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Genome-Wide Identification of Target Genes for the Key B Cell Transcription Factor Ets1

View Article: PubMed Central - PubMed

ABSTRACT

Background: The 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.

Results: To 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.

Conclusion: We 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.

No MeSH data available.


Identification of Ets1-binding sites in mouse B cells. (A) Western blot to show phosphorylation of Ets1 in freshly isolated B cells versus rested B cells. GAPDH serves as loading control. (B) Pie chart of location of Ets1 sites in the genome. (C) Motifs enriched in Ets1-bound regions. Shown are overrepresented transcription factor-binding motifs localized in the Ets1 peaks and the percent of sites with that motif. (D) Gene ontology biological terms associated with Ets1-binding peaks in B cells. (E) Analysis of epigenetic features surrounding Ets1-bound regions by mapping adjacent histone modifications. Data come from the ENCODE Consortium or from the studies described in Ref. (50, 51).
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Figure 1: Identification of Ets1-binding sites in mouse B cells. (A) Western blot to show phosphorylation of Ets1 in freshly isolated B cells versus rested B cells. GAPDH serves as loading control. (B) Pie chart of location of Ets1 sites in the genome. (C) Motifs enriched in Ets1-bound regions. Shown are overrepresented transcription factor-binding motifs localized in the Ets1 peaks and the percent of sites with that motif. (D) Gene ontology biological terms associated with Ets1-binding peaks in B cells. (E) Analysis of epigenetic features surrounding Ets1-bound regions by mapping adjacent histone modifications. Data come from the ENCODE Consortium or from the studies described in Ref. (50, 51).

Mentions: To gain insight into how Ets1 mechanistically regulates B cell differentiation, we assessed its genome-wide occupancy by chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) using chromatin derived from mouse mature B cells. Purified splenic B cells showed enhanced phosphorylation of Ets1 based on SDS-PAGE mobility when compared to whole spleen (Figure 1A). This shift in mobility arises from calcium-induced CAM kinase-dependent serine phosphorylation (34, 60) and results in inhibition of Ets1 DNA binding (61). In order to restore Ets1-binding activity, we rested the B cells for 1–2 h, which resulted in normalization of Ets1 phosphorylation status (Figure 1A). After the resting period, purified B cells were fixed and sonicated.


Genome-Wide Identification of Target Genes for the Key B Cell Transcription Factor Ets1
Identification of Ets1-binding sites in mouse B cells. (A) Western blot to show phosphorylation of Ets1 in freshly isolated B cells versus rested B cells. GAPDH serves as loading control. (B) Pie chart of location of Ets1 sites in the genome. (C) Motifs enriched in Ets1-bound regions. Shown are overrepresented transcription factor-binding motifs localized in the Ets1 peaks and the percent of sites with that motif. (D) Gene ontology biological terms associated with Ets1-binding peaks in B cells. (E) Analysis of epigenetic features surrounding Ets1-bound regions by mapping adjacent histone modifications. Data come from the ENCODE Consortium or from the studies described in Ref. (50, 51).
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Related In: Results  -  Collection

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Figure 1: Identification of Ets1-binding sites in mouse B cells. (A) Western blot to show phosphorylation of Ets1 in freshly isolated B cells versus rested B cells. GAPDH serves as loading control. (B) Pie chart of location of Ets1 sites in the genome. (C) Motifs enriched in Ets1-bound regions. Shown are overrepresented transcription factor-binding motifs localized in the Ets1 peaks and the percent of sites with that motif. (D) Gene ontology biological terms associated with Ets1-binding peaks in B cells. (E) Analysis of epigenetic features surrounding Ets1-bound regions by mapping adjacent histone modifications. Data come from the ENCODE Consortium or from the studies described in Ref. (50, 51).
Mentions: To gain insight into how Ets1 mechanistically regulates B cell differentiation, we assessed its genome-wide occupancy by chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) using chromatin derived from mouse mature B cells. Purified splenic B cells showed enhanced phosphorylation of Ets1 based on SDS-PAGE mobility when compared to whole spleen (Figure 1A). This shift in mobility arises from calcium-induced CAM kinase-dependent serine phosphorylation (34, 60) and results in inhibition of Ets1 DNA binding (61). In order to restore Ets1-binding activity, we rested the B cells for 1–2 h, which resulted in normalization of Ets1 phosphorylation status (Figure 1A). After the resting period, purified B cells were fixed and sonicated.

View Article: PubMed Central - PubMed

ABSTRACT

Background: The 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.

Results: To 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.

Conclusion: We 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.

No MeSH data available.