Negative regulation of CD8 expression via Cd8 enhancer–mediated recruitment of the zinc finger protein MAZR

Abstract

Coreceptor expression is tightly regulated during thymocyte development. Deletion of specific Cd8 enhancers leads to variegated expression of CD8αβ heterodimers in double-positive thymocytes. Here we show CD8 variegation is correlated with an epigenetic 'off' state, linking Cd8 enhancer function with chromatin remodeling of the adjacent genes Cd8a and Cd8b1 (Cd8). The zinc finger protein MAZR bound the Cd8 enhancer and interacted with the nuclear receptor corepressor N-CoR complex in double-negative thymocytes. MAZR was downregulated in double-positive and CD8 single-positive thymocytes. 'Enforced' expression of MAZR led to impaired Cd8 activation and variegated CD8 expression. Our results demonstrate epigenetic control of the Cd8 loci and identify MAZR as an important regulator of Cd8 expression.

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Figure 1: Epigenetic changes at the Cd8aCd8b1 complex in thymocytes from mice doubly deficient in E8I and E8II.
Figure 2: DNA methylation of the promoter, exon and intron regions of Cd8a and Cd8b1.
Figure 3: E8II binding properties and expression pattern of MAZR.
Figure 4: Enforced expression of MAZR in thymocytes induces variegation of CD8 expression.
Figure 5: MAZR-induced variegation in wild-type, E8I-deficient and E8II-deficient thymocytes.
Figure 6: MAZR is recruited to multiple sites in the Cd8aCd8b1 complex.
Figure 7: BTB domain–dependent interaction of MAZR and N-CoR.
Figure 8: The BTB domain alone is not sufficient to induce variegated expression of CD8 in DP thymocytes.

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Acknowledgements

We thank V.J. Bardwell for N-CoR expression constructs; E. Pfeiffer and E. Selzev for help with irradiation; and S. Sakaguchi, P. Kinross and R. Herbst for critical reading of the manuscript. Supported by the Austrian Science Fund (P14261 and P16708), the START Program (Project Y-163) of the Austrian Ministry of Education, Science and Culture, the K-plus Competence Center Biomolecular Therapeutics, the Federal Bank of Austria (Project 10530), the Austrian Program for Advanced Research and Technology of the Austrian Academy of Sciences (W.E.) and the National Institutes of Health (HD39454 and HD18184 to C.B.W.).

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Correspondence to Wilfried Ellmeier.

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Supplementary information

Supplementary Fig. 1

Loss of Dnmt1 leads to a partial reactivation of CD8 expression in variegated DP cells. (PDF 80 kb)

Supplementary Fig. 2

Map of the clone isolated in the YOH screen and localization of the bait RE-1 sequence. (PDF 123 kb)

Supplementary Fig. 3

Specificity of the anti-MAZR serum. (PDF 84 kb)

Supplementary Fig. 4

Retroviral constructs used for the transduction of hematopoietic stem cells. (PDF 95 kb)

Supplementary Fig. 5

CD8-negative DP thymocytes develop upon forced expression of MAZR in Tcra/ thymocytes. (PDF 140 kb)

Supplementary Table 1

PCR primers used for bisulfite sequencing analysis. (PDF 12 kb)

Supplementary Table 2

PCR primers used for ChIP assays. (PDF 16 kb)

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Bilic, I., Koesters, C., Unger, B. et al. Negative regulation of CD8 expression via Cd8 enhancer–mediated recruitment of the zinc finger protein MAZR. Nat Immunol 7, 392–400 (2006). https://doi.org/10.1038/ni1311

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