and 5-6 (shown as blue boxes in Figure 8).
The second set of constructs was more focused and included a smaller fragment of the Ets1 promoter (-114 bp to +60 bp from the TSS) along with shorter segments of the putative regulatory elements (shown in green boxes in Figure 8).For the second set of constructs, we chose elements that were located in regions of H3K27ac enrichment and that were more accessible in either resting B cells or plasma cells (as highlighted in Figure 5B), assuming that differences in chromatin accessibility would reflect the high and low expression of Ets1 in these cell types.
The reporter constructs were transfected into the A20 mouse B lymphoma cell line, which expresses Ets1 and downregulates it transcriptionally in response to BCR stimulation similar to primary B cells (16,17).Out of the putative regulatory elements tested, ranging from -110 kb upstream to +49 kb downstream of the Ets1 TSS, none showed a strong ability to activate or repress expression (Figure 8C-D).The strongest activity was found with sequences in Site 2 that were able to stimulate transcription modestly at approximately 1.5-fold over the promoter alone (Figure 8C).Given that none of these sequences alone was able to direct high level expression of a reporter gene in B cells, these data suggest that Ets1 may be regulated by a combinatorial network of response elements that cooperate to stimulate gene expression in B cells.located downstream of the 3' end of the BAC that are required in T cells, but not B cells, to prevent heterochromatin spread.A previously-described super-enhancer segment located ~250 kb downstream of Ets1 was shown to be essential for maximal Ets1 expression in double positive thymocytes and in Th1 cells and for proper Th1 cell differentiation (31).This super-enhancer region is not present in the BACtg and its absence may lead to variegated expression of the GFP marker in T cells.However, it is important to note that there is also a region of accessible chromatin in T cells located at approximately +120 kb from the Ets1 TSS that is missing from the BACtg (see Figure S2).This sequence could also play a role in regulation of Ets1 expression in T cells.
To identify potential regulatory elements of Ets1, we examined epigenetic datasets from B cells and defined seven broad regions that have properties of regulatory sequences (Sites 1-7).All seven of these sites have regions with high DNA sequence homology between the mouse and human genomes.However, these large regions of sequence showed only weak activity in a luciferase reporter assay in A20 B lymphoma cells.To refine our analysis, we compared open chromatin regions in follicular B cells, which express high levels of Ets1, to open chromatin regions in plasma cells, which have very low levels of Ets1.This analysis identified 6 regions with differential accessibility, two of which overlapped with sequences in Sites 1-7.
We tested these six shorter segments in luciferase assays, but they also failed to mediate strong activation of the promoter.Therefore, we did not identify any regions that were able to strongly activate or repress expression of a reporter gene in transient transfection assays.This result suggests the possibility that transcription of the Ets1 gene is more complicated and relies on the combinatorial action of multiple regulatory regions that must function cooperatively.
Since sequences in the BACtg were sufficient to mediate down-regulation of Ets1 gene transcription upon BCR stimulation, we identified differentially-accessible regions (DARs) in the chromatin of stimulated and unstimulated B cells.In the region surrounding the Ets1 locus, four DAR regions were identified and three of these overlapped with sequences in Sites 1-7.The two sites that gained accessibility after stimulation contained motifs for AP-1 family members.AP-1 motifs were also enriched globally in sites that gained accessibility with stimulation, in line with other studies examining stimulation-responsive chromatin and gene expression that have implicated AP-1 in mediating early genome-wide changes (60,61).AP-1 is activated downstream of MAPK pathways, particularly JNK and ERK, and we have previously demonstrated that JNK activity is required for Ets1 downregulation (16).Binding of AP-1 subunits c-Jun and JunD was detected at the +49 DAR in CH12 B lymphoma cells.While AP-1 proteins often serve to activate gene expression, considerable evidence shows that they can also serve as repressors of transcription in appropriate contexts (62)(63)(64)(65)(66). Thus, recruitment of AP-1 proteins to the -85 kb and +49 kb DAR regions might serve to inhibit Ets1 gene transcription after BCR stimulation.Further studies will be needed to fully determine the role of AP-1 family proteins in the regulation of Ets1 expression.
The two regions that lost accessibility upon stimulation could represent regulatory elements bound by transcription factors responsible for maintaining Ets1 expression in resting B cells.These two regions contained potential binding sites for a variety of transcription factors including Smad proteins, NFκB family members, and ETS family member SpiC.SpiC is a member of the ETS family that has been shown to be a negative regulator of B cell function (67).However, examining ChIP-seq data for SpiC in a mouse pro-B cell line (68) did not reveal any binding of SpiC to its putative site in the DAR at +39 kb (data not shown).

Therefore,
SpiC seems to be a less likely candidate for promoting basal expression of Ets1 in resting B cells.Canonical NFκB proteins are sequestered in the cytoplasm in the resting state and hence also seem unlikely to mediate basal expression of Ets1 in resting B cells.On the other hand, it is possible that noncanonical NFκB signaling, such as that triggered by the BAFF receptor or CD40 receptor, contributes to the maintaining Ets1 expression in resting B cells.The Smad2/3/4 complex is activated downstream of TGFβ signaling, which has been shown to suppress B cell responses through upregulation of inhibitory signaling components(69), and mice lacking the receptor TGFβ RII have hyper-responsive B cells(70).Therefore, the TFGβ signaling pathway is an attractive possibility for regulating basal levels of Ets1 in resting B cells.Future studies on potential roles for Smad proteins and non-canonical NFκB proteins will be important to determine whether they are involved in maintaining Ets1 in resting B cells.Overall, in this report we have analyzed the regions surrounding the mouse Ets1 gene and determined that a BAC transgene carrying sequences from -113 kb to +105 kb from the TSS is sufficient for high level and reproducible expression of a reporter gene in B cells.Unexpectedly, this region is not sufficient for maintaining consistent expression of the reporter gene in T cells and instead resulted in variegated expression in both CD4 and CD8 T cells.Therefore, there are differences in the regulatory sequences required for optimal expression in B and T cells.Further analysis of epigenetic marks and the comparison of open chromatin in resting B cells versus activated B cells as well as in follicular B cells versus plasma cells resulted in the identification of numerous regions with features of regulatory elements that could participate in controlling Ets1 expression in B cells.However, none of the elements was sufficient by itself to strongly activate or repress transcription in transient transfection assays, suggesting that Ets1 may be regulated by a network of several regulatory elements located upstream and within its introns.One limitation worth pointing out is the episomal nature and the lack of intact chromatin conformations in plasmid-based reporters that were utilized in this report.Further studies are needed to fully dissect the Ets1 cis-regulatory network and the role it may play in B cell activation.

Figure 1 .
Figure 1.Identification of the Ets1 TAD and sub-TAD.Chromatin interaction maps of mouse B cell Hi-

Figure 2 .
Figure 2. Design of a BAC-derived reporter construct to identify sequences required for lymphoid

Figure 3 .
Figure 3. Expression of the BACtg eGFP in lymphoid cells.(A) Flow cytometry analysis of intracellular

Figure 4 .
Figure 4.The Ets1 sub-TAD region contains several putative response elements highly enriched for

Figure 5 .
Figure 5. Differences in Ets1 expression in different B cell differentiation stages correlate with

Fig. 7 .
Fig. 7. DARs in the Ets1 gene locus contain motifs for B cell-relevant transcription factors.(A) Shown

Fig. 8 .
Fig. 8. Individual putative response elements do not mediate strong activation or repression in