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The transcription factor Ets1 is important for CD4 repression and Runx3 up-regulation during CD8 T cell differentiation in the thymus.

Zamisch M, Tian L, Grenningloh R, Xiong Y, Wildt KF, Ehlers M, Ho IC, Bosselut R - J. Exp. Med. (2009)

Bottom Line: We further show that Ets1 promotes expression of Runx3, a transcription factor important for CD8 T cell differentiation and the cessation of Cd4 gene expression.Finally, we document that Ets1 binds at least two evolutionarily conserved regions within the Runx3 gene in vivo, supporting the possibility that Ets1 directly contributes to Runx3 transcription.These findings identify Ets1 as a key player during CD8 lineage differentiation and indicate that it acts, at least in part, by promoting Runx3 expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Immune Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
The transcription factor Ets1 contributes to the differentiation of CD8 lineage cells in the thymus, but how it does so is not understood. In this study, we demonstrate that Ets1 is required for the proper termination of CD4 expression during the differentiation of major histocompatability class 1 (MHC I)-restricted thymocytes, but not for other events associated with their positive selection, including the initiation of cytotoxic gene expression, corticomedullary migration, or thymus exit. We further show that Ets1 promotes expression of Runx3, a transcription factor important for CD8 T cell differentiation and the cessation of Cd4 gene expression. Enforced Runx3 expression in Ets1-deficient MHC I-restricted thymocytes largely rescued their impaired Cd4 silencing, indicating that Ets1 is not required for Runx3 function. Finally, we document that Ets1 binds at least two evolutionarily conserved regions within the Runx3 gene in vivo, supporting the possibility that Ets1 directly contributes to Runx3 transcription. These findings identify Ets1 as a key player during CD8 lineage differentiation and indicate that it acts, at least in part, by promoting Runx3 expression.

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Ets1 promotes Runx3 expression. (A and B) Expression of Runx3 was evaluated in mice carrying a BAC transgene in which a tRFP cDNA has been inserted within the second exon of Runx3. (A) Two-parameter contour plots of CD4 and CD8 expression (top) are gated on TCRhi CD24lo thymocytes from Ets1+/− and Ets1−/− mice. Subsets defined by boxes are numbered and analyzed for tRFP expression. Overlaid histograms (bottom) depict tRFP fluorescence in indicated subsets of tRFP-transgenic Ets1+/− and Ets1−/− mice. Gray-shaded histogram show background fluorescence in CD8 SP thymocytes from control Ets1+/+ nontransgenic mice. The mean intensity of tRFP fluorescence in subset 1 (maturelike DP cells from Ets1−/− mice) was 49% of that in subset 4 (CD8 SP cells from tRFP-transgenic Ets1+/− controls; mean on all three experiments). (B) Two parameter plots of tRFP and CD24 expression (bottom) are shown on TCRhi gated cells (histograms, top). Data (A and B) is representative of three mice of each genotype analyzed in three separate experiments. (C) Expression of Runx3 was assessed as in Fig. 4 on the same mRNA preparations and is shown relative to that in Ets1+/+ P14 CD4−CD8+ cells. The difference between Ets1−/− Vα2hi CD24lo DP and Ets1+/+ CD8 SP thymocytes for Runx3 expression was statistically significant (*, P < 10−4, two tailed Student's t test). Data are from more than three experiments. (D) Expression of Runx proteins was assessed in sorted thymocyte subsets by immunoblotting with an antibody directed against the Runt domain and recognizing both Runx1 and Runx3. CD4 SP thymocytes were sorted from wild-type mice and used as positive and negative controls for Runx1 and Runx3 expression, respectively. MW marker sizes are indicated on the left. Numbers underneath indicate expression of β-actin in each samples, quantified on the same membrane and expressed relative to that of wild-type CD8 SP thymocyte. The β-actin signal was consistently lower in DP thymocytes than in other cell subsets, but was not reproducibly affected by Ets1 disruption. The figure is a composite of two parts of a single blot (separated as indicated by the vertical black bar). Data are from three determinations performed from two distinct sets of sorted cells.
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fig6: Ets1 promotes Runx3 expression. (A and B) Expression of Runx3 was evaluated in mice carrying a BAC transgene in which a tRFP cDNA has been inserted within the second exon of Runx3. (A) Two-parameter contour plots of CD4 and CD8 expression (top) are gated on TCRhi CD24lo thymocytes from Ets1+/− and Ets1−/− mice. Subsets defined by boxes are numbered and analyzed for tRFP expression. Overlaid histograms (bottom) depict tRFP fluorescence in indicated subsets of tRFP-transgenic Ets1+/− and Ets1−/− mice. Gray-shaded histogram show background fluorescence in CD8 SP thymocytes from control Ets1+/+ nontransgenic mice. The mean intensity of tRFP fluorescence in subset 1 (maturelike DP cells from Ets1−/− mice) was 49% of that in subset 4 (CD8 SP cells from tRFP-transgenic Ets1+/− controls; mean on all three experiments). (B) Two parameter plots of tRFP and CD24 expression (bottom) are shown on TCRhi gated cells (histograms, top). Data (A and B) is representative of three mice of each genotype analyzed in three separate experiments. (C) Expression of Runx3 was assessed as in Fig. 4 on the same mRNA preparations and is shown relative to that in Ets1+/+ P14 CD4−CD8+ cells. The difference between Ets1−/− Vα2hi CD24lo DP and Ets1+/+ CD8 SP thymocytes for Runx3 expression was statistically significant (*, P < 10−4, two tailed Student's t test). Data are from more than three experiments. (D) Expression of Runx proteins was assessed in sorted thymocyte subsets by immunoblotting with an antibody directed against the Runt domain and recognizing both Runx1 and Runx3. CD4 SP thymocytes were sorted from wild-type mice and used as positive and negative controls for Runx1 and Runx3 expression, respectively. MW marker sizes are indicated on the left. Numbers underneath indicate expression of β-actin in each samples, quantified on the same membrane and expressed relative to that of wild-type CD8 SP thymocyte. The β-actin signal was consistently lower in DP thymocytes than in other cell subsets, but was not reproducibly affected by Ets1 disruption. The figure is a composite of two parts of a single blot (separated as indicated by the vertical black bar). Data are from three determinations performed from two distinct sets of sorted cells.

Mentions: To distinguish between these possibilities, we first examined Runx3 expression in Ets1−/− thymocytes. In wild-type mice, there is little or no Runx3 gene expression in preselection DP thymocytes, and its preferential up-regulation during the DP to CD8 SP transition results in higher mRNA levels in CD8 than in CD4 SP thymocytes (Taniuchi et al., 2002a; Liu and Bosselut, 2004; Egawa et al., 2007). Analyses of Runx3 expression in maturelike DP thymocytes selected by endogenously rearranged TCRs are hampered by the small numbers of these cells; an additional level of complexity comes from alternative promoter usage in the Runx3 gene, resulting in mRNA species that appear to not equally contribute to Runx3 protein synthesis (Egawa et al., 2007; Egawa and Littman, 2008). To overcome these obstacles, we introduced into Ets1−/− mice a transgenic BAC reporter in which the sequence coding for the tandem-dimer-tomato red fluorescent protein (tRFP ; Shaner et al., 2005) had been inserted into the second exon of the Runx3 gene (Fig. S4 A and unpublished data). As the tRFP cDNA insertion respects all Runx3 noncoding sequences, and as tRFP translation is initiated from endogenous Runx3 ATG codons, expression of tRFP in the thymus matched expression of endogenous Runx3 protein (Woolf et al., 2003; Egawa et al., 2007). In wild-type thymi, we readily detected tRFP in a subset of DN cells and in CD8 lineage thymocytes, whereas little or no expression was seen in DP and CD4 lineage cells (Fig. S4 B and unpublished data). Similarly, there was little tRFP fluorescence in Ets1−/− CD4 lineage cells. However, fluorescence intensities in CD8 SP thymocytes were slightly lower in Ets1−/− than in their wild-type counterparts and tRFP expression in maturelike DP thymocytes was half of that in wild-type CD8 SP thymocytes (Fig. 6 A). In fact, the fraction of positively selected (TCRhi) thymocytes that expressed Runx3, as well as their level of expression, were lower in Ets1-deficient than Ets1-sufficient thymocytes, indicating that the low expression observed on maturelike DP cells did not result from a gating bias (Fig. 6 B). These experiments indicated that Ets1 is important for appropriate Runx3 expression.


The transcription factor Ets1 is important for CD4 repression and Runx3 up-regulation during CD8 T cell differentiation in the thymus.

Zamisch M, Tian L, Grenningloh R, Xiong Y, Wildt KF, Ehlers M, Ho IC, Bosselut R - J. Exp. Med. (2009)

Ets1 promotes Runx3 expression. (A and B) Expression of Runx3 was evaluated in mice carrying a BAC transgene in which a tRFP cDNA has been inserted within the second exon of Runx3. (A) Two-parameter contour plots of CD4 and CD8 expression (top) are gated on TCRhi CD24lo thymocytes from Ets1+/− and Ets1−/− mice. Subsets defined by boxes are numbered and analyzed for tRFP expression. Overlaid histograms (bottom) depict tRFP fluorescence in indicated subsets of tRFP-transgenic Ets1+/− and Ets1−/− mice. Gray-shaded histogram show background fluorescence in CD8 SP thymocytes from control Ets1+/+ nontransgenic mice. The mean intensity of tRFP fluorescence in subset 1 (maturelike DP cells from Ets1−/− mice) was 49% of that in subset 4 (CD8 SP cells from tRFP-transgenic Ets1+/− controls; mean on all three experiments). (B) Two parameter plots of tRFP and CD24 expression (bottom) are shown on TCRhi gated cells (histograms, top). Data (A and B) is representative of three mice of each genotype analyzed in three separate experiments. (C) Expression of Runx3 was assessed as in Fig. 4 on the same mRNA preparations and is shown relative to that in Ets1+/+ P14 CD4−CD8+ cells. The difference between Ets1−/− Vα2hi CD24lo DP and Ets1+/+ CD8 SP thymocytes for Runx3 expression was statistically significant (*, P < 10−4, two tailed Student's t test). Data are from more than three experiments. (D) Expression of Runx proteins was assessed in sorted thymocyte subsets by immunoblotting with an antibody directed against the Runt domain and recognizing both Runx1 and Runx3. CD4 SP thymocytes were sorted from wild-type mice and used as positive and negative controls for Runx1 and Runx3 expression, respectively. MW marker sizes are indicated on the left. Numbers underneath indicate expression of β-actin in each samples, quantified on the same membrane and expressed relative to that of wild-type CD8 SP thymocyte. The β-actin signal was consistently lower in DP thymocytes than in other cell subsets, but was not reproducibly affected by Ets1 disruption. The figure is a composite of two parts of a single blot (separated as indicated by the vertical black bar). Data are from three determinations performed from two distinct sets of sorted cells.
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fig6: Ets1 promotes Runx3 expression. (A and B) Expression of Runx3 was evaluated in mice carrying a BAC transgene in which a tRFP cDNA has been inserted within the second exon of Runx3. (A) Two-parameter contour plots of CD4 and CD8 expression (top) are gated on TCRhi CD24lo thymocytes from Ets1+/− and Ets1−/− mice. Subsets defined by boxes are numbered and analyzed for tRFP expression. Overlaid histograms (bottom) depict tRFP fluorescence in indicated subsets of tRFP-transgenic Ets1+/− and Ets1−/− mice. Gray-shaded histogram show background fluorescence in CD8 SP thymocytes from control Ets1+/+ nontransgenic mice. The mean intensity of tRFP fluorescence in subset 1 (maturelike DP cells from Ets1−/− mice) was 49% of that in subset 4 (CD8 SP cells from tRFP-transgenic Ets1+/− controls; mean on all three experiments). (B) Two parameter plots of tRFP and CD24 expression (bottom) are shown on TCRhi gated cells (histograms, top). Data (A and B) is representative of three mice of each genotype analyzed in three separate experiments. (C) Expression of Runx3 was assessed as in Fig. 4 on the same mRNA preparations and is shown relative to that in Ets1+/+ P14 CD4−CD8+ cells. The difference between Ets1−/− Vα2hi CD24lo DP and Ets1+/+ CD8 SP thymocytes for Runx3 expression was statistically significant (*, P < 10−4, two tailed Student's t test). Data are from more than three experiments. (D) Expression of Runx proteins was assessed in sorted thymocyte subsets by immunoblotting with an antibody directed against the Runt domain and recognizing both Runx1 and Runx3. CD4 SP thymocytes were sorted from wild-type mice and used as positive and negative controls for Runx1 and Runx3 expression, respectively. MW marker sizes are indicated on the left. Numbers underneath indicate expression of β-actin in each samples, quantified on the same membrane and expressed relative to that of wild-type CD8 SP thymocyte. The β-actin signal was consistently lower in DP thymocytes than in other cell subsets, but was not reproducibly affected by Ets1 disruption. The figure is a composite of two parts of a single blot (separated as indicated by the vertical black bar). Data are from three determinations performed from two distinct sets of sorted cells.
Mentions: To distinguish between these possibilities, we first examined Runx3 expression in Ets1−/− thymocytes. In wild-type mice, there is little or no Runx3 gene expression in preselection DP thymocytes, and its preferential up-regulation during the DP to CD8 SP transition results in higher mRNA levels in CD8 than in CD4 SP thymocytes (Taniuchi et al., 2002a; Liu and Bosselut, 2004; Egawa et al., 2007). Analyses of Runx3 expression in maturelike DP thymocytes selected by endogenously rearranged TCRs are hampered by the small numbers of these cells; an additional level of complexity comes from alternative promoter usage in the Runx3 gene, resulting in mRNA species that appear to not equally contribute to Runx3 protein synthesis (Egawa et al., 2007; Egawa and Littman, 2008). To overcome these obstacles, we introduced into Ets1−/− mice a transgenic BAC reporter in which the sequence coding for the tandem-dimer-tomato red fluorescent protein (tRFP ; Shaner et al., 2005) had been inserted into the second exon of the Runx3 gene (Fig. S4 A and unpublished data). As the tRFP cDNA insertion respects all Runx3 noncoding sequences, and as tRFP translation is initiated from endogenous Runx3 ATG codons, expression of tRFP in the thymus matched expression of endogenous Runx3 protein (Woolf et al., 2003; Egawa et al., 2007). In wild-type thymi, we readily detected tRFP in a subset of DN cells and in CD8 lineage thymocytes, whereas little or no expression was seen in DP and CD4 lineage cells (Fig. S4 B and unpublished data). Similarly, there was little tRFP fluorescence in Ets1−/− CD4 lineage cells. However, fluorescence intensities in CD8 SP thymocytes were slightly lower in Ets1−/− than in their wild-type counterparts and tRFP expression in maturelike DP thymocytes was half of that in wild-type CD8 SP thymocytes (Fig. 6 A). In fact, the fraction of positively selected (TCRhi) thymocytes that expressed Runx3, as well as their level of expression, were lower in Ets1-deficient than Ets1-sufficient thymocytes, indicating that the low expression observed on maturelike DP cells did not result from a gating bias (Fig. 6 B). These experiments indicated that Ets1 is important for appropriate Runx3 expression.

Bottom Line: We further show that Ets1 promotes expression of Runx3, a transcription factor important for CD8 T cell differentiation and the cessation of Cd4 gene expression.Finally, we document that Ets1 binds at least two evolutionarily conserved regions within the Runx3 gene in vivo, supporting the possibility that Ets1 directly contributes to Runx3 transcription.These findings identify Ets1 as a key player during CD8 lineage differentiation and indicate that it acts, at least in part, by promoting Runx3 expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Immune Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
The transcription factor Ets1 contributes to the differentiation of CD8 lineage cells in the thymus, but how it does so is not understood. In this study, we demonstrate that Ets1 is required for the proper termination of CD4 expression during the differentiation of major histocompatability class 1 (MHC I)-restricted thymocytes, but not for other events associated with their positive selection, including the initiation of cytotoxic gene expression, corticomedullary migration, or thymus exit. We further show that Ets1 promotes expression of Runx3, a transcription factor important for CD8 T cell differentiation and the cessation of Cd4 gene expression. Enforced Runx3 expression in Ets1-deficient MHC I-restricted thymocytes largely rescued their impaired Cd4 silencing, indicating that Ets1 is not required for Runx3 function. Finally, we document that Ets1 binds at least two evolutionarily conserved regions within the Runx3 gene in vivo, supporting the possibility that Ets1 directly contributes to Runx3 transcription. These findings identify Ets1 as a key player during CD8 lineage differentiation and indicate that it acts, at least in part, by promoting Runx3 expression.

Show MeSH