Study the regulation of murine CD5 gene expression in lymphocytes
The central focus of our laboratory is to characterize gene expression in, and function of, phenotypically distinct lymphocyte subsets in mouse and human. My current project aims at defining the cellular and molecular mechanism(s) that regulate the expression of the murine CD5 gene on lymphocytes.
The CD5 gene encodes a transmembrane glycoprotein expressed in all T-lineage cells and a unique small B cell subpopulation, called B-1a, which differs from more abundant convention B cells with respect to development, phenotype, growth and function. CD5 expression is always lymphocyte-specific; however, levels of expression on phenotypically and functionally distinct lymphocyte subsets vary up to 30 fold. Importantly, the expression profile characteristic of CD5 is similar both in mice and human, indicating an evolutionarily conserved regulatory mechanism, which reflects its potentially important function in the immune system. Our lab cloned CD5 gene several years ago. Recently, characterization of 3kb long sequence upstream of CD5 gene first translation codon in a T lymphoma cell line, we identified a regulatory promoter approximately 200bp in length and defined several cis-elements centering in a 50bp of region within this regulatory promoter. Initial work also showed that several well-known transcription factors bind to these cis-elements and appear to functionally control the transcription of CD5 promoter in either positive or negative manner in T and B lymphocytes. In addition, it showed that the CD5 expression detected on primary lymphocytes is mainly regulated at the transcriptional level in that the amount of message is proportional to the amount of surface CD5.My current studies extend the previous work by focusing on an in-depth investigation of the genetic regulatory mechanisms that mediate the qualitative (on/off) and quantitative (precise level) constitutive expression of CD5 on primary lymphocytes. We are working towards generation of two transgenic (Tg) mouse strains that will express a luciferase-GFP-dual reporter gene detectable both by FACS and whole animal imaging that is driven, in individual strains, either by the 3kb full length CD5 5' flanking sequence or the 200bp CD5 regulatory promoter. Facilitated by imaging in intact living animal, this system enables us to monitor the reporter gene expression in a temporal-and-spatial way, particularly, allow us to determine whether reporter-expressing cells home to appropriate sites in the animal. In addition, we will be able to measure both the endogenous CD5 expression and the reporter signal (e.g. GFP) on individual cells in the transgenic mice. Thus, this system will allow us to extend the functional study of promoter classically carried out in culture cell lines in vitro to primary populations in vivo by determining whether the 3Kb CD5 5' fragment and even the 200bp CD5 regulatory promoter contains enough genetic information capable of rendering the characteristic expression pattern observed for the endogenous CD5 gene. In parallel with these studies, I plan to characterize which and how transcription factors are functionally involved in regulating CD5 gene expression through interaction with their cognate cis-elements located in 50bp central region of 200bp CD5 regulatory promoter by using FACS staining for intracellular proteins, cell sorting coupled with other molecular tools.
The significance of these studies lies not only in elucidating the mechanisms underlying the qualitative and quantitative control of CD5 expression but also in pioneering methods for studying promoter or other genetic regulatory elements studies in vivo. This work also provides a good model to understand the strategies the organism explored to achieve the proper gene expression required for appropriate biological events.
Publications:
Yang Y., Contag C.H., Felsher D., Shachaf C.M., Cao Y., Herzenberg L.A. & Tung J.W. (2004) The E47 transcription factor negatively regulates CD5 expression during thymocyte development. Proc Natl Acad Sci U S A, 101, 3898.
Yang Yang, Hu Hailiang, Hong Guofan. px2, the newly identified gene in Rhizobium leguminosarum is characterized to enhance its adjacent nodF expression. Biochemical and Biophysical Research communications 275, 91-96 (2000).
Yang Yang, Hu HaiLiang, Hong Guofan. A promoter overlapping with that of nodF is responsible for a new RNA molecule transcription in Rhizobium. ACTA Biochimica et Biophysica Sinica, 2000, 32 (6)
Hu Hailiang, Liu Songtao, Yang Yang, Chang Weizhong, Hong Guofan. In Rhrizobium leguminosarum, NodD represses its own transcription by competing with RNA polymerase for binding sites. Nucleic Acids Research, 2000, 28(14)2784
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