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Bcl11a is essential for lymphoid development and negatively regulates p53.

Yu Y, Wang J, Khaled W, Burke S, Li P, Chen X, Yang W, Jenkins NA, Copeland NG, Zhang S, Liu P - J. Exp. Med. (2012)

Bottom Line: Myeloid development, in contrast, is not obviously affected by the loss of Bcl11a.Bcl11a regulates expression of Bcl2, Bcl2-xL, and Mdm2, which inhibits p53 activities.Bcl11a is therefore essential for lymphopoiesis and negatively regulates p53 activities.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China.

ABSTRACT
Transcription factors play important roles in lymphopoiesis. We have previously demonstrated that Bcl11a is essential for normal lymphocyte development in the mouse embryo. We report here that, in the adult mouse, Bcl11a is expressed in most hematopoietic cells and is highly enriched in B cells, early T cell progenitors, common lymphoid progenitors (CLPs), and hematopoietic stem cells (HSCs). In the adult mouse, Bcl11a deletion causes apoptosis in early B cells and CLPs and completely abolishes the lymphoid development potential of HSCs to B, T, and NK cells. Myeloid development, in contrast, is not obviously affected by the loss of Bcl11a. Bcl11a regulates expression of Bcl2, Bcl2-xL, and Mdm2, which inhibits p53 activities. Overexpression of Bcl2 and Mdm2, or p53 deficiency, rescues both lethality and proliferative defects in Bcl11a-deficient early B cells and enables the mutant CLPs to differentiate to lymphocytes. Bcl11a is therefore essential for lymphopoiesis and negatively regulates p53 activities. Deletion of Bcl11a may represent a new approach for generating a mouse model that completely lacks an adaptive immune system.

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Analysis of Bcl11a binding sites in mouse pro–B cells. (A) qPCR validation of Bcl11a binding at the Mdm2, Mdm4, and Bcl2 loci in ChIP assay. Potential Bcl11a binding sites (red boxes) at the Mdm2 and Mdm4 loci, predicted as GGCCGG-containing sequences. Black bars indicate the qPCR-amplified regions of the validated sites. Potential Bcl11a binding sites (arrows) at the mouse Bcl2 locus, predicted as homologous regions to BCL11A-binding sites at the human BCL2 locus (ENCODE project), are analyzed by ChIP assay. The fold-enrichments of the amplified regions are (from 5′ to 3′) are as follows: 1.31 ± 0.40, 1.69 ± 0.32, 5.32 ± 0.92 (red arrow), 1.87 ± 0.001, 2.37 ± 0.60, 2.80 ± 0.43, 1.32 ± 0.21, 2.12 ± 0.44, 1.88 ± 0.21, 2.29 ± 0.40, 3.00 ± 0.16, and 2.68 ± 0.72. (B) Distribution of Bcl11a-binding sites in the genome from ChIP-Seq analysis. The 10-kb region upstream from transcription start site is defined as Proximal Promoter, and the 10-kb region downstream from transcription stop site is defined as downstream. (C) qPCR validation of Bcl11a binding at the genomic loci of Bcl-xL, p21, Pax5, and E2A. Genomic DNA pull-down using IgG was used as a control. A–C represent three independent experiments. *, P < 0.05; **, P < 0.01.
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fig8: Analysis of Bcl11a binding sites in mouse pro–B cells. (A) qPCR validation of Bcl11a binding at the Mdm2, Mdm4, and Bcl2 loci in ChIP assay. Potential Bcl11a binding sites (red boxes) at the Mdm2 and Mdm4 loci, predicted as GGCCGG-containing sequences. Black bars indicate the qPCR-amplified regions of the validated sites. Potential Bcl11a binding sites (arrows) at the mouse Bcl2 locus, predicted as homologous regions to BCL11A-binding sites at the human BCL2 locus (ENCODE project), are analyzed by ChIP assay. The fold-enrichments of the amplified regions are (from 5′ to 3′) are as follows: 1.31 ± 0.40, 1.69 ± 0.32, 5.32 ± 0.92 (red arrow), 1.87 ± 0.001, 2.37 ± 0.60, 2.80 ± 0.43, 1.32 ± 0.21, 2.12 ± 0.44, 1.88 ± 0.21, 2.29 ± 0.40, 3.00 ± 0.16, and 2.68 ± 0.72. (B) Distribution of Bcl11a-binding sites in the genome from ChIP-Seq analysis. The 10-kb region upstream from transcription start site is defined as Proximal Promoter, and the 10-kb region downstream from transcription stop site is defined as downstream. (C) qPCR validation of Bcl11a binding at the genomic loci of Bcl-xL, p21, Pax5, and E2A. Genomic DNA pull-down using IgG was used as a control. A–C represent three independent experiments. *, P < 0.05; **, P < 0.01.

Mentions: Bcl11a is a zinc finger transcription factor that binds to its target sites in the genome (Avram et al., 2002; Liu et al., 2006). The aforementioned genetic studies indicate that Bcl11a regulates p53 via Mdm2 or Mdm4 in lymphocyte development. To confirm this regulation, we performed chromatin immunoprecipitation (ChIP) in mouse B cells with an anti-Bcl11a antibody. Bcl11a was reported to have a consensus binding site as GGCCGG (Avram et al., 2002). Computational prediction indicated that there were several putative binding sites in the Mdm2 and Mdm4 loci (Fig. 8 A). Quantitative real-time PCR (qPCR) of ChIP pull-down DNA showed a 4.95-fold enrichment using the Bcl11a antibody compared with the IgG control in the promoter region of Mdm4 locus. A 3.08-fold enrichment of Bcl11a binding was also found at the Mdm2 locus (Fig. 8 A). Bcl11a deletion caused rapid down-regulation of Bcl2 in early B cells. Analysis of ENCODE (The Encyclopedia of DNA Elements) data shows that in human B lymphocytes, there are 15 putative Bcl11a binding sites at the human BCL2 intronic and downstream regions. We analyzed these genomic regions and found that 12 of them were conserved in the mouse (Fig. 8 A). qPCR results showed that a higher than twofold enrichment of Bcl11a binding was found at seven regions, with the highest fold enrichment (fivefold) being identified in an intronic region (Fig. 8 A).


Bcl11a is essential for lymphoid development and negatively regulates p53.

Yu Y, Wang J, Khaled W, Burke S, Li P, Chen X, Yang W, Jenkins NA, Copeland NG, Zhang S, Liu P - J. Exp. Med. (2012)

Analysis of Bcl11a binding sites in mouse pro–B cells. (A) qPCR validation of Bcl11a binding at the Mdm2, Mdm4, and Bcl2 loci in ChIP assay. Potential Bcl11a binding sites (red boxes) at the Mdm2 and Mdm4 loci, predicted as GGCCGG-containing sequences. Black bars indicate the qPCR-amplified regions of the validated sites. Potential Bcl11a binding sites (arrows) at the mouse Bcl2 locus, predicted as homologous regions to BCL11A-binding sites at the human BCL2 locus (ENCODE project), are analyzed by ChIP assay. The fold-enrichments of the amplified regions are (from 5′ to 3′) are as follows: 1.31 ± 0.40, 1.69 ± 0.32, 5.32 ± 0.92 (red arrow), 1.87 ± 0.001, 2.37 ± 0.60, 2.80 ± 0.43, 1.32 ± 0.21, 2.12 ± 0.44, 1.88 ± 0.21, 2.29 ± 0.40, 3.00 ± 0.16, and 2.68 ± 0.72. (B) Distribution of Bcl11a-binding sites in the genome from ChIP-Seq analysis. The 10-kb region upstream from transcription start site is defined as Proximal Promoter, and the 10-kb region downstream from transcription stop site is defined as downstream. (C) qPCR validation of Bcl11a binding at the genomic loci of Bcl-xL, p21, Pax5, and E2A. Genomic DNA pull-down using IgG was used as a control. A–C represent three independent experiments. *, P < 0.05; **, P < 0.01.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3526365&req=5

fig8: Analysis of Bcl11a binding sites in mouse pro–B cells. (A) qPCR validation of Bcl11a binding at the Mdm2, Mdm4, and Bcl2 loci in ChIP assay. Potential Bcl11a binding sites (red boxes) at the Mdm2 and Mdm4 loci, predicted as GGCCGG-containing sequences. Black bars indicate the qPCR-amplified regions of the validated sites. Potential Bcl11a binding sites (arrows) at the mouse Bcl2 locus, predicted as homologous regions to BCL11A-binding sites at the human BCL2 locus (ENCODE project), are analyzed by ChIP assay. The fold-enrichments of the amplified regions are (from 5′ to 3′) are as follows: 1.31 ± 0.40, 1.69 ± 0.32, 5.32 ± 0.92 (red arrow), 1.87 ± 0.001, 2.37 ± 0.60, 2.80 ± 0.43, 1.32 ± 0.21, 2.12 ± 0.44, 1.88 ± 0.21, 2.29 ± 0.40, 3.00 ± 0.16, and 2.68 ± 0.72. (B) Distribution of Bcl11a-binding sites in the genome from ChIP-Seq analysis. The 10-kb region upstream from transcription start site is defined as Proximal Promoter, and the 10-kb region downstream from transcription stop site is defined as downstream. (C) qPCR validation of Bcl11a binding at the genomic loci of Bcl-xL, p21, Pax5, and E2A. Genomic DNA pull-down using IgG was used as a control. A–C represent three independent experiments. *, P < 0.05; **, P < 0.01.
Mentions: Bcl11a is a zinc finger transcription factor that binds to its target sites in the genome (Avram et al., 2002; Liu et al., 2006). The aforementioned genetic studies indicate that Bcl11a regulates p53 via Mdm2 or Mdm4 in lymphocyte development. To confirm this regulation, we performed chromatin immunoprecipitation (ChIP) in mouse B cells with an anti-Bcl11a antibody. Bcl11a was reported to have a consensus binding site as GGCCGG (Avram et al., 2002). Computational prediction indicated that there were several putative binding sites in the Mdm2 and Mdm4 loci (Fig. 8 A). Quantitative real-time PCR (qPCR) of ChIP pull-down DNA showed a 4.95-fold enrichment using the Bcl11a antibody compared with the IgG control in the promoter region of Mdm4 locus. A 3.08-fold enrichment of Bcl11a binding was also found at the Mdm2 locus (Fig. 8 A). Bcl11a deletion caused rapid down-regulation of Bcl2 in early B cells. Analysis of ENCODE (The Encyclopedia of DNA Elements) data shows that in human B lymphocytes, there are 15 putative Bcl11a binding sites at the human BCL2 intronic and downstream regions. We analyzed these genomic regions and found that 12 of them were conserved in the mouse (Fig. 8 A). qPCR results showed that a higher than twofold enrichment of Bcl11a binding was found at seven regions, with the highest fold enrichment (fivefold) being identified in an intronic region (Fig. 8 A).

Bottom Line: Myeloid development, in contrast, is not obviously affected by the loss of Bcl11a.Bcl11a regulates expression of Bcl2, Bcl2-xL, and Mdm2, which inhibits p53 activities.Bcl11a is therefore essential for lymphopoiesis and negatively regulates p53 activities.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China.

ABSTRACT
Transcription factors play important roles in lymphopoiesis. We have previously demonstrated that Bcl11a is essential for normal lymphocyte development in the mouse embryo. We report here that, in the adult mouse, Bcl11a is expressed in most hematopoietic cells and is highly enriched in B cells, early T cell progenitors, common lymphoid progenitors (CLPs), and hematopoietic stem cells (HSCs). In the adult mouse, Bcl11a deletion causes apoptosis in early B cells and CLPs and completely abolishes the lymphoid development potential of HSCs to B, T, and NK cells. Myeloid development, in contrast, is not obviously affected by the loss of Bcl11a. Bcl11a regulates expression of Bcl2, Bcl2-xL, and Mdm2, which inhibits p53 activities. Overexpression of Bcl2 and Mdm2, or p53 deficiency, rescues both lethality and proliferative defects in Bcl11a-deficient early B cells and enables the mutant CLPs to differentiate to lymphocytes. Bcl11a is therefore essential for lymphopoiesis and negatively regulates p53 activities. Deletion of Bcl11a may represent a new approach for generating a mouse model that completely lacks an adaptive immune system.

Show MeSH
Related in: MedlinePlus