Molecular Characterization of the Transcription Factor MAZR

Cell fate specifications during thymocyte differentiation are linked with the tightly controlled expression of CD4 and CD8 coreceptor molecules. Therefore, understanding the regulation of Cd4 and Cd8 gene expression might not only provide insight into transcriptional control mechanisms during T cell development, but also may help to explain the molecular basis of lineage commitment and function. The expression of the adjacent genes Cd8a and Cd8b is controlled by at least five developmental stage-, subset- and lineage-specific cis-regulatory elements. Combined deletion of Cd8 enhancers E8I and E8II leads to variegated expression of CD8 in DP thymocytes, suggesting that chromatin remodeling is required for activation of Cd8 gene loci. We therefore compared epigenetic modifications such as histone acetylation and methylation, and DNA methylation at the Cd8 gene loci of variegated and non-variegated CD8 alleles. We could show that CD8 variegation is correlated with an epigenetic "off" state. These data link Cd8 enhancer function with chromatin remodeling of the Cd8 gene loci. To study the molecular basis of the epigenetic regulation of CD8 expression in more detail, we have employed yeast one-hybrid screens to search for factors that bind to Cd8 enhancers. We show that the zinc finger protein MAZR, encoded by the Zfp278 gene, bound the Cd8 enhancers and interacted with the corepressor N-CoR complex in DN thymocytes. MAZR expression was down-regulated during thymocyte development, leading to reduced recruitment of MAZR to the Cd8 gene loci in DP thymocytes as compared to DN cells. Enforced expression of MAZR led to impaired Cd8 activation and variegated CD8 expression in DP thymocytes. Our results demonstrate epigenetic control of the Cd8 gene loci and identify MAZR as an important regulator of Cd8 gene expression. Within this FWF Project, we propose to study molecular mechanisms of MAZR function. We want to determine functional domains in MAZR required for the induction of CD8 variegation and we want to investigate the role of N-CoR and N-CoR binding to MAZR for the regulation of Cd8 gene expression. Furthermore, in order to test whether MAZR is essential for the proper regulation of CD8 expression in DN thymocytes and to learn more about the in vivo role of MAZR in the T cell lineage (and in other lineages), we are proposing to generate (conditional) MAZR knockout mice using the Cre/LoxP system. Our studies will provide a molecular link between structural domains and biological activity and will also provide important insight into the physiological role of MAZR.

T cells for a subgroup of white blood cells and have important functions in the control of immune responses. They can be subdivided into two major subsets, the so-called CD4+ helper T cells and the CD8+ cytotoxic T cells. Both T cells subsets develop in the thymus from a so-called CD4 and CD8 double-positive thymocytes. We are interested to understand how this CD4 (i.e. helper) versus CD8 (i.e. "cytotoxic") "cell fate" decision of double- positive (DP) thymocytes is regulated. The helper versus cytotoxic lineage specification of thymocytes is linked to coreceptor expression. We previously showed that the transcription factor MAZR is an important regulator of Cd8 expression. By generating MAZR-deficient mice we now showed with the support of this FWF project that MAZR is also regulating CD4/CD8 cell fate choice. Loss of MAZR led to partial "re-programming" of cytotoxic T cells into CD4+ helper-like T cells during thymocyte development, which correlated with derepression of Th-POK, a central transcription factor for helper-lineage development. MAZR bound the silencer of the gene encoding Th- POK, which indicated direct regulation of this locus by MAZR. Thus, MAZR is part of the transcription factor network that regulates the CD4/CD8 lineage differentiation of DP thymocytes.