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 CD4+ helper or CD8+ cytotoxic T 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 numerous 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 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 was unable to upregulate CD4, demonstrating that heritable Cd4 silencing following cell division becomes independent of the original DNA sequence that was 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.
Recently, we identified DNA methylation as critical mechanism in mediating CD4 silencing in mature CD8+ T cells. Our work also implicated an important role for active DNA demethylation through the TET enzymes in order to promote CD4 expression in the helper lineage during development. We are currently exploring how cis-regulatory elements establish patterns of DNA methylation and direct Cd4 locus specific demethylation. Furthermore, we are interested in determining whether lineage specific transcription factors act to recruit DNA methyltransferases or TET enzymes, which would suggest a crucial role for DNA methylation in lineage commitment. Another recent focus has been to clarify the role of the Cd4 maturation enhancer, an element specifically active following positive selection and in mature CD4+ T cells, as this may lead to insight into helper T cell lineage commitment that occurs following the DP cell stage. We have also performed additional shRNA and CRISPR based screens to identify other regulators of heritable silencing of Cd4 in CD8+ T cells and found a critical role for histone chaperones and histone modifying enzymes. We are currently exploring how DNA methylation, histone modifications and the DNA replication machinery are coordinated to maintain repressed chromatin states following cell division. 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.
Recent Publications
Issuree, P.D., Ng, C.P., Littman, D.R. (2017) Heritable Gene Regulation in the CD4:CD8 T Cell Lineage Choice. Front. Immunol. 8:291. PMCID: PMC5360760.
Sellars M, Huh JR, Day K, Issuree PD, Galan C, Gobeil S, Absher D, Green MR, Littman DR. (2015) Regulation of DNA methylation dictates Cd4 expression during the development of helper and cytotoxic T cell lineages. Nat Immunol. 16(7):746-54. [Pubmed]
Knuckles, P., Vogt, M.A., Lugert, S., Milo, M., Chong, M.M., Hautbergue, G.M., Wilson, S.A., Littman, D.R. & Taylor, V. (2012) Drosha regulates neurogenesis by controlling Neurogenin 2 expression independent of microRNAs. Nat. Neurosci. 15(7): 962-9.
Teta, M., Choi, Y.S., Okegbe, T., Wong, G., Tam, O.H., Chong, M.M., Seykora, J.T., Nagy, A., Littman, D.R., Andl, T. & Millar, S.E. (2012) Inducible deletion of epidermal Dicer and Drosha reveals multiple functions for miRNAs in postnatal skin. Development 139(8), 1405-16. PMC3308177.
Kirigin, F.F., Lindstedt, K., Sellars, M., Ciofani, M., Low, S.L., Jones, L., Bell, F., Pauli, F., Bonneau, R., Myers, R.M., Littman, D.R. & Chong, M.M.W. (2012) Dynamic microRNA gene transcription and processing during T cell development. J. Immunol. 188(7), 3257-67.
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.
Gialitakis, M., Sellars, M. and Littman, D.R. (2011) The epigenetic landscape of lineage choice: lessons from the heritability of CD4 and CD8 expression. Curr. Top. Microbiol. Immunol. 356, 165-88.
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.
Kaneko, H., Dridi, S., Tarallo, V., Gelfand, B.D., Fowler, B.J., Cho, W.G., Kleinman, M.E., Ponicsan, S.L., Hauswirth, W.W., Chiodo, V.A., Karikó, K., Yoo, J.W., Lee, D.K., Hadziahmetovic, M., Song, Y., Misra, S., Chaudhuri, G., Buaas, F.W., Braun, R.E., Hinton, D.R., Zhang, Q., Grossniklaus, H.E., Provis, J.M., Madigan, M.C., Milam, A.H., Justice, N.L., Albuquerque, R.J., Blandford, A.D., Bogdanovich, S., Hirano, Y., Witta, J., Fuchs, E., Littman, D.R., Ambati, B.K., Rudin, C.M., Chong, M.M., Provost, P., Kugel, J.F., Goodrich, J.A., Dunaief, J.L., Baffi, J.Z., & Ambati, J. (2011) DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration. Nature. 471(7338), 325-30. PMC3077055.
Zhdanova, O., Srivastava, S., Di, L., Li, Z., Tchelebi, L., Dworkin, S., Johnstone, D.B., Zavadil, J., Chong, M.M., Littman, D.R., Holzman, L.B., Barisoni, L. & Skolnik, E.Y. (2011) The inducible deletion of Drosha and microRNAs in mature podocytes results in a collapsing glomerulopathy. Kidney Int. 80(7), 719-30. PMC3246347.