The transcription factor MafA/RIPE3b1 is an important regulator of insulin gene expression. MafA binds to the insulin enhancer element RIPE3b (C1-A2), now designated as insulin MARE (Maf response element). The insulin MARE element shares an overlapping DNA-binding region with another insulin enhancer element A2. A2.2, a β-cell-specific activator, like the MARE-binding factor MafA, binds to the overlapping A2 element. Our previous results demonstrated that two nucleotides in the overlapping region are required for the binding of both factors. Surprisingly, instead of interfering with each other's binding activity, the MafA and the A2-binding factors co-operatively activated insulin gene expression. To understand the molecular mechanisms responsible for this functional co-operation, we have determined the nucleotides essential for the binding of the A2.2 factor. Using this information, we have constructed non-overlapping DNA-binding elements and their derivatives, and subsequently analysed the effect of these modifications on insulin gene expression. Our results demonstrate that the overlapping binding site is essential for maximal insulin gene expression. Furthermore, the overlapping organization is critical for MafA-mediated transcriptional activation, but has a minor effect on the activity of A2-binding factors. Interestingly, the binding affinities of both MafA and A2.2 to the overlapping or non-overlapping binding sites were not significantly different, implying that the overlapping binding organization may increase the activation potential of MafA by physical/functional interactions with A2-binding factors. Thus our results demonstrate a novel mechanism for the regulation of MafA activity, and in turn β-cell function, by altering expression and/or binding of the A2.2 factor. Our results further suggest that the major downstream targets of MafA will in addition to the MARE element have a binding site for the A2.2 factor.
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Research Article|
November 08 2005
Regulation of insulin gene expression by overlapping DNA-binding elements
Wataru Nishimura;
Wataru Nishimura
*Section of Islet Transplantation and Cell Biology, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215, U.S.A.
†Department of Medicine, Harvard Medical School, Boston, MA 02215, U.S.A.
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Therese Salameh;
Therese Salameh
*Section of Islet Transplantation and Cell Biology, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215, U.S.A.
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Takuma Kondo;
Takuma Kondo
*Section of Islet Transplantation and Cell Biology, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215, U.S.A.
†Department of Medicine, Harvard Medical School, Boston, MA 02215, U.S.A.
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Arun Sharma
Arun Sharma
1
*Section of Islet Transplantation and Cell Biology, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215, U.S.A.
†Department of Medicine, Harvard Medical School, Boston, MA 02215, U.S.A.
1To whom correspondence should be addressed, at Research Division, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215, U.S.A. (email arun.sharma@joslin.harvard.edu).
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Publisher: Portland Press Ltd
Received:
June 17 2005
Revision Received:
July 26 2005
Accepted:
July 28 2005
Accepted Manuscript online:
July 28 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 392 (1): 181–189.
Article history
Received:
June 17 2005
Revision Received:
July 26 2005
Accepted:
July 28 2005
Accepted Manuscript online:
July 28 2005
Citation
Wataru Nishimura, Therese Salameh, Takuma Kondo, Arun Sharma; Regulation of insulin gene expression by overlapping DNA-binding elements. Biochem J 15 November 2005; 392 (1): 181–189. doi: https://doi.org/10.1042/BJ20050970
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