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J. Biol. Chem., Vol. 280, Issue 42, 35417-35423, October 21, 2005

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Identification of a Repressor in the First Intron of the Human 2(I) Collagen Gene (COL1A2)^*

Taras T. Antoniv1, Shizuko Tanaka1, Bayan Sudan, Sarah De Val, Ke Liu¶, Lu Wang, Dominic J. Wells¶, George Bou-Gharios, and Francesco Ramirez||2

From the Laboratory of Genetics and Organogenesis, Research Division of the Hospital for Special Surgery, and Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, New York 10021, the ^¶Gene Targeting Unit, Division of Neuroscience and Mental Health, Imperial College School of Medicine, Charing Cross Hospital, London W6 8RP, United Kingdom, Renal Medicine, Imperial College School of Medicine, Hammersmith Campus, London W12 ONN, United Kingdom, and ^||CEINGE-Biotecnologie Avanzate, 80131 Naples, Italy

The human and mouse genes that code for the 2 chain of collagen I (COL1A2 and Col1a2, respectively) share a common chromatin structure and nearly identical proximal promoter and far upstream enhancer sequences. Despite these homologies, species-specific differences have been reported regarding the function of individual cis-acting elements, such as the first intron sequence. In the present study, we have investigated the transcriptional contribution of the unique open chromatin site in the first intron of COL1A2 using a transgenic mouse model. DNase I footprinting identified a cluster of three distinct areas of nuclease protection (FI1-3) that span from nucleotides +647 to +760, relative to the transcription start site, and which contain consensus sequences for GATA and interferon regulatory factor (IRF) transcription factors. Gel mobility shift and chromatin immunoprecipitation assays corroborated this last finding by documenting binding of GATA-4 and IRF-1 and IRF-2 to the first intron sequence. Moreover, a short sequence encompassing the three footprints was found to inhibit expression of transgenic constructs containing the COL1A2 proximal promoter and far upstream enhancer in a position-independent manner. Mutations inserted into each of the footprints restored transgenic expression to different extents. These results therefore indicated that the unique open chromatin site of COL1A2 corresponds to a repressor, the activity of which seems to be mediated by the concerted action of GATA and IRF proteins. More generally, the study reiterated the existence of species-specific difference in the regulatory networks of the mammalian 2(I) collagen coding genes.

Received for publication, March 10, 2005 , and in revised form, June 23, 2005.

^* This work was supported by National Institutes of Health Grant AR386481, the St. Giles Foundation, the James D. Farley Family, and the Arthritis Research Campaign. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Laboratory of Genetics and Organogenesis Hospital for Special Surgery, 535 East 70th St., New York, NY 10021. Tel.: 212-774-7554; Fax: 212-774-7864; E-mail: ramirezf{at}hss.edu.

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