The surface proteins CD8 and CD4 mark distinct stages of T cell development. In the thymus CD8+CD4+ double-positive (DP) thymocytes extinguish expression of CD8 or CD4 during lineage commitment into peptide/MHC class II-specific CD4+ helper (and regulatory) or peptide/MHC class I-specific CD8+ cytotoxic T cells (single-positive, or SP cells). Due to the coupling of CD4/CD8 expression with the helper or cytotoxic T cell fates, respectively, we have studied their transcriptional regulation to gain insight into the mechanism of lineage commitment. We have identified multiple stage-specific enhancers that direct expression of CD8 and CD4 during T cell development. Our studies of Cd4 regulation led to the identification of one of the first bona fide silencer elements in vertebrates. The Cd4 silencer (S4) is required for reversible silencing in CD8-CD4- double-negative (DN) thymocytes as well as for the establishment of heritable silencing during differentiation of DP cells into CD8+ T cells. Subsequently, we identified Runx family members Runx1 and Runx3 as the critical transcription factors that mediate S4 activity. We also found that the Runx proteins form an antagonistic regulatory loop with the essential transcription factor of the CD4+ lineage, ThPOK, highlighting how regulators of CD4/CD8 expression also have critical roles in lineage commitment. Stage-specific requirements of the silencer element were investigated using Cre-Lox technology in genetically engineered mouse strains. S4 deletion in mature CD8+ T cells did not result in upregulation of CD4, demonstrating that heritable Cd4 silencing following cell division becomes independent of the original DNA sequence required for its establishment. In an analogous study with the Cd4 proximal enhancer, we found that conditional enhancer deletion in mature CD4+ T cells did not affect expression of CD4, in contrast with defective expression induced by germline deletion. Thus, the Cd4 locus offers a model to study the establishment and heritable maintenance of repressed and active chromatin states.

We identified DNA methylation as a critical mechanism mediating maintenance of Cd4 silencing in mature CD8+ T cells. Surprisingly, not only the maintenance methylase DNMT1, but also DNMT3a and DNMT3b, which are typically de novo methyltransferases, have important roles the maintenance of Cd4 silencing. The DNMT3s appear to do so in a methylation-independent manner, by cooperating with histone-modifying enzymes that are recruited to the replication fork by the histone chaperone CAF-1. We are exploring how DNA methylation, histone modifications and the DNA replication machinery are coordinated to maintain repressed chromatin states at diverse loci throughout the genome following cell division. In CD8+ T cells, targeted inactivation of Chaf1a and Dnmts resulted in Cd4 de-repression, but the genomewide profiles of changes in gene expression differed markedly. It will be very interesting to determine if multiple mechanisms of heritability operate at different regions of the genome. Another major question that remains to be addressed is how S4-binding factors initiate the heritable repressive state in CD8-lineage cells.

In cells directed towards the CD4 lineage, we found that active DNA demethylation mediated by TET enzymes is required for continued CD4 expression after selection of MHC class II-specific thymocytes and subsequent activation of the cells in the periphery. Two different enhancers cooperate to mediate the demethylation: the proximal enhancer (E4p) directs CD4 expression in DP thymocytes and is required for demethylation of the locus during the DP to CD4 SP transition; and an intronic “maturation” enhancer, E4m, contributes to establishing and maintaining the TET-dependent demethylated state after selection. We are investigating how the cis-regulatory elements establish patterns of DNA methylation and direct Cd4 locus-specific demethylation. It will be important to determine whether lineage specific transcription factors act to recruit DNA methyltransferases or TET enzymes. By studying the regulation of Cd4 we hope to gain further insights into lineage commitment during development and elucidate fundamental mechanisms of gene regulation in differentiated cell types.

Related Publications:

  • Ng, C., Aichinger, M., Nguyen, T., Wu, L., Mesa, K.R., Quivy, J-P., Almouzni, G., Zuber, J. & Littman, D.R. (2019) The histone chaperone CAF-1 cooperates with the DNA methyltransferases to maintain Cd4 silencing in cytotoxic T cells.  Genes and Development, 33, 669-82. PMID:  30975723

  • Issuree, P.D., Day, K., Au, C., Raviram, R., Zappile, P., Skok, J.A., Xue, H-H., Myers, R.M. & Littman, D.R. (2018). Stage-specific epigenetic regulation of CD4 expression by coordinated enhancer elements during T cell development.  Nature Communications, 9, 3594.  PMID:  30185805

  • Issuree, P.D., Ng, C.P., & Littman, D.R. (2017) Heritable gene regulation in the CD4:CD8 lineage choice. Front Immunol. 8:291. PMID: 28382035

  • Sellars, M., Huh, J.R., Day, K., Galan, C., Gobeil, S., Absher, D., Green, M.R. & Littman, D.R.  (2015) Regulation of DNA methylation dictates Cd4 gene expression during development of helper and cytotoxic T cell lineages.  Nature Immunology, 16, 746-54. PMID: 26030024

  • Egawa, T. and Littman, D.R. (2011) Transcription factor AP4 modulates reversible and epigenetic silencing of the Cd4 gene. Proc. Natl. Acad. Sci. U.S.A. 108(36), 14873-8. PMC3169121.

  • Collins, A., Hewitt, S.L., Chaumeil, J., Sellars, M., Micsinai, M., Allinne, J., Parisi, F., Nora, E.P., Bolland, D.J., Corcoran, A.E., Kluger, Y., Bosselut, R., Ellmeier, W., Chong, M.M., Littman, D.R., & Skok, J.A. (2011) RUNX Transcription Factor-Mediated Association of Cd4 and Cd8 Enables Coordinate Gene Regulation. Immunity 34(3), 303-14.  PMC3101577.

  • Chong, M.M.W., Simpson, N., Ciofani, M., Chen, G., Collins, A. & Littman, D.R. (2010) Epigenetic propagation of CD4 expression is established by the Cd4 proximal enhancer in helper T cells.  Genes & Dev. 24(7), 659-69.  PMC2849123.

  • Egawa, T. & Littman, D.R. (2008) ThPOK acts late in specification of the helper T cell lineage and suppresses Runx-mediated commitment to the cytotoxic T cell lineage.  Nat. Immunol.  9(10), 1131-9.  Epub 2008 Sept 7. PMC2666788.

  • Eberl, G. & Littman, D.R. (2004) Thymic origin of intestinal ab T cells revealed by fate mapping of RORgt+ cells.  Science, 305, 248-251. PMID: 15247480

  • Taniuchi, I., Osato, M., Egawa, T., Sunshine, M.J., Bae, S.-C., Komori, T., Ito, Y. & Littman, D.R. (2002) Differential requirements for Runx proteins in CD4 repression and epigenetic silencing during T lymphocyte development.  Cell 111, 621-633. PMID: 12464175

  • Taniuchi, I., Sunshine, J.J., Festenstein, R., & Littman, D.R. (2002) Evidence for distinct CD4 silencer functions at different stages of thymocyte differentiation.  Mol. Cell 10, 1083-96. PMID: 12453416

  • Zou, Y.R., Sunshine, M.J., Taniuchi, I., Hatam, F., Killeen, N., & Littman, D.R. (2001) The CD4 gene is epigenetically silenced in T cells committed to the cytotoxic lineage.  Nature Genetics 29, 332-336. PMID: 11687799

  • Sun, Z., Unutmaz, D., Zou, Y.R., Sunshine, M.J., Pierani, A., Brenner-Morton, S., Mebius, R.E., & Littman, D.R. (2000) Requirement for RORg in thymocyte survival and lymphoid organ development.  Science 288, 2369-2373. PMID:  10875923

  • Ellmeier, W., Sawada, S., & Littman, D.R. (1999)  The regulation of CD4 and CD8 coreceptor gene expression during T cell development.  Ann. Rev. Immunol. 17, 523-554.

  • Ellmeier, W., Sunshine, M.J.,  Losos, K., & Littman, D.R. (1998)  Multiple developmental stage-specific enhancers regulate CD8 expression in developing thymocytes and in thymus-independent T cells.  Immunity 9, 485-496.

  • Ellmeier, W., Sunshine, M.J.,  Losos, K., Hatam, F. & Littman, D.R. (1997) Identification of an enhancer that directs lineage-specific expression of CD8 in positively selected thymocytes and mature T cells.  Immunity 7, 537-547.

  • Sawada, S., Scarborough, J.D., Killeen, N., and Littman, D.R.  (1994)  A lineage-specific transcriptional silencer regulates CD4 gene expression during T lymphocyte development.  Cell 77, 917-929.

  • Sawada, S., and Littman, D.R.  (1993)  A Heterodimer of HEB and an E12-Related protein interacts with the CD4 enhancer and regulates its activity in T-cell lines.  Mol. Cell. Biol. 13, 5620-5628.

  • Sawada, S., & Littman, D.R.  (1991)  Identification and characterization of a T cell specific enhancer adjacent to the murine CD4 gene.  Mol. Cell. Biol. 11, 5506-5515.