Roles of ZF5 and CGGBP-20 transcription factors in regulating expression of human FMR1 gene responsible for fragile X-syndrome

• Published in: Cell and tissue biology 2010-03, Vol.4 (1), p.54-62
• Main Authors: Gulyy, P. V., Orlov, S. V., Dizhe, E. B., Kuteikin-Teplyakov, K. B., Ignatovich, I. A., Zhuk, S. V., Perevozchikov, A. P.
• Format: Article
• Language: English
• Subjects: Biomedical and Life Sciences; Cell Biology; Life Sciences
• ISSN: 1990-519X; 1990-5203
• DOI: 10.1134/S1990519X10010050

The human FMR1 gene encodes an RNA-binding protein taking part in translation regulation. The 5′-untranslated region of FMR1 gene contains a large number of tandem repeats of GCC triplets (5–50); an increase in their number by more then 200 times is responsible for fragile X syndrome (human congenital anomaly). At least two transcription factors are thought to be able to interact specifically with triplet repeats (GCC)n, i.e., CGGBP-20 and ZF5 in regulatory regions of some genes. In this work, roles of these factors in the regulation of FMR1 gene expression was studied. Using the method of gel retardation, the selective binding of recombinant protein ZF5 with the sequence (GCC)9 has been shown. The tissue-specific distribution of protein ZF5 in mammalian cells overlaps with the known expression regions of the FMR1 . By using specific interfering RNA (RNA-i ZF5 ), it has been shown that the suppression of activity for the expression of the ZF5 gene in cells of the human HepG2 hepatoma stimulates at least 1.5 times the expression of the endogenous FMR1 gene in these cells, which indicates the negative character of the regulation of the expression of the FMR1 gene by the ZF5 transcription factor. In addition GCC repeats, the 5′ regulatory region FMR1 contains classic (canonical) sites of ZF5 binding located near the points of initiation of transcription. Two genetic constructions have been created to elucidate the contribution of the GCC triplet repeats to FMR1 gene regulation by transcription factors ZF5 and CGGBP-20; these constructions contain the reporter gene of luciferase under the control of the cloned deletional variant of the 5′ regulatory region of the human FMR1 gene and differ by the presence of (GCC) 9 triplet repeats in this region. In experiments on the cotransfection of the human HepG2 hepatoma cells by these genetic constructions, together with vectors of expression of the ZF5 and/or CGGBP-20 genes, ZF5 has been established to suppress the activity of the 5′ regulatory FMR1 region through classic sites of binding. Moreover, the presence of GCC repeats attenuates the inhibitory ZF5 effect on the activity of the 5′ regulatory FMR1 region. CGGBP-20 suppresses activity of the 5′ regulatory FMR1 region only in the presence of GCC triplet repeats. The obtained data indicate the differently directed action of the transcription factor ZF5 on the FMR1 gene, which can explain the previously shown increase in the level of FMR1 mRNA in carriers of premut