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. INTRODUCTIONFragile X syndrome (FXS, OMIM #300624) is the leading cause of heritable intellectual disability and autism, belonging to the group of the so-called FRAXopathies. 1,2 FXS affects~1 in 4000 males and 1 in 9000 females. 3,4 It is almost invariably due to a large expansion (full mutation, FM) of an instable CGG repeat in the 5′ untranslated region (5′-UTR) of the FMR1 gene (in Xq27.3). CGG expansion over 200 repeats (FM) is followed by epigenetic modifications of the 5′-UTR of the FMR1 gene, including primarily DNA methylation in both the expanded CGG tract and in the neighboring CpG island, as well as other heterochromatic histone modifications (ie, methylation of H3K9, trimethylation of H3K27, H3 and H4 hypoacetylation). These epigenetic modifications cause the transcriptional silencing of the FMR1 gene and consequently the lack of the FMRP protein. [5][6][7] FMRP is an RNA-binding protein that is involved in multiple pathways of mRNA metabolism, essentially as an inhibitor of translation particularly at synaptic level. 8 Several years ago have been described rare individuals of normal intelligence, carrying a transcriptionally active unmethylated full mutation (UFM). 9 The epigenetic characterization of these rare UFM cell lines showed histone marks similar to euchromatin with absence of DNA methylation in both the CpG island of the promoter region and the CGG repeat itself. Levels of FMR1 transcription are sl...

The CGG triplet repeat binding protein 1 counteracts DNA secondary structure-induced transcription-replication conflicts

“…CGGBP1 is a 20kDa protein with a nuclear localization signal and a predicted C2H2-type Zn finger DNA-binding domain (Müller-Hartmann et al, 2000;Singh and Westermark, 2015). Electrophoretic mobility shift assays with CGG-repeat containing oligonucleotides confirmed strong affinity to CGG-repeat sequences in vitro (Deissler et al, 1996;Müller-Hartmann et al, 2000), which has also been verified in vivo at selected loci, such as the CGG repeats in the 5' UTR of the FMR1 gene (Goracci et al, 2016). Overexpression of CGGBP1 promotes the binding onto the 5' UTR of the FMR1 gene leading to transcriptional repression (Müller-Hartmann et al, 2000), suggesting that an excess of CGGBP1-bound repeats may have a dominant negative effect on transcription elongation.…”

DNA Methylation, Mechanisms of FMR1 Inactivation and Therapeutic Perspectives for Fragile X Syndrome

“…Another interesting candidate protein is the CGG-binding protein 1, CGGBP1 [ 43 ], which specifically binds unmethylated CGG repeats in a length-dependent manner [ 43 ]. Though its depletion does not change FMR1 transcription levels [ 44 ], its overexpression could be associated with an increased binding to longer CGG repeat tracts that may guide local H3K9 methylation.…”

CGGBP1-regulated cytosine methylation at CTCF-binding motifs resists stochasticity

“…Interestingly, like CTCF, CGGBP1 itself is a cytosine methylation-sensitive DNA-binding protein [ 20 – 25 ]. However, there is evidence that CGGBP1 binding to the target sequences prevents cytosine methylation from taking place [ 25 , 26 ]. WGBS experiments have shown that CGGBP1 depletion leads to genome-wide disturbances in cytosine methylation [ 27 , 28 ].…”