Article

Novel repressor of the human FMR1 gene - identification of p56 human (GCC)(n)-binding protein as a Krüppel-like transcription factor ZF5.

Department of Biochemistry, Institute of Experimental Medicine, Russian Academy of Medical Sciences, St Petersburg, Russia.
FEBS Journal (Impact Factor: 4.25). 10/2007; 274(18):4848-62. DOI: 10.1111/j.1742-4658.2007.06006.x
Source: PubMed

ABSTRACT 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.

0 Bookmarks
 · 
77 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Genome-wide chromatin maps have permitted the systematic mapping of putative regulatory elements across multiple human cell types, revealing tens of thousands of candidate distal enhancer regions. However, until recently, their experimental dissection by directed regulatory motif disruption has remained unfeasible at the genome scale, due to the technological lag in large-scale DNA synthesis. Here, we employ a massively parallel reporter assay (MPRA) to measure the transcriptional levels induced by 145bp DNA segments centered on evolutionarily-conserved regulatory motif instances and found in 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 2,104 wild-type sequences and an additional 3,314 engineered enhancer variants containing targeted motif disruptions, each using 10 barcode tags in two cell lines and 2 replicates. The resulting data strongly confirm the enhancer activity and cell type specificity of enhancer chromatin states, the ability of 145bp 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) scrambling, removing, or 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 all associated with wild-type enhancer activity; (3) scrambling repressor motifs leads to aberrant reporter expression in cell lines where the enhancers are usually not active. Our results suggest a general strategy for deciphering cis-regulatory elements by systematic large-scale experimental manipulation, and provide quantitative enhancer activity measurements across thousands of constructs that can be mined to generate and test predictive models of gene expression.
    Genome Research 03/2013; · 14.40 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: While FMR1 is silenced in Fragile X syndrome (FXS) patients carrying the full mutation, its expression is elevated (2-8 fold) in premutated individuals. These people may develop the Fragile X-associated Tremor/Ataxia syndrome (FXTAS), a late onset neurodegenerative disorder characterized by ataxia and parkinsonism. In addition, people carrying the premutation can be affected by a set of neurological and behavioural disorders during young age. Problems of memory have been detected in these patients as well as in the mouse models for FXTAS. To date little is known concerning the metabolism of FMR1 mRNA, notwithstanding the importance of the finely tuned regulation of the expression of this gene. In the present study we identified three microRNAs that specifically target the 3'UTR of FMR1 and can modulate its expression throughout the brain particularly at the synapse where their expression is very high. The expression level of miR-221 is reduced in synaptosomal preparations of young FXTAS mice suggesting a general deregulation of transcripts located at the synapse of these mice. By transcriptome analysis we show here a robust deregulation of the expression levels of genes involved in learning, memory and autistic behavior, Parkinson disease and neurodegeneration. These findings suggest the presence of a 'synapthopathy' in these animals. Interestingly, many of those deregulated mRNAs are target of the same miRNAs that modulate the expression of FMR1 at the synapse.
    Human Molecular Genetics 02/2013; · 7.69 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Complement C3 is involved in various protective and regulatory mechanisms of immune system. Recently it was established that C3 expression is regulated by nuclear receptors. Hepatic nuclear factor 4α (HNF4α) is a nuclear receptor critical for hepatic development and metabolism. We have shown that HNF4α is a positive regulator of C3 gene expression, realizing its effects through binding to two HNF4-response elements within the C3 promoter in HepG2 cells. TNFα is a well established positive regulator of C3 expression in hepatocytes during acute phase of inflammation. TNFα decreases the amount of HNF4α protein in HepG2 cells through NF-κB and MEK1/2 pathways thereby leading to decrease in HNF4α bound to the C3 promoter. TNFα and HNF4α act in a synergetic way resulting in the potent activation of C3 transcription. These results suggest a novel mechanism of C3 regulation during acute phase response in HepG2 cells and display the mechanism of interaction of TNFα-induced pathways and HNF4α in transcriptional regulation of C3 gene.
    Gene 04/2013; · 2.20 Impact Factor

Full-text (2 Sources)

View
32 Downloads
Available from
Jun 3, 2014