The overall goals of our program are to study in detail the properties and functional role of the lung Kruppel-like factor (LKLF/KLF2). This transcription factor is a member of a closely related family of KLF transcription factors. We have shown that LKLF in mice is necessary for embryos to survive past 12.5 days gestation and in addition have shown using chimeric animals that this transcription factor is necessary for normal lung development. We have also studied LKLF expression during development using whole mount tissue hybridization, and characterized its DNA binding and activating domains, as well as identified cis elements responsible for the regulated LKLF gene expression. The cis elements responsible for tissue specific expression of LKLF are conserved between mouse and human LKLF promoter. During the next granting period, we wish to further explore the inhibitory domain of LKLF. Interestingly, this domain inhibits transcription when bound to DNA next to a strong transcriptional activator. Preliminary studies suggest that a co-repressor exists and exerts its activity by binding to LKLF. This is demonstrated by the finding that overexpression of the inhibitory domain, without a DNA binding domain, alleviates the inhibition of LKLF presumably by sequestering a co-repressor. Using a two-hybrid yeast screen, we have identified a potential co-repressor molecule and this will be studied in detail during the next granting period. Modification of the inhibitory domain will be investigated including ubiquination and acetylation. In addition, we will investigate the regulation of the LKLF gene. We have already identified one region that is important for expression and will use transgenic mouse assays to define other elements. Once these elements are identified, the nuclear factors which bind to these elements will be defined, cloned and their activity studied. Finally, we will search for targets of the LKLF transcription factor. We will use gene microarray analysis of cells with and without the LKLF gene and develop conditional expression of LKLF to define downstream target genes in tissues such as lung, macrophages and T-lymphocytes.
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