Novel repressor of the human FMR1 gene—Identification of p56 human (GCC)(n)-binding protein as a Kruppel-like transcription factor ZF5

Department of Biochemistry, Institute of Experimental Medicine, Russian Academy of Medical Sciences, St Petersburg, Russia.
FEBS Journal (Impact Factor: 4). 10/2007; 274(18):4848-62. DOI: 10.1111/j.1742-4658.2007.06006.x
Source: PubMed


A series of relatively short (GCC)(n) triplet repeats (n = 3-30) located within regulatory regions of many mammalian genes may be considered as putative cis-acting transcriptional elements (GCC-elements). Fragile X-mental retardation syndrome is caused by an expansion of (GCC)(n) triplet repeats within the 5'-untranslated region of the human fragile X-mental retardation 1 (FMR1) gene. The present study aimed to characterize a novel human (GCC)(n)-binding protein and investigate its possible role in the regulation of the FMR1 gene. A novel human (GCC)(n)-binding protein, p56, was isolated and identified as a Krüppel-like transcription factor, ZF5, by MALDI-TOF analysis. The capacity of ZF5 to specifically interact with (GCC)(n) triplet repeats was confirmed by the electrophoretic mobility shift assay with purified recombinant ZF5 protein. In cotransfection experiments, ZF5 overexpression repressed activity of the GCC-element containing mouse ribosomal protein L32 gene promoter. Moreover, RNA interference assay results showed that endogenous ZF5 acts as a repressor of the human FMR1 gene. Thus, these data identify a new class of ZF5 targets, a subset of genes containing GCC-elements in their regulatory regions, and raise the question of whether transcription factor ZF5 is implicated in the pathogenesis of fragile X syndrome.

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Available from: Konstantin Kuteikin Kuteykin-Teplyakov,
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    • "Conversely, we reasoned that motif depletion is a signature of a repressor because it suggests that motif absence is a condition for enhancer activity: GFI1 showed motif depletion in K562 enhancers and is indeed a known hematopoietic repressor (Hock and Orkin 2006); ZFP161, another known repressor (Sobek-Klocke et al. 1997; Orlov et al. 2007), showed motif depletion in HepG2 enhancers. While the sharing of motifs across factors and post-translation modifications limit the interpretability of expression in this context, we found that for five of these seven motifs the corresponding factor had higher expression in the cell line, where motif enrichment or depletion was noted (Fig. 1A; Supplemental Fig. S2, right). "
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    ABSTRACT: Genome-wide chromatin annotations have permitted the mapping of putative regulatory elements across multiple human cell types. However, their experimental dissection by directed regulatory motif disruption has remained unfeasible at the genome scale. Here, we use a massively parallel reporter assay (MPRA) to measure the transcriptional levels induced by 145-bp DNA segments centered on evolutionarily conserved regulatory motif instances within enhancer chromatin states. We select five predicted activators (HNF1, HNF4, FOXA, GATA, NFE2L2) and two predicted repressors (GFI1, ZFP161) and measure reporter expression in erythroleukemia (K562) and liver carcinoma (HepG2) cell lines. We test 2104 wild-type sequences and 3314 engineered enhancer variants containing targeted motif disruptions, each using 10 barcode tags and two replicates. The resulting data strongly confirm the enhancer activity and cell-type specificity of enhancer chromatin states, the ability of 145-bp segments to recapitulate both, the necessary role of regulatory motifs in enhancer function, and the complementary roles of activator and repressor motifs. We find statistically robust evidence that (1) disrupting the predicted activator motifs abolishes enhancer function, while silent or motif-improving changes maintain enhancer activity; (2) evolutionary conservation, nucleosome exclusion, binding of other factors, and strength of the motif match are predictive of enhancer activity; (3) scrambling repressor motifs leads to aberrant reporter expression in cell lines where the enhancers are usually inactive. Our results suggest a general strategy for deciphering cis-regulatory elements by systematic large-scale manipulation and provide quantitative enhancer activity measurements across thousands of constructs that can be mined to develop predictive models of gene expression.
    Genome Research 03/2013; 23(5). DOI:10.1101/gr.144899.112 · 14.63 Impact Factor
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    • "AP2-α, ZF5 and Pax-2, all function in the regulation of distinct transcriptional programs. ZF5 is a kruppel-type TF which has been shown to regulate the expression of the fragile-X causative gene FMR1 (46). In addition, binding sites for ZF5 are enriched in the genetic promoters of clock-regulated genes, as have binding sites for the known BRCA1 interacting TFs; E2F, AP-1 and NF-Y, SP1 and Oct-1 (14,15,47). "
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    Nucleic Acids Research 08/2011; 39(22):9536-48. DOI:10.1093/nar/gkr679 · 9.11 Impact Factor
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    ABSTRACT: 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) which increasing (more then 200) is responsible for X-fragile syndrome (human congenital anomaly). As it has been shown earlier, al least two transcription factors (ZF5 and CGGBP-20) are capable of interacting specifically with GCC-repeats in regulatory regions of some genes. In this work, their roles in FMR1 gene expression regulation were studied. It was demonstrated by electrophoretic mobility shift assay that ZF5 recombinant protein specifically bound with GCC-triplet repeats (GCC 9). Tissue-specific distributions of ZF5 and FMR1 proteins are very overlapped in mammalian. Inhibition of ZF5 expression in HepG2 cells (by RNA interference) leads to at least 1.5 times stimulations of FMR1 gene expression in these cells. To estimate the contribution of GCC-triplet repeats in FMR1 gene expression regulation we used two alternative variants of genetic construction: containing Iuciferase reporter gene under 5′-regulatory region fragment devoid of GCC-triplet repeats or including the GCC9 nucleotide sequence. HepG2 cells were co-transfected by these constructions and expressions vectors of ZF5 or (and) CGGBP-20 respectively. It was found that ZF5 downregulated the activity of 5′-regulatory region of FMR1 gene in both cases (acting probably through canonic 5′-GCGCGC3′ sites). The presence of GCC-triplet repeats in the construction weakens this ZF5 effect. CGGBP-20 downregulates the activity of 5'-region of FMR1 gene in the presence of GCC-tripIets only. The data obtained evidently indicate differently directed ZF5 effects on FMR1 gene expression and suggest the mechanism to explain the earlier demonstrated phenomenon about increasing of mRNA level in permutation FMR1 allele carries.
    Cell and Tissue Biology 01/2009; 4(1):54-62. DOI:10.1134/S1990519X10010050
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