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Lack of the ubiquitin-editing enzyme A20 results in loss of hematopoietic stem cell quiescence.

Nakagawa MM, Thummar K, Mandelbaum J, Pasqualucci L, Rathinam CV - J. Exp. Med. (2015)

Bottom Line: Lack of A20 in HSCs results in diminished pool size, impaired radioprotection, defective repopulation, and loss of quiescence.Strikingly, deletion of both IFN-γ and A20 in hematopoietic cells results in partial rescue of the HSC phenotype.We anticipate that our experiments will facilitate the understanding of mechanisms through which A20-mediated inflammatory signals control HSC quiescence and functions.

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Affiliation: Department of Genetics and Development, Institute for Cancer Genetics, and Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY 10032.

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Increased IFN-γ signaling in A20-deficient HSCs. (A) Real-time PCR data indicating expression levels of Ifn-γ transcripts in the BM cells and splenocytes of 14-d-old A20Hem-KO and control mice. Expression levels of Ifn-γ were normalized to Hprt levels. (B) FACS plots indicating YFP expression in the BM cells and splenocytes of IFN-γ–YFP reporter (GREAT) mice, crossed with either control or A20Hem-KO mice. (A and B) Data are representative of three independent experiments. (C) Diagrammatic representation of the Ifn-γ gene indicating the presence of five NF-κB–binding sites in its promoter. (D) NF-κB–binding site in the promoter of Ifn-γ gene of the indicated species. (E) ChIP analysis of NF-κB (p65) binding to the Ifn-γ promoter in the BM cells of 14-d-old A20Hem-KO and control mice. Shown are the real-time PCR data of p65 immunoprecipitates, which were normalized to IgG control immunoprecipitates. Data are representative of two independent experiments. (F) Real-time PCR data indicating expression of IFN-γ target genes in CD150+LSK cells from 14-d-old A20Hem-KO and control mice. Expression levels of target genes were normalized to Hprt levels. Data are pooled from two independent experiments with six mice per group. (G and H) Representative FACS plots (G) and cumulative frequencies (H) indicating expression levels of Sca1 in Lin−c-Kit+CD150+ cells of the BM from 14-d-old A20Hem-KO and control mice. Data are representative of 10 independent experiments (G) and are pooled from six mice per group (H). (I) Real-time PCR data indicating expression levels of IFN-γ–controlled cell cycle regulators in HSCs of 14-d-old A20Hem-KO and control mice. Expression levels of target genes were normalized to Hprt levels. Data are representative of two independent experiments with six mice per group. All data represent mean ± SEM. Two-tailed Student’s t tests were used to assess statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
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fig6: Increased IFN-γ signaling in A20-deficient HSCs. (A) Real-time PCR data indicating expression levels of Ifn-γ transcripts in the BM cells and splenocytes of 14-d-old A20Hem-KO and control mice. Expression levels of Ifn-γ were normalized to Hprt levels. (B) FACS plots indicating YFP expression in the BM cells and splenocytes of IFN-γ–YFP reporter (GREAT) mice, crossed with either control or A20Hem-KO mice. (A and B) Data are representative of three independent experiments. (C) Diagrammatic representation of the Ifn-γ gene indicating the presence of five NF-κB–binding sites in its promoter. (D) NF-κB–binding site in the promoter of Ifn-γ gene of the indicated species. (E) ChIP analysis of NF-κB (p65) binding to the Ifn-γ promoter in the BM cells of 14-d-old A20Hem-KO and control mice. Shown are the real-time PCR data of p65 immunoprecipitates, which were normalized to IgG control immunoprecipitates. Data are representative of two independent experiments. (F) Real-time PCR data indicating expression of IFN-γ target genes in CD150+LSK cells from 14-d-old A20Hem-KO and control mice. Expression levels of target genes were normalized to Hprt levels. Data are pooled from two independent experiments with six mice per group. (G and H) Representative FACS plots (G) and cumulative frequencies (H) indicating expression levels of Sca1 in Lin−c-Kit+CD150+ cells of the BM from 14-d-old A20Hem-KO and control mice. Data are representative of 10 independent experiments (G) and are pooled from six mice per group (H). (I) Real-time PCR data indicating expression levels of IFN-γ–controlled cell cycle regulators in HSCs of 14-d-old A20Hem-KO and control mice. Expression levels of target genes were normalized to Hprt levels. Data are representative of two independent experiments with six mice per group. All data represent mean ± SEM. Two-tailed Student’s t tests were used to assess statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Mentions: Earlier studies identified IFN-γ as a direct target of NF-κB proteins (Sica et al., 1997), and based on the critical role of IFN-γ in the maintenance of HSCs (Baldridge et al., 2011; King and Goodell, 2011), we hypothesized that deregulated IFN-γ signaling might be potentially responsible for the HSC phenotype of A20Hem-KO mice. To test this, we measured the expression levels of IFN-γ in A20Hem-KO mice. In line with our hypothesis, we detected elevated levels of Ifn-γ mRNA in both BM and spleen of A20-deficient mice (Fig. 6 A). To substantiate this finding and to identify the cellular source of IFN-γ in A20 mutant mice, we crossed A20Hem-KO mice with the IFN-γ–YFP reporter GREAT (IFN-γ reporter with endogenous polyA transcript) mice (Reinhardt et al., 2009). These transgenic mice have an IRES-eYFP reporter cassette inserted between the translational stop codon and the 3′ UTR/polyA tail of the Ifn-γ gene (Reinhardt et al., 2009). Our analysis of hematopoietic compartments from GREAT-A20Hem-KO mice suggested an elevated expression of YFP and, therefore, IFN-γ in CD11C+, CD3+CD4+, and CD3+CD8+ cells of BM and spleen (Fig. 6 B). In addition, myeloid cells, such as granulocytes and monocytes, from GREAT-A20Hem-KO mice also expressed YFP, albeit at lower levels (Fig. 6 B). Next, we tested whether Ifn-γ is a direct target of NF-κB in A20 mutant BM cells. Our analysis of mouse Ifn-γ gene sequences indicated the presence of five (AAGGACTTCCTC) NF-κB–binding sites (Wong et al., 2011) in its promoter region. Among these sites, one NF-κB–binding site (from −541 to −530) is highly conserved across species (Fig. 6, C and D), and chromatin immunoprecipitation (ChIP) data revealed an increased binding of NF-κB to the Ifn-γ promoter in A20Hem-KO BM cells (Fig. 6 E). To understand the physiological relevance of deregulated IFN-γ expression on the HSC compartment, we investigated whether IFN-γ signaling was augmented in the HSCs from A20Hem-KO mice. Real-time PCR data indicated that the expression levels of IFN-γ target genes, such as Irf1, Irgm1, and STAT1, were elevated in A20 mutant HSCs (Fig. 6 F). Deregulated IFN-γ signaling results in up-regulated surface expression of Sca1 in HSPCs, as Sca1 is a direct target of IFN-γ (Snapper et al., 1991; King and Goodell, 2011). Our analysis of the LSK compartment indicated increased surface expression of Sca1 in hematopoietic progenitors and in total BM cells of A20Hem-KO mice (Fig. 6, G and H; and not depicted). Intriguingly, up-regulation of Sca1 in HSPCs has been connected with increased cell cycle activity (Essers et al., 2009).


Lack of the ubiquitin-editing enzyme A20 results in loss of hematopoietic stem cell quiescence.

Nakagawa MM, Thummar K, Mandelbaum J, Pasqualucci L, Rathinam CV - J. Exp. Med. (2015)

Increased IFN-γ signaling in A20-deficient HSCs. (A) Real-time PCR data indicating expression levels of Ifn-γ transcripts in the BM cells and splenocytes of 14-d-old A20Hem-KO and control mice. Expression levels of Ifn-γ were normalized to Hprt levels. (B) FACS plots indicating YFP expression in the BM cells and splenocytes of IFN-γ–YFP reporter (GREAT) mice, crossed with either control or A20Hem-KO mice. (A and B) Data are representative of three independent experiments. (C) Diagrammatic representation of the Ifn-γ gene indicating the presence of five NF-κB–binding sites in its promoter. (D) NF-κB–binding site in the promoter of Ifn-γ gene of the indicated species. (E) ChIP analysis of NF-κB (p65) binding to the Ifn-γ promoter in the BM cells of 14-d-old A20Hem-KO and control mice. Shown are the real-time PCR data of p65 immunoprecipitates, which were normalized to IgG control immunoprecipitates. Data are representative of two independent experiments. (F) Real-time PCR data indicating expression of IFN-γ target genes in CD150+LSK cells from 14-d-old A20Hem-KO and control mice. Expression levels of target genes were normalized to Hprt levels. Data are pooled from two independent experiments with six mice per group. (G and H) Representative FACS plots (G) and cumulative frequencies (H) indicating expression levels of Sca1 in Lin−c-Kit+CD150+ cells of the BM from 14-d-old A20Hem-KO and control mice. Data are representative of 10 independent experiments (G) and are pooled from six mice per group (H). (I) Real-time PCR data indicating expression levels of IFN-γ–controlled cell cycle regulators in HSCs of 14-d-old A20Hem-KO and control mice. Expression levels of target genes were normalized to Hprt levels. Data are representative of two independent experiments with six mice per group. All data represent mean ± SEM. Two-tailed Student’s t tests were used to assess statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
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fig6: Increased IFN-γ signaling in A20-deficient HSCs. (A) Real-time PCR data indicating expression levels of Ifn-γ transcripts in the BM cells and splenocytes of 14-d-old A20Hem-KO and control mice. Expression levels of Ifn-γ were normalized to Hprt levels. (B) FACS plots indicating YFP expression in the BM cells and splenocytes of IFN-γ–YFP reporter (GREAT) mice, crossed with either control or A20Hem-KO mice. (A and B) Data are representative of three independent experiments. (C) Diagrammatic representation of the Ifn-γ gene indicating the presence of five NF-κB–binding sites in its promoter. (D) NF-κB–binding site in the promoter of Ifn-γ gene of the indicated species. (E) ChIP analysis of NF-κB (p65) binding to the Ifn-γ promoter in the BM cells of 14-d-old A20Hem-KO and control mice. Shown are the real-time PCR data of p65 immunoprecipitates, which were normalized to IgG control immunoprecipitates. Data are representative of two independent experiments. (F) Real-time PCR data indicating expression of IFN-γ target genes in CD150+LSK cells from 14-d-old A20Hem-KO and control mice. Expression levels of target genes were normalized to Hprt levels. Data are pooled from two independent experiments with six mice per group. (G and H) Representative FACS plots (G) and cumulative frequencies (H) indicating expression levels of Sca1 in Lin−c-Kit+CD150+ cells of the BM from 14-d-old A20Hem-KO and control mice. Data are representative of 10 independent experiments (G) and are pooled from six mice per group (H). (I) Real-time PCR data indicating expression levels of IFN-γ–controlled cell cycle regulators in HSCs of 14-d-old A20Hem-KO and control mice. Expression levels of target genes were normalized to Hprt levels. Data are representative of two independent experiments with six mice per group. All data represent mean ± SEM. Two-tailed Student’s t tests were used to assess statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
Mentions: Earlier studies identified IFN-γ as a direct target of NF-κB proteins (Sica et al., 1997), and based on the critical role of IFN-γ in the maintenance of HSCs (Baldridge et al., 2011; King and Goodell, 2011), we hypothesized that deregulated IFN-γ signaling might be potentially responsible for the HSC phenotype of A20Hem-KO mice. To test this, we measured the expression levels of IFN-γ in A20Hem-KO mice. In line with our hypothesis, we detected elevated levels of Ifn-γ mRNA in both BM and spleen of A20-deficient mice (Fig. 6 A). To substantiate this finding and to identify the cellular source of IFN-γ in A20 mutant mice, we crossed A20Hem-KO mice with the IFN-γ–YFP reporter GREAT (IFN-γ reporter with endogenous polyA transcript) mice (Reinhardt et al., 2009). These transgenic mice have an IRES-eYFP reporter cassette inserted between the translational stop codon and the 3′ UTR/polyA tail of the Ifn-γ gene (Reinhardt et al., 2009). Our analysis of hematopoietic compartments from GREAT-A20Hem-KO mice suggested an elevated expression of YFP and, therefore, IFN-γ in CD11C+, CD3+CD4+, and CD3+CD8+ cells of BM and spleen (Fig. 6 B). In addition, myeloid cells, such as granulocytes and monocytes, from GREAT-A20Hem-KO mice also expressed YFP, albeit at lower levels (Fig. 6 B). Next, we tested whether Ifn-γ is a direct target of NF-κB in A20 mutant BM cells. Our analysis of mouse Ifn-γ gene sequences indicated the presence of five (AAGGACTTCCTC) NF-κB–binding sites (Wong et al., 2011) in its promoter region. Among these sites, one NF-κB–binding site (from −541 to −530) is highly conserved across species (Fig. 6, C and D), and chromatin immunoprecipitation (ChIP) data revealed an increased binding of NF-κB to the Ifn-γ promoter in A20Hem-KO BM cells (Fig. 6 E). To understand the physiological relevance of deregulated IFN-γ expression on the HSC compartment, we investigated whether IFN-γ signaling was augmented in the HSCs from A20Hem-KO mice. Real-time PCR data indicated that the expression levels of IFN-γ target genes, such as Irf1, Irgm1, and STAT1, were elevated in A20 mutant HSCs (Fig. 6 F). Deregulated IFN-γ signaling results in up-regulated surface expression of Sca1 in HSPCs, as Sca1 is a direct target of IFN-γ (Snapper et al., 1991; King and Goodell, 2011). Our analysis of the LSK compartment indicated increased surface expression of Sca1 in hematopoietic progenitors and in total BM cells of A20Hem-KO mice (Fig. 6, G and H; and not depicted). Intriguingly, up-regulation of Sca1 in HSPCs has been connected with increased cell cycle activity (Essers et al., 2009).

Bottom Line: Lack of A20 in HSCs results in diminished pool size, impaired radioprotection, defective repopulation, and loss of quiescence.Strikingly, deletion of both IFN-γ and A20 in hematopoietic cells results in partial rescue of the HSC phenotype.We anticipate that our experiments will facilitate the understanding of mechanisms through which A20-mediated inflammatory signals control HSC quiescence and functions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics and Development, Institute for Cancer Genetics, and Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY 10032.

Show MeSH
Related in: MedlinePlus