Limits...
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.


Reduction of selected target genes in Ets1−/− B cells. (A) Analysis of Blimp1 expression in lipopolysaccharide-stimulated Ets1+/+, Ets1−/−, and Ets1−/−Prdm1+/gfp B cells. The location of the full-length wild-type and the truncated proteins is indicated on the panel. (B) ELISPOT analysis of the numbers of IgM- and IgG-secreting cells in the spleens of unchallenged mice of the indicated genotypes (n = 4–5 for each genotype). *p < 0.05, **p < 0.01, and ***p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5383717&req=5

Figure 7: Reduction of selected target genes in Ets1−/− B cells. (A) Analysis of Blimp1 expression in lipopolysaccharide-stimulated Ets1+/+, Ets1−/−, and Ets1−/−Prdm1+/gfp B cells. The location of the full-length wild-type and the truncated proteins is indicated on the panel. (B) ELISPOT analysis of the numbers of IgM- and IgG-secreting cells in the spleens of unchallenged mice of the indicated genotypes (n = 4–5 for each genotype). *p < 0.05, **p < 0.01, and ***p < 0.001.

Mentions: Unlike Stat4 and Ptpn22, Egr1 and Prdm1 were upregulated in Ets1−/− B cells by about twofold to threefold, suggesting that Ets1 represses expression of these genes. We used a retroviral vector encoding shRNAs to knockdown expression of these genes in B cells (Figure 6A) (59). Expression of shRNA against Egr1 was effective in reducing levels of Egr1 protein in stimulated B cells (Figure 6B), but did not impair formation of B220lowCD138+ plasmablasts (Figures 6C–E). The Prdm1 shRNA was also able to knockdown expression, although it was less efficient in Ets1−/− B cells (Figure 6B and data not shown). The Prdm1 shRNA did not alter ASC differentiation in WT or Ets1−/− B cells (Figures 6C–E), despite the fact that Blimp1 is known to be essential for plasma cell generation. This is likely because sufficient Blimp1 is still expressed to allow ASC differentiation. To further assess the role of Prdm1 in the phenotype of Ets1−/− B cells, we crossed Ets1−/− mice to mice carrying a GFP knock in allele in the Prdm1 locus that disrupts expression of the Prdm1 gene and leads to reduced levels of full-length functional Blimp1 protein (104). Homozygous Prdm1 knockout mice carrying this allele die embryonically due to a combination of developmental defects, but heterozygous mice are viable (104). We generated Ets1−/−Prdm1gfp/+ mice that carry a single copy of Prdm1 and express reduced levels of Blimp1 (Figure 7A). The numbers of ASCs in these mice and control mice was quantitated using ELISPOT, which showed that reduced levels of Prdm1 was not sufficient by itself to restrain excess ASC formation in the absence of Ets1 (Figure 7B). In summary, the changes in expression of the genes we tested (Stat4, Ptpn22, Egr1 and Prdm1) cannot by themselves explain the effects of Ets1 on ASC formation.


Genome-Wide Identification of Target Genes for the Key B Cell Transcription Factor Ets1
Reduction of selected target genes in Ets1−/− B cells. (A) Analysis of Blimp1 expression in lipopolysaccharide-stimulated Ets1+/+, Ets1−/−, and Ets1−/−Prdm1+/gfp B cells. The location of the full-length wild-type and the truncated proteins is indicated on the panel. (B) ELISPOT analysis of the numbers of IgM- and IgG-secreting cells in the spleens of unchallenged mice of the indicated genotypes (n = 4–5 for each genotype). *p < 0.05, **p < 0.01, and ***p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5383717&req=5

Figure 7: Reduction of selected target genes in Ets1−/− B cells. (A) Analysis of Blimp1 expression in lipopolysaccharide-stimulated Ets1+/+, Ets1−/−, and Ets1−/−Prdm1+/gfp B cells. The location of the full-length wild-type and the truncated proteins is indicated on the panel. (B) ELISPOT analysis of the numbers of IgM- and IgG-secreting cells in the spleens of unchallenged mice of the indicated genotypes (n = 4–5 for each genotype). *p < 0.05, **p < 0.01, and ***p < 0.001.
Mentions: Unlike Stat4 and Ptpn22, Egr1 and Prdm1 were upregulated in Ets1−/− B cells by about twofold to threefold, suggesting that Ets1 represses expression of these genes. We used a retroviral vector encoding shRNAs to knockdown expression of these genes in B cells (Figure 6A) (59). Expression of shRNA against Egr1 was effective in reducing levels of Egr1 protein in stimulated B cells (Figure 6B), but did not impair formation of B220lowCD138+ plasmablasts (Figures 6C–E). The Prdm1 shRNA was also able to knockdown expression, although it was less efficient in Ets1−/− B cells (Figure 6B and data not shown). The Prdm1 shRNA did not alter ASC differentiation in WT or Ets1−/− B cells (Figures 6C–E), despite the fact that Blimp1 is known to be essential for plasma cell generation. This is likely because sufficient Blimp1 is still expressed to allow ASC differentiation. To further assess the role of Prdm1 in the phenotype of Ets1−/− B cells, we crossed Ets1−/− mice to mice carrying a GFP knock in allele in the Prdm1 locus that disrupts expression of the Prdm1 gene and leads to reduced levels of full-length functional Blimp1 protein (104). Homozygous Prdm1 knockout mice carrying this allele die embryonically due to a combination of developmental defects, but heterozygous mice are viable (104). We generated Ets1−/−Prdm1gfp/+ mice that carry a single copy of Prdm1 and express reduced levels of Blimp1 (Figure 7A). The numbers of ASCs in these mice and control mice was quantitated using ELISPOT, which showed that reduced levels of Prdm1 was not sufficient by itself to restrain excess ASC formation in the absence of Ets1 (Figure 7B). In summary, the changes in expression of the genes we tested (Stat4, Ptpn22, Egr1 and Prdm1) cannot by themselves explain the effects of Ets1 on ASC formation.

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 &gt;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.