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Identification of two elements involved in regulating expression of the murine leukaemia inhibitory factor gene.

ABSTRACT: Mouse leukaemia inhibitory factor (LIF) is a polyfunctional cytokine which exhibits multiple functions in vitro and in vivo. Two forms of LIF cDNA, differing at their 5' ends, have been described encoding either diffusible (D-LIF) or matrix-associated (M-LIF) forms of the protein [Rathjen, Toth, Willis, Heath and Smith (1990) (Cell 62, 1105-1114]. The present report describes the DNA sequence and functional characterization of the murine LIF gene and its surrounding transcriptional regulatory elements. Transient transfection of constructs containing the LIF gene and various amounts of 5'-non-coding sequence failed to give detectable levels of expression, suggesting the presence of inhibitory sequences within the LIF gene. Stable cell lines were produced by transfection of experimental constructs containing various lengths of 5'-non-coding sequence of the LIF gene, or the heterologous phosphoglycerate kinase promoter, linked to an LIF/neomycin-resistance-hybrid-coding sequence. The frequency of recovery of stable clones indicated that sequences located in the first intron between the transcriptional start sites for D-LIF and M-LIF act to suppress expression of the gene in most genomic locations. This region is rich in GC residues and has been shown to be hypomethylated in vitro [Kaspar, Dvorak and Bartunek (1993) FEBS Lett. 319, 159-162]. Analysis of the LIF/neomycin-resistance transgene expression in these stable cell clones demonstrated that transcripts containing the M-LIF or D-LIF exons required the presence of sequences located between -1200 and -3200 in the LIF gene. In the absence of these sequences, transcription is initiated elsewhere within the first intron. These sequences can be replaced by the heterologous phosphoglycerate kinase promoter. Deletion of the GC-rich region between the D-LIF and M-LIF transcriptional start sites results in the appearance of transcripts that do not splice out the first intron of the LIF gene. These may result from gene or promoter trapping of the LIF gene. Sequence analysis of the region between -1200 and -3200 revealed a number of minimal steroid-response elements, regions of similarity to DNAase I-hypersensitive sites in the uteroglobin gene and a region of alternating purine/pyrimidine sequence. This study therefore defines two important regulatory regions in the LIF gene: a GC-rich region in the first intron and a distal 'enhancer' located between -3200 and -1200.

SUBMITTER: Hsu LW

PROVIDER: S-EPMC1137196 | biostudies-other | 1994 Aug

REPOSITORIES: biostudies-other

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Project description:Proper development of vertebrate embryos depends not only on the crucial funtions of key evolutionarily conserved transcriptional regulators, but also on the precisely spatiotemporal expression of these transcriptional regulators. The mouse Nolz-1/Znf503/Zfp503 gene is a mammalian member of the conserved zinc-finger containing NET family. The expression pattern of Nolz-1 in mouse embryos is highly correlated with that of its homologues in different species. To study the spatiotemporal regulation of Nolz-1, we first identified two evolutionarily conserved cis-elements, UREA and UREB, in 5' upstream regions of mouse Nolz-1 locus. We then generated UREA-LacZ and UREB-LacZ transgenic reporter mice to characterize the putative enhancer activity of UREA and UREB. The results indicated that both UREA and UREB contained tissue-specific enhancer activity for directing LacZ expression in selective tissue organs during mouse embryogensis. UREA directed LacZ expression preferentially in selective regions of developing central nervous system, including the forebrain, hindbrain and spinal cord, whereas UREB directed LacZ expression mainly in other developing tissue organs such as the Nolz-1 expressing branchial arches and its derivatives, the apical ectodermal ridge of limb buds and the urogenital tissues. Both UREA and UREB directed strong LacZ expression in the lateral plate mesoderm where endogenous Nolz-1 was also expressed. Despite that the LacZ expression pattern did not full recapitulated the endogenous Nolz-1 expression and some mismatched expression patterns were observed, co-expression of LacZ and Nolz-1 did occur in many cells of selective tissue organs, such as in the ventrolateral cortex and ventral spinal cord of UREA-LacZ embryos, and the urogenital tubes of UREB-LacZ embryos. Taken together, our study suggests that UREA and UREB may function as evolutionarily conserved cis-regulatory elements that coordinate with other cis-elements to regulate spatiotemporal expression of Nolz-1 in different tissue organs during mouse embryogenesis.

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Identification of two elements involved in regulating expression of the murine leukaemia inhibitory factor gene.

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