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RUNX transcription factor-mediated association of Cd4 and Cd8 enables coordinate gene regulation.

Collins A, Hewitt SL, Chaumeil J, Sellars M, Micsinai M, Allinne J, Parisi F, Nora EP, Bolland DJ, Corcoran AE, Kluger Y, Bosselut R, Ellmeier W, Chong MM, Littman DR, Skok JA - Immunity (2011)

Bottom Line: Here we found that nuclear organization was altered by interplay among members of this transcription factor circuitry: RUNX binding mediated association of Cd4 and Cd8 whereas ThPOK binding kept the loci apart.Moreover, targeted deletions within Cd4 modulated CD8 expression and pericentromeric repositioning of Cd8.Communication between Cd4 and Cd8 thus appears to enable long-range epigenetic regulation to ensure that expression of one excludes the other in mature CD4 or CD8 single-positive (SP) cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathogenesis Program, The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA.

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The Cd4 Proximal Enhancer Inhibits Cd4-Cd8 Association(A) Flow cytometry analysis of wild-type and Cd4 PE-deficient thymocytes (Cd4 PE Δ/Δ).(B) Cd4-Cd8 association in wild-type and Cd4 PE-deficient cells, including statistical analysis. Association is higher in Cd4 PE-deficient DN, CD4+CD8lo, and CD4 SP cells than in wild-type cells. n = 196–286 alleles.(C) Confocal microscopy sections of Cd4-Cd8 distances representative of each genotype. Scale bars represent 1 μm.(D) Recruitment of Cd4 to pericentromeric heterochromatin in wild-type and Cd4 PE-deficient cells. Recruitment is higher in Cd4 PE-deficient DN, CD4+CD8lo and CD4 SP than in wild-type cells.(E) RT-PCR analysis of Cd4 or Cd8a expression in wild-type and Cd4 PE-deficient cells. Standard error bars were calculated from three independent experiments.See also Figure S5 and Table S4.
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fig5: The Cd4 Proximal Enhancer Inhibits Cd4-Cd8 Association(A) Flow cytometry analysis of wild-type and Cd4 PE-deficient thymocytes (Cd4 PE Δ/Δ).(B) Cd4-Cd8 association in wild-type and Cd4 PE-deficient cells, including statistical analysis. Association is higher in Cd4 PE-deficient DN, CD4+CD8lo, and CD4 SP cells than in wild-type cells. n = 196–286 alleles.(C) Confocal microscopy sections of Cd4-Cd8 distances representative of each genotype. Scale bars represent 1 μm.(D) Recruitment of Cd4 to pericentromeric heterochromatin in wild-type and Cd4 PE-deficient cells. Recruitment is higher in Cd4 PE-deficient DN, CD4+CD8lo and CD4 SP than in wild-type cells.(E) RT-PCR analysis of Cd4 or Cd8a expression in wild-type and Cd4 PE-deficient cells. Standard error bars were calculated from three independent experiments.See also Figure S5 and Table S4.

Mentions: The experiments we have described above indicate that the transcription factor RUNX could bring Cd4 and Cd8 together to streamline their regulation (Schoenfelder et al., 2010). It is also possible that Cd4 and Cd8 could exert a more direct influence over each other. If so, alterations of key regulatory elements within the Cd4 locus would translate into changes in Cd8 regulation. To address this question, we made use of gene-targeted mice. Cd4 expression is regulated by a silencer element and at least one stage-specific enhancer element (Chong et al., 2010; Kioussis and Ellmeier, 2002). The proximal enhancer Cd4 PE, located 13 Kb upstream of the Cd4 start site, is absolutely required for transcription, and therefore expression, of Cd4 in DP thymocytes (Chong et al., 2010). The position of this enhancer is diagrammed in Figure S5A. After positive selection in Cd4 proximal enhancer (PE)-deficient mice, CD4-expressing single-positive thymocytes and CD4+ peripheral T cells were detected, albeit at reduced numbers, and levels of CD4 expression were comparable to wild-type mice, suggesting that one or more putative enhancer elements rescue Cd4 expression (Figure 5A and data not shown). DNA FISH and confocal microscopy analysis of sorted thymocyte populations from Cd4 PE-deficient mice (Figure S2B) revealed that the Cd4 PE, and therefore Cd4 transcription, is not required for either the Cd4-Cd8 association at the DP stage or for the repositioning away from PCH, because the degree of Cd4-Cd8 association and pericentromeric localization were comparable to wild-type (Figures 5B–5D; Table S4).


RUNX transcription factor-mediated association of Cd4 and Cd8 enables coordinate gene regulation.

Collins A, Hewitt SL, Chaumeil J, Sellars M, Micsinai M, Allinne J, Parisi F, Nora EP, Bolland DJ, Corcoran AE, Kluger Y, Bosselut R, Ellmeier W, Chong MM, Littman DR, Skok JA - Immunity (2011)

The Cd4 Proximal Enhancer Inhibits Cd4-Cd8 Association(A) Flow cytometry analysis of wild-type and Cd4 PE-deficient thymocytes (Cd4 PE Δ/Δ).(B) Cd4-Cd8 association in wild-type and Cd4 PE-deficient cells, including statistical analysis. Association is higher in Cd4 PE-deficient DN, CD4+CD8lo, and CD4 SP cells than in wild-type cells. n = 196–286 alleles.(C) Confocal microscopy sections of Cd4-Cd8 distances representative of each genotype. Scale bars represent 1 μm.(D) Recruitment of Cd4 to pericentromeric heterochromatin in wild-type and Cd4 PE-deficient cells. Recruitment is higher in Cd4 PE-deficient DN, CD4+CD8lo and CD4 SP than in wild-type cells.(E) RT-PCR analysis of Cd4 or Cd8a expression in wild-type and Cd4 PE-deficient cells. Standard error bars were calculated from three independent experiments.See also Figure S5 and Table S4.
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fig5: The Cd4 Proximal Enhancer Inhibits Cd4-Cd8 Association(A) Flow cytometry analysis of wild-type and Cd4 PE-deficient thymocytes (Cd4 PE Δ/Δ).(B) Cd4-Cd8 association in wild-type and Cd4 PE-deficient cells, including statistical analysis. Association is higher in Cd4 PE-deficient DN, CD4+CD8lo, and CD4 SP cells than in wild-type cells. n = 196–286 alleles.(C) Confocal microscopy sections of Cd4-Cd8 distances representative of each genotype. Scale bars represent 1 μm.(D) Recruitment of Cd4 to pericentromeric heterochromatin in wild-type and Cd4 PE-deficient cells. Recruitment is higher in Cd4 PE-deficient DN, CD4+CD8lo and CD4 SP than in wild-type cells.(E) RT-PCR analysis of Cd4 or Cd8a expression in wild-type and Cd4 PE-deficient cells. Standard error bars were calculated from three independent experiments.See also Figure S5 and Table S4.
Mentions: The experiments we have described above indicate that the transcription factor RUNX could bring Cd4 and Cd8 together to streamline their regulation (Schoenfelder et al., 2010). It is also possible that Cd4 and Cd8 could exert a more direct influence over each other. If so, alterations of key regulatory elements within the Cd4 locus would translate into changes in Cd8 regulation. To address this question, we made use of gene-targeted mice. Cd4 expression is regulated by a silencer element and at least one stage-specific enhancer element (Chong et al., 2010; Kioussis and Ellmeier, 2002). The proximal enhancer Cd4 PE, located 13 Kb upstream of the Cd4 start site, is absolutely required for transcription, and therefore expression, of Cd4 in DP thymocytes (Chong et al., 2010). The position of this enhancer is diagrammed in Figure S5A. After positive selection in Cd4 proximal enhancer (PE)-deficient mice, CD4-expressing single-positive thymocytes and CD4+ peripheral T cells were detected, albeit at reduced numbers, and levels of CD4 expression were comparable to wild-type mice, suggesting that one or more putative enhancer elements rescue Cd4 expression (Figure 5A and data not shown). DNA FISH and confocal microscopy analysis of sorted thymocyte populations from Cd4 PE-deficient mice (Figure S2B) revealed that the Cd4 PE, and therefore Cd4 transcription, is not required for either the Cd4-Cd8 association at the DP stage or for the repositioning away from PCH, because the degree of Cd4-Cd8 association and pericentromeric localization were comparable to wild-type (Figures 5B–5D; Table S4).

Bottom Line: Here we found that nuclear organization was altered by interplay among members of this transcription factor circuitry: RUNX binding mediated association of Cd4 and Cd8 whereas ThPOK binding kept the loci apart.Moreover, targeted deletions within Cd4 modulated CD8 expression and pericentromeric repositioning of Cd8.Communication between Cd4 and Cd8 thus appears to enable long-range epigenetic regulation to ensure that expression of one excludes the other in mature CD4 or CD8 single-positive (SP) cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathogenesis Program, The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA.

Show MeSH
Related in: MedlinePlus