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Combined heterozygous loss of Ebf1 and Pax5 allows for T-lineage conversion of B cell progenitors.

Ungerbäck J, Åhsberg J, Strid T, Somasundaram R, Sigvardsson M - J. Exp. Med. (2015)

Bottom Line: Whereas combined reduction of Pax5 and Ebf1 had minimal impact on the development of the earliest CD19(+) progenitors, these cells displayed an increased T cell potential in vivo and in vitro.This report stresses the importance of the levels of transcription factor expression during lymphocyte development, and suggests that Pax5 and Ebf1 collaborate to modulate the transcriptional response to Notch signaling.This provides an insight on how transcription factors like Ebf1 and Pax5 preserve cellular identity during differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical and Experimental Medicine, Experimental Hematopoiesis Unit, Faculty of Health Sciences, Linköping University, 58183 Linköping, Sweden.

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Combined dose reduction of Ebf1 and Pax5 results in alterations in gene expression patterns and Ebf1 binding but does not result in collapse of the B-lineage transcriptional program. (A) Heatmap of 204 Wt versus TH(Ebf1+/−Pax5+/−) differentially expressed genes in primary ex vivo sorted pro–B cells. Log2 RPKM values of Wt (n = 4), TH (n = 4), Ebf1+/− (n = 2), and Pax5+/− (n = 2), respectively, are displayed. Differentially expressed genes have been hierarchically clustered with the Cluster 3.0 software (Euclidean distance with average linkage). (B) Heatmap of 204 Wt versus TH differentially expressed genes in primary sorted probe cells. Log2 fold-changes between Wt (n = 4) and TH (n = 4), Wt and Ebf1+/− (n = 2), Wt and Pax5+/− (n = 2), respectively, are displayed. Differentially expressed genes have been clustered on Ebf1+/− and Pax5+/− (Revilla-I-Domingo et al., 2012; Vilagos et al., 2012) binding categories and sorted after increasing P values (top to bottom) as defined by Student’s t test (Benjamini-Hoeschberg corrected) for TH versus Wt differential gene expression. (C) Pie-charts displaying overlapping and nonoverlapping Ebf1+/− and Pax5+/− peaks (Revilla-I-Domingo et al., 2012; Vilagos et al., 2012) in the 204 TH versus Wt differentially regulated genes, as well as the 109 up-regulated and 95 down-regulated genes. Numbers in the pie-charts are percent of total peaks in each group. (D) Venn diagram representing the total number of differentially and shared Ebf1 bound sites determined by ChIP-seq analysis of Wt and TH pro–B cells. The bottom pie-charts represent the number of peaks detected in Wt cells displaying statistically significant changed binding in TH cells and the number of sites with P < 0.05 and fourfold of differentially bound Ebf1 peaks (444 peaks in total). Chromosomes X, Y, M, and Random were filtered out from the Wt and TH peak lists before analysis. The heatmap shows Ebf1 binding on the 444 peaks differentially bound in TH centered on the Wt Ebf1 peaks with a window of 5-kb and a bin size of 100 bp. Data were collected from two independent experiments from each genotype. (E) A list of genes (1467) closest to the 1635 differentially bound (P < 0.05) Ebf1 peaks was compared with the list of genes that are differentially expressed between Wt and TH and co-bound by both Pax5 and Ebf1 as defined by ChIP seq from (Revilla-I-Domingo et al., 2012; Vilagos et al., 2012). (F) Tags from Ebf1 ChIP-seq in Wt and TH samples were plotted centered on peaks defined by the P < 0.05 differentially bound peak list, found within 100 kb of genes that were up or down-regulated in TH versus Wt as indicated.
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fig3: Combined dose reduction of Ebf1 and Pax5 results in alterations in gene expression patterns and Ebf1 binding but does not result in collapse of the B-lineage transcriptional program. (A) Heatmap of 204 Wt versus TH(Ebf1+/−Pax5+/−) differentially expressed genes in primary ex vivo sorted pro–B cells. Log2 RPKM values of Wt (n = 4), TH (n = 4), Ebf1+/− (n = 2), and Pax5+/− (n = 2), respectively, are displayed. Differentially expressed genes have been hierarchically clustered with the Cluster 3.0 software (Euclidean distance with average linkage). (B) Heatmap of 204 Wt versus TH differentially expressed genes in primary sorted probe cells. Log2 fold-changes between Wt (n = 4) and TH (n = 4), Wt and Ebf1+/− (n = 2), Wt and Pax5+/− (n = 2), respectively, are displayed. Differentially expressed genes have been clustered on Ebf1+/− and Pax5+/− (Revilla-I-Domingo et al., 2012; Vilagos et al., 2012) binding categories and sorted after increasing P values (top to bottom) as defined by Student’s t test (Benjamini-Hoeschberg corrected) for TH versus Wt differential gene expression. (C) Pie-charts displaying overlapping and nonoverlapping Ebf1+/− and Pax5+/− peaks (Revilla-I-Domingo et al., 2012; Vilagos et al., 2012) in the 204 TH versus Wt differentially regulated genes, as well as the 109 up-regulated and 95 down-regulated genes. Numbers in the pie-charts are percent of total peaks in each group. (D) Venn diagram representing the total number of differentially and shared Ebf1 bound sites determined by ChIP-seq analysis of Wt and TH pro–B cells. The bottom pie-charts represent the number of peaks detected in Wt cells displaying statistically significant changed binding in TH cells and the number of sites with P < 0.05 and fourfold of differentially bound Ebf1 peaks (444 peaks in total). Chromosomes X, Y, M, and Random were filtered out from the Wt and TH peak lists before analysis. The heatmap shows Ebf1 binding on the 444 peaks differentially bound in TH centered on the Wt Ebf1 peaks with a window of 5-kb and a bin size of 100 bp. Data were collected from two independent experiments from each genotype. (E) A list of genes (1467) closest to the 1635 differentially bound (P < 0.05) Ebf1 peaks was compared with the list of genes that are differentially expressed between Wt and TH and co-bound by both Pax5 and Ebf1 as defined by ChIP seq from (Revilla-I-Domingo et al., 2012; Vilagos et al., 2012). (F) Tags from Ebf1 ChIP-seq in Wt and TH samples were plotted centered on peaks defined by the P < 0.05 differentially bound peak list, found within 100 kb of genes that were up or down-regulated in TH versus Wt as indicated.

Mentions: Lineage plasticity in Ebf1−/− or Pax5−/− B cell progenitors has been linked to rather drastic changes in gene expression patterns, including increased expression of T-lineage associated genes such as Notch1 (Souabni et al., 2002; Nechanitzky et al., 2013). To investigate how the combined dose reduction of Pax5 and Ebf1 would impact the transcriptional program in the pro–B cell compartment, we sorted pro–B cells from Wt, Ebf1+/−, Pax5+/−, and Pax5+/−Ebf1+/− animals and performed RNA sequencing analysis. Analysis of the data suggested that even though the transcriptome of Wt and Pax5+/−Ebf1+/− pro–B cells differed in expression at 204 genes (Fig. 3 A), the levels of classical B- or T-lineage genes, including Ebf1 and Pax5 target genes such as CD79α, CD79β, Notch1, or Gata3, were not significantly altered in mice carrying combined heterozygous deletion of Ebf1 and Pax5. K.E.G.G pathway analysis of the differentially expressed genes highlighted a significant difference (Benjamini-Hochberg corrected, P < 0.05) in the categories Cytokine–cytokine receptor interactions, Acute Myeloid Leukemia, Nod-like receptor signaling pathway, Leukocyte transendothelial migration, and NK cell–mediated cytotoxicity. Even though genes involved in cellular signaling were differentially expressed, we did not detect any obvious changes that could be linked to the cellular plasticity observed in Pax5+/−Ebf1+/− pro–B cells. Analysis of the gene expression data suggested that a major part of the genes differentially expressed in the Pax5+/−Ebf1+/− cells was dependent on the combined loss of Ebf1 and Pax5, as the RNA levels were not significantly altered in the single heterozygous cells. To investigate if the differentially expressed genes were direct targets for Ebf1, Pax5, or both, we investigated the binding of these transcription factors in the proximity of the differentially expressed genes using existing Rag2−/− pro–B cell ChIP-sequencing data (Revilla-I-Domingo et al., 2012; Vilagos et al., 2012). This indicated that 85 (42%) of the differentially expressed genes (≥2-fold) contained either overlapping or nonoverlapping binding sites for both Ebf1 and Pax5 (Fig. 3, B and C), whereas an additional 32% contained binding sites for either Ebf1 or Pax5 (Fig. 3 B). Hence, the majority of the differentially regulated genes are direct targets for Ebf1, Pax5, or both. 29% of the up-regulated and 33% of the down-regulated genes contained overlapping peaks (Fig. 3 C), supporting the notion that Pax5 and Ebf1 share a substantial number of regulatory elements.


Combined heterozygous loss of Ebf1 and Pax5 allows for T-lineage conversion of B cell progenitors.

Ungerbäck J, Åhsberg J, Strid T, Somasundaram R, Sigvardsson M - J. Exp. Med. (2015)

Combined dose reduction of Ebf1 and Pax5 results in alterations in gene expression patterns and Ebf1 binding but does not result in collapse of the B-lineage transcriptional program. (A) Heatmap of 204 Wt versus TH(Ebf1+/−Pax5+/−) differentially expressed genes in primary ex vivo sorted pro–B cells. Log2 RPKM values of Wt (n = 4), TH (n = 4), Ebf1+/− (n = 2), and Pax5+/− (n = 2), respectively, are displayed. Differentially expressed genes have been hierarchically clustered with the Cluster 3.0 software (Euclidean distance with average linkage). (B) Heatmap of 204 Wt versus TH differentially expressed genes in primary sorted probe cells. Log2 fold-changes between Wt (n = 4) and TH (n = 4), Wt and Ebf1+/− (n = 2), Wt and Pax5+/− (n = 2), respectively, are displayed. Differentially expressed genes have been clustered on Ebf1+/− and Pax5+/− (Revilla-I-Domingo et al., 2012; Vilagos et al., 2012) binding categories and sorted after increasing P values (top to bottom) as defined by Student’s t test (Benjamini-Hoeschberg corrected) for TH versus Wt differential gene expression. (C) Pie-charts displaying overlapping and nonoverlapping Ebf1+/− and Pax5+/− peaks (Revilla-I-Domingo et al., 2012; Vilagos et al., 2012) in the 204 TH versus Wt differentially regulated genes, as well as the 109 up-regulated and 95 down-regulated genes. Numbers in the pie-charts are percent of total peaks in each group. (D) Venn diagram representing the total number of differentially and shared Ebf1 bound sites determined by ChIP-seq analysis of Wt and TH pro–B cells. The bottom pie-charts represent the number of peaks detected in Wt cells displaying statistically significant changed binding in TH cells and the number of sites with P < 0.05 and fourfold of differentially bound Ebf1 peaks (444 peaks in total). Chromosomes X, Y, M, and Random were filtered out from the Wt and TH peak lists before analysis. The heatmap shows Ebf1 binding on the 444 peaks differentially bound in TH centered on the Wt Ebf1 peaks with a window of 5-kb and a bin size of 100 bp. Data were collected from two independent experiments from each genotype. (E) A list of genes (1467) closest to the 1635 differentially bound (P < 0.05) Ebf1 peaks was compared with the list of genes that are differentially expressed between Wt and TH and co-bound by both Pax5 and Ebf1 as defined by ChIP seq from (Revilla-I-Domingo et al., 2012; Vilagos et al., 2012). (F) Tags from Ebf1 ChIP-seq in Wt and TH samples were plotted centered on peaks defined by the P < 0.05 differentially bound peak list, found within 100 kb of genes that were up or down-regulated in TH versus Wt as indicated.
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fig3: Combined dose reduction of Ebf1 and Pax5 results in alterations in gene expression patterns and Ebf1 binding but does not result in collapse of the B-lineage transcriptional program. (A) Heatmap of 204 Wt versus TH(Ebf1+/−Pax5+/−) differentially expressed genes in primary ex vivo sorted pro–B cells. Log2 RPKM values of Wt (n = 4), TH (n = 4), Ebf1+/− (n = 2), and Pax5+/− (n = 2), respectively, are displayed. Differentially expressed genes have been hierarchically clustered with the Cluster 3.0 software (Euclidean distance with average linkage). (B) Heatmap of 204 Wt versus TH differentially expressed genes in primary sorted probe cells. Log2 fold-changes between Wt (n = 4) and TH (n = 4), Wt and Ebf1+/− (n = 2), Wt and Pax5+/− (n = 2), respectively, are displayed. Differentially expressed genes have been clustered on Ebf1+/− and Pax5+/− (Revilla-I-Domingo et al., 2012; Vilagos et al., 2012) binding categories and sorted after increasing P values (top to bottom) as defined by Student’s t test (Benjamini-Hoeschberg corrected) for TH versus Wt differential gene expression. (C) Pie-charts displaying overlapping and nonoverlapping Ebf1+/− and Pax5+/− peaks (Revilla-I-Domingo et al., 2012; Vilagos et al., 2012) in the 204 TH versus Wt differentially regulated genes, as well as the 109 up-regulated and 95 down-regulated genes. Numbers in the pie-charts are percent of total peaks in each group. (D) Venn diagram representing the total number of differentially and shared Ebf1 bound sites determined by ChIP-seq analysis of Wt and TH pro–B cells. The bottom pie-charts represent the number of peaks detected in Wt cells displaying statistically significant changed binding in TH cells and the number of sites with P < 0.05 and fourfold of differentially bound Ebf1 peaks (444 peaks in total). Chromosomes X, Y, M, and Random were filtered out from the Wt and TH peak lists before analysis. The heatmap shows Ebf1 binding on the 444 peaks differentially bound in TH centered on the Wt Ebf1 peaks with a window of 5-kb and a bin size of 100 bp. Data were collected from two independent experiments from each genotype. (E) A list of genes (1467) closest to the 1635 differentially bound (P < 0.05) Ebf1 peaks was compared with the list of genes that are differentially expressed between Wt and TH and co-bound by both Pax5 and Ebf1 as defined by ChIP seq from (Revilla-I-Domingo et al., 2012; Vilagos et al., 2012). (F) Tags from Ebf1 ChIP-seq in Wt and TH samples were plotted centered on peaks defined by the P < 0.05 differentially bound peak list, found within 100 kb of genes that were up or down-regulated in TH versus Wt as indicated.
Mentions: Lineage plasticity in Ebf1−/− or Pax5−/− B cell progenitors has been linked to rather drastic changes in gene expression patterns, including increased expression of T-lineage associated genes such as Notch1 (Souabni et al., 2002; Nechanitzky et al., 2013). To investigate how the combined dose reduction of Pax5 and Ebf1 would impact the transcriptional program in the pro–B cell compartment, we sorted pro–B cells from Wt, Ebf1+/−, Pax5+/−, and Pax5+/−Ebf1+/− animals and performed RNA sequencing analysis. Analysis of the data suggested that even though the transcriptome of Wt and Pax5+/−Ebf1+/− pro–B cells differed in expression at 204 genes (Fig. 3 A), the levels of classical B- or T-lineage genes, including Ebf1 and Pax5 target genes such as CD79α, CD79β, Notch1, or Gata3, were not significantly altered in mice carrying combined heterozygous deletion of Ebf1 and Pax5. K.E.G.G pathway analysis of the differentially expressed genes highlighted a significant difference (Benjamini-Hochberg corrected, P < 0.05) in the categories Cytokine–cytokine receptor interactions, Acute Myeloid Leukemia, Nod-like receptor signaling pathway, Leukocyte transendothelial migration, and NK cell–mediated cytotoxicity. Even though genes involved in cellular signaling were differentially expressed, we did not detect any obvious changes that could be linked to the cellular plasticity observed in Pax5+/−Ebf1+/− pro–B cells. Analysis of the gene expression data suggested that a major part of the genes differentially expressed in the Pax5+/−Ebf1+/− cells was dependent on the combined loss of Ebf1 and Pax5, as the RNA levels were not significantly altered in the single heterozygous cells. To investigate if the differentially expressed genes were direct targets for Ebf1, Pax5, or both, we investigated the binding of these transcription factors in the proximity of the differentially expressed genes using existing Rag2−/− pro–B cell ChIP-sequencing data (Revilla-I-Domingo et al., 2012; Vilagos et al., 2012). This indicated that 85 (42%) of the differentially expressed genes (≥2-fold) contained either overlapping or nonoverlapping binding sites for both Ebf1 and Pax5 (Fig. 3, B and C), whereas an additional 32% contained binding sites for either Ebf1 or Pax5 (Fig. 3 B). Hence, the majority of the differentially regulated genes are direct targets for Ebf1, Pax5, or both. 29% of the up-regulated and 33% of the down-regulated genes contained overlapping peaks (Fig. 3 C), supporting the notion that Pax5 and Ebf1 share a substantial number of regulatory elements.

Bottom Line: Whereas combined reduction of Pax5 and Ebf1 had minimal impact on the development of the earliest CD19(+) progenitors, these cells displayed an increased T cell potential in vivo and in vitro.This report stresses the importance of the levels of transcription factor expression during lymphocyte development, and suggests that Pax5 and Ebf1 collaborate to modulate the transcriptional response to Notch signaling.This provides an insight on how transcription factors like Ebf1 and Pax5 preserve cellular identity during differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical and Experimental Medicine, Experimental Hematopoiesis Unit, Faculty of Health Sciences, Linköping University, 58183 Linköping, Sweden.

Show MeSH
Related in: MedlinePlus