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Volume 271, Number 22, Issue of May 31, 1996 pp. 12944-12950
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Sp1 Family Proteins Recognize the U5 Repressive Element of the Long Terminal Repeat of Human T Cell Leukemia Virus Type I through Binding to the CACCC Core Motif

(Received for publication, September 12, 1995, and in revised form, February 8, 1996)

Koichi Okumura , Gaku Sakaguchi , Shin Takagi , Kazumi Naito , Tsuneyo Mimori ^§ and Hisanaga Igarashi

From the  Shionogi Institute for Medical Science, 2-5-1 Mishima, Settsu, Osaka 566 and ^§ Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo 160, Japan

We have identified several nuclear proteins binding to the U5 repressive element (U5RE) at the U5 region of the human T cell leukemia virus type I (HTLV-I) long terminal repeat (LTR). In gel mobility shift assays with the U5RE DNA probe, Jurkat T cell nuclear proteins generated five different complexes, named U5RE binding protein complexes (U5RP)-A1, -A2, -A3, -B, and -C. Only U5RP-C was affected by pretreatment with an excess of poly(dI-dC) and was immunodepressed by anti-Ku/p80 antibodies, suggesting that U5RP-C is a nonspecific complex involving Ku antigen. UV cross-linking showed at least six nuclear proteins involved in the other complexes, including U5RP-A1, -A2, -A3, and -B. The sequence of the binding core element of these specific complexes, determined by competition assays and gel mobility shift assays using a series of the U5RE mutants, is CACCC which is identical to that for the Sp1 transcription factor. LTR with a mutant U5RE, which has no ability to bind with the nuclear proteins, showed stronger promoter activity than LTR with the wild U5RE, suggesting that the specific interaction of these U5RE-binding proteins might result in the U5-mediated repression. U5RP-A1 was supershifted by anti-Sp1 antibodies and U5RP-A2 and -B were supershifted by anti-Sp3 antibodies, suggesting that Sp1 or Sp3 is involved in U5RP-A1 or U5RP-A2 and -B, respectively. Although the other nuclear proteins remain to be characterized, these findings suggest that U5RE-binding proteins in U5RP-A1, -A2, -A3, and -B are involved in HTLV-I gene repression.

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