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. 2015 May 16;16(1):390.
doi: 10.1186/s12864-015-1593-2.

CGGBP1 mitigates cytosine methylation at repetitive DNA sequences

Prasoon Agarwal  1 , Paul Collier  2 , Markus Hsi-Yang Fritz  3 , Vladimir Benes  4 , Helena Jernberg Wiklund  5 , Bengt Westermark  6 , Umashankar Singh  7
Affiliations

CGGBP1 mitigates cytosine methylation at repetitive DNA sequences

Prasoon Agarwal et al. BMC Genomics. .

Abstract

Background: CGGBP1 is a repetitive DNA-binding transcription regulator with target sites at CpG-rich sequences such as CGG repeats and Alu-SINEs and L1-LINEs. The role of CGGBP1 as a possible mediator of CpG methylation however remains unknown. At CpG-rich sequences cytosine methylation is a major mechanism of transcriptional repression. Concordantly, gene-rich regions typically carry lower levels of CpG methylation than the repetitive elements. It is well known that at interspersed repeats Alu-SINEs and L1-LINEs high levels of CpG methylation constitute a transcriptional silencing and retrotransposon inactivating mechanism.

Results: Here, we have studied genome-wide CpG methylation with or without CGGBP1-depletion. By high throughput sequencing of bisulfite-treated genomic DNA we have identified CGGBP1 to be a negative regulator of CpG methylation at repetitive DNA sequences. In addition, we have studied CpG methylation alterations on Alu and L1 retrotransposons in CGGBP1-depleted cells using a novel bisulfite-treatment and high throughput sequencing approach.

Conclusions: The results clearly show that CGGBP1 is a possible bidirectional regulator of CpG methylation at Alus, and acts as a repressor of methylation at L1 retrotransposons.

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Figures

Figure 1
Figure 1
Global methylation changes upon CGGBP1-depletion. A: Colorimetric analysis reveals an increase in CpG methylation. Y-axis shows colorimetry signal from 3 independent assays (mean ± SEM). B: GeneSpring output showing changes in CpG methylation. The increase in methylation is significant between CGGBP1-depleted and Control samples. Y-axis shows C count (%, [calculated as C count x100/total number of nucleotides]). C: Frequency plotting of CpG methylation changes across different ranges of methylation. This plot shows binning of data depicted in 1B. X-axis shows methylation frequency bins and Y-axis shows C count (%).
Figure 2
Figure 2
Alu and LINE-1 repeats exhibit methylation changes upon CGGBP1-depletion. A: Mean methylation increase on Alu repeats measured by CG frequency per PCR product sequence. Y-axis shows nucleotide frequency calculated per sequence. X-axis shows the samples and treatments. B: Frequency distribution of Alu methylation across different ranges shows decrease (<8%) and increase (>12%) at extremes in CGGBP1 shmiR sample as compared to Control shmiR sample. Y-axis shows relative frequencies of CG (a measure of methylation; normalized for different number of sequences per sample). C: Plotting of the >12% and <8% methylation subset from B shows the tailing of differentially methylated sequences at both extremes clearly. D: Frequency plot of data plotted in C and best curve fit shows sum-of-two-Gaussian fit for CGGBP1-depleted sample and a single Gaussian distribution for control sample suggesting that indeed there are two groups of methylation levels for Alus in CGGBP1 shmiR but only one group of methylation level in Control shmiR sample. E and F: Increase in CpG content negatively correlated with TpG frequency in both samples establishing the fact that the changes in cytosine content was indeed due to bisulfite conversion of unmethylated cytosines. G: Increase in methylation on LINE-1 elements was significant with no bidirectional heterogeneity as seen for the Alus. H: Frequency plotting showed that <7.5% (marked with dotted line) methylation was prevalent in control sample, but >7.5% methylation was prevalent in CGGBP1-depleted sample (values normalized for different number of sequences per sample). I and J: CpG and TpG frequencies on LINE-1 exhibited inverse correlations in CGGBP1 and control shmiR samples establishing the fact that the changes in cytosine content was indeed due to bisulfite conversion of unmethylated cytosines.
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References

    1. Naumann F, Remus R, Schmitz B, Doerfler W. Gene structure and expression of the 5’-(CGG) (n)-3’-binding protein (CGGBP1) Genomics. 2004;83:106–118. doi: 10.1016/S0888-7543(03)00212-X. - DOI - PubMed
    1. Deissler H, Wilm M, Genc B, Schmitz B, Ternes T, Naumann F, et al. Rapid protein sequencing by tandem mass spectrometry and cDNA cloning of p20-CGGBP. A novel protein that binds to the unstable triplet repeat 5’-d (CGG) n-3’ in the human FMR1 gene. J Biol Chem. 1997;272:16761–16768. doi: 10.1074/jbc.272.27.16761. - DOI - PubMed
    1. Muller-Hartmann H, Deissler H, Naumann F, Schmitz B, Schroer J, Doerfler W. The human 20-kDa 5’-(CGG) (n)-3’-binding protein is targeted to the nucleus and\ affects the activity of the FMR1 promoter. J Biol Chem. 2000;275:6447–6452. doi: 10.1074/jbc.275.9.6447. - DOI - PubMed
    1. Deissler H, Behn-Krappa A, Doerfler W. Purification of nuclear proteins from human HeLa cells that bind specifically to the unstable tandem repeat (CGG) n in the human FMR1 gene. J Biol Chem. 1996;271:4327–4334. doi: 10.1074/jbc.271.8.4327. - DOI - PubMed
    1. Agarwal P, Enroth S, Teichmann M, Wiklund HJ, Smit A, Westermark B, et al. Growth signals employ CGGBP1 to suppress transcription of Alu-SINEs. Cell cycle. 2014:0. [Epub ahead of print]. - PMC - PubMed

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