| MOLECULAR MECHANISMS OF T CELL DEVELOPMENT
We are interested to understand how the CD4/CD8 cell fate decision of double-positive (DP) thymocytes into helper or cytotxic T cells is regulated. The transcriptional regulation of the Cd4 and Cd8 genes is tightly linked to the functional program of T cells. It is conceivable that factors that regulate CD4 and CD8 expression are also involved in directing DP thymocytes towards the helper or cytotoxic lineage, respectively. Therefore, it is important to understand how the coreceptor genes are transcriptionally regulated during T cell development and to identify cis -regulatory elements and trans -acting factors involved in their regulation. This will not only provide insights into transcriptional control mechanism in T cells, but may also lead to the identification of molecular factors which are involved in cell fate specifications during T cell development.
Ongoing studies in the laboratory aim to further characterize Cd8 cis-regulatory elements by performing (combinatorial) enhancer knockout experiments, to isolate additional Cd8 enhancer binding factors, and to study in detail the role of the BTB zinc finger transcription factors MAZR and PLZF during T cell development. Moreover, we are investigating the role of members of the histone deacetylase family in T cells.
Our research is supported by:
• Austrian Science Fund (FWF; stand alone project P19930)
• Austrian Science Fund (FWF; International collaboration project I698)
• FWF/MedUni Wien Doktoratskolleg "Inflammation and Immunity" (W1212)
• Vienna Science and Technology Fund (WWTF, Project LS09-031)
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Epigenetic and transciptional control of Cd8 gene expression during T cell development
Our laboratory is studying the regulation of CD8 coreceptor expression, a key molecule in the immune system for the development of the cytotoxic T cell lineage. CD8 coreceptor expression is tightly regulated during thymocyte development by the activity of at least five different cis -regulatory elements. We recently linked Cd8 enhancer function with chromatin remodeling of the adjacent genes Cd8a and Cd8b1 (Cd8) and demonstrated epigenetic control of the Cd8 gene complex (Bilic et al., 2006, Nature Immunology, 7, 392).
We further aim to identify Cd8 enhancer binding factors. Since important Cd8 cis-regulatory elements are expected to be evolutionary conserved, a cross-species comparison of the Cd8a and Cd8b genomic loci to search for evolutionary conserved regions (ECR) was performed. This approach revealed several ECRs ranging from 200bp to 500bp within known Cd8 enhancers, which can be used as molecular baits for the isolation of binding factors. Moreover, this approach led to the identification of a novel evolutionary conserved Cd8 enhancer element that is currently analyzed in more detail. Our results demonstrate that a combination of bioinformatic and biological approaches is a powerful tool to identify new cis -regulatory elements at complex regulated gene loci.
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Molecular analysis of the zinc finger transcription factor MAZR
We have recently identified that the BTB domain-containing zinc finger protein MAZR is an important regulator of CD8 expression. MAZR binds to Cd8 enhancers and forced expression of MAZR during T cell development induced variegated CD8 expression, most likely due to co-recruitment of repressor complexes (Bilic et al., 2006, Nature Immunology). This indicates that MAZR regulates chromatin remodeling at the Cd8 gene complex. We recently generated MAZR-deficient mice and could show that MAZR is part of the transcription factor network that regulates CD4/CD8 cell fate choice (Sakaguchi et al., 2010, Nature Immunology). Ongoing studies focus on the further characterization of MAZR function in T cells and in other cells of the hematopoietic system.
In addition, to understand the molecular mechanisms of how MAZR regulates the expression of CD8, we are aiming to identify MAZR interacting factor using tandem affinity purification strategies and mass spectroscopy approaches.
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The role of Histone Deacetylases in T cell development and function
Reversible changes in histone acetylation patterns have been shown to accompany many important processes in T cells ranging from VDJ recombination during T cell development to the induction of cytokine expression during Th1/Th2 effector differentiation. Modification of core histones by lysine acetylation is controlled by histone acetyltransferases and histone deacetylases, which are considered as transcriptional co-activators and co-repressors, respectively. Eighteen histone deacetylases (HDACs) have been identified in mammalian organisms, however dissecting individual roles for each member of the HDAC family in specific cell lineages and tissues remains a major scientific challenge. In a close collaboration with the research group of Christian Seiser (Max F. Perutz Laboratories, Vienna) we are analyzing the role of certain members of the HDAC family in T cells.
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Transcriptional control of memory-phenotype T cell development by PLZF
Peripheral CD4+ and CD8+ T cell subsets have been traditionally divided into naïve CD44low CD62L+ and memory CD44high CD62L– populations, while the surface expression phenotype of the latter population also resembles recently activated T cells. However, the memory T cell subset is not a population consisting only of true antigen-specific memory cells that developed in response to a foreign antigen. Rather, the memory population contains in addition a variety of different T lymphocyte subsets, some of which acquired their memory phenotype through homeostatic proliferation, while others have immediate effector function and may play a role in the front-line defense against certain bacterial infections ("innate-like" T cells). The signaling pathways and transcriptional networks that regulate the developmental cell fate decisions between conventional and innate-like memory-phenotype T cell subsets are not well understood.
We recently observed that the BTB domain-containing protein promyelocytic leukemia zinc finger (PLZF) is expressed in CD44high memory-phenotype but not in naïve CD44low CD4+ T cells. To investigate a potential role of PLZF in either the generation and/or function of the CD44high CD4+ T cell subset, we performed gain-of-function experiments and generated PLZF transgenic mice. With this experimental approach we could show that PLZF is a crucial transcriptional regulator that induces the development of CD44high memory-phenotype T cells with innate-like characteristics. Ongoing studies address mechanistic aspects of how PLZF regulates the development of CD44high memory-phenotype T cells.
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