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Defining the role of the CGGBP1 protein in FMR1 gene expression.

ABSTRACT: Fragile X syndrome is the most common heritable form of intellectual disability and is caused by the expansion over 200 repeats and subsequent methylation of the CGG triplets at the 5' UTR of the FMR1 gene, leading to its silencing. The epigenetic and molecular mechanisms responsible for FMR1 gene silencing are not fully clarified. To identify structure-specific proteins that could recruit components of the silencing machinery we investigated the role of CGGBP1 in FMR1 gene transcription. CGGBP1 is a highly conserved protein that binds specifically to unmethylated CGG tracts. Its role on FMR1 transcription is yet to be defined. Sequencing analysis and expression studies through quantitative PCR of CGGBP1 were performed in cell lines with different allele expansions: wild type, premutation, methylated full mutation and unmethylated full mutation, demonstrating no differences between them. ChIP assays clearly demonstrated that CGGBP1 binds to unmethylated CGG triplets of the FMR1 gene, but not to methylated CGGs. We also observed that CGGBP1 binding to the FMR1 locus was restored after pharmacological demethylation, with 5-azadC, of alleles, carriers of methylated full mutation, suggesting a possible role for CGGBP1 in FMR1 expression. CGGBP1 silencing with shRNAs (reaching ~98% of CGGBP1-mRNA depletion) did not affect FMR1 transcription and CGG expansion stability in expanded alleles. Although the strong binding to the CGG tract could suggest a relevant role of CGGBP1 on FMR1 gene expression, our results demonstrate that CGGBP1 has no direct effect on FMR1 transcription and CGG repeat stability.

SUBMITTER: Goracci M

PROVIDER: S-EPMC4930089 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Defining the role of the CGGBP1 protein in FMR1 gene expression.

Goracci Martina M Lanni Stella S Mancano Giorgia G Palumbo Federica F Chiurazzi Pietro P Neri Giovanni G Tabolacci Elisabetta E

European journal of human genetics : EJHG 20150826 5

Fragile X syndrome is the most common heritable form of intellectual disability and is caused by the expansion over 200 repeats and subsequent methylation of the CGG triplets at the 5' UTR of the FMR1 gene, leading to its silencing. The epigenetic and molecular mechanisms responsible for FMR1 gene silencing are not fully clarified. To identify structure-specific proteins that could recruit components of the silencing machinery we investigated the role of CGGBP1 in FMR1 gene transcription. CGGBP1 ...[more]

PMID: 26306647

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Project description:BACKGROUND:Adhesion G protein-coupled receptors (aGPCRs) are the second largest of the five GPCR families and are essential for a wide variety of physiological processes. Zebrafish have proven to be a very effective model for studying the biological functions of aGPCRs in both developmental and adult contexts. However, aGPCR repertoires have not been defined in any fish species, nor are aGPCR expression profiles in adult tissues known. Additionally, the expression profiles of the aGPCR family have never been extensively characterized over a developmental time-course in any species. RESULTS:Here, we report that there are at least 59 aGPCRs in zebrafish that represent homologs of 24 of the 33 aGPCRs found in humans; compared to humans, zebrafish lack clear homologs of GPR110, GPR111, GPR114, GPR115, GPR116, EMR1, EMR2, EMR3, and EMR4. We find that several aGPCRs in zebrafish have multiple paralogs, in line with the teleost-specific genome duplication. Phylogenetic analysis suggests that most zebrafish aGPCRs cluster closely with their mammalian homologs, with the exception of three zebrafish-specific expansion events in Groups II, VI, and VIII. Using quantitative real-time PCR, we have defined the expression profiles of 59 zebrafish aGPCRs at 12 developmental time points and 10 adult tissues representing every major organ system. Importantly, expression profiles of zebrafish aGPCRs in adult tissues are similar to those previously reported in mouse, rat, and human, underscoring the evolutionary conservation of this family, and therefore the utility of the zebrafish for studying aGPCR biology. CONCLUSIONS:Our results support the notion that zebrafish are a potentially useful model to study the biology of aGPCRs from a functional perspective. The zebrafish aGPCR repertoire, classification, and nomenclature, together with their expression profiles during development and in adult tissues, provides a crucial foundation for elucidating aGPCR functions and pursuing aGPCRs as therapeutic targets.

Project description:Low FMR1 variants (CGGn<26) have been associated with premature ovarian aging, female infertility and poor IVF treatment success. Until now, there is little published information concerning possible molecular mechanisms for this effect. We wished to examine whether relative expression of RNA and the FMR1 gene's fragile X mental retardation protein (FMRP) RNA isoforms differ in women with various FMR1 sub-genotypes (normal, low CGGn<26 and/or high CGGn≥34). This prospective cohort study was conducted between 2014 and 2017 in a clinical research unit of the Center for Human Reproduction in New York City. The study involved a total of 98 study subjects, including 18 young oocyte donors and 80 older infertility patients undergoing routine in vitro fertilization (IVF) cycles. The main outcome measure was RNA expression in human luteinized granulosa cells of 5 groups of FMRP isoforms. The relative expression of FMR1 RNA in human luteinized granulosa cells was measured by real-time PCR and a possible association with CGGn was explored. All 5 groups of FMRP RNA isoforms examined were found to be differentially expressed in human luteinized granulosa cells. The relative expression of four FMR1 RNA isoforms showed significant differences among 6 FMR1 sub-genotypes. Women with at least one low allele expressed significantly lower levels of all 5 sets of FRMP isoforms in comparison to the non-low group. While it would be of interest to see whether FMRP is also decreased in the low-group we recognize that in recent years it has been increasingly documented that information flow of genetics may be regulated by non-coding RNA, that is, without translation to a protein product. We, thus, conclude that various CGG expansions of FMR1 allele may lead to changes of RNA levels and ratios of distinct FMRP RNA isoforms, which could regulate the translation and/or cellular localization of FMRP, affect the expression of steroidogenic enzymes and hormonal receptors, or act in some other epigenetic process and therefore result in the ovarian dysfunction in infertility.

Dataset Information

Defining the role of the CGGBP1 protein in FMR1 gene expression.

Publications

Defining the role of the CGGBP1 protein in FMR1 gene expression.

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