CpG islands (CGIs) function as promoters for greater than 60% of human genes. Importantly, these elements remain mostly free of CpG methylation - an epigenetic mark associated with stable transcriptional silencing - despite the fact that the vast majority of CpG sites in the genome are methylated. While the relative immunity of CGIs against epigenetic silencing has been noted for years and is critical to their function, its underlying mechanism has remained elusive. Here, we report three novel and key observations relevant to this process: (i) a large fraction of CGI promoters, while GC-rich overall, display marked strand asymmetry, or skew, in the distribution of G and C residues;(ii) transcription through such regions of high GC-skew leads to the formation of long R-loop structures in which the newly transcribed G-rich RNA remains hybridized to the template C-rich DNA strand, forcing the non-template DNA strand into a largely single-stranded conformation;and (iii) R-loop formation protects the underlying DNA sequence from the action of DNA methyltransferases (DNMTs). Based on this knowledge, we hypothesize that R-loop formation at mammalian CGI promoters serves to protect these regions against epigenetic silencing. We propose to further test this hypothesis through three Specific Aims combining computational, genomics, biochemical, and molecular genetics approaches in human and mouse cells.
Specific Aim 1 : To test the hypothesis that R-loop formation is a widespread and conserved property of mammalian CGI promoters.
Specific Aim 2 : To test the hypothesis that R-loop formation protects against DNA methylation.
Specific Aim 3 : To test the hypothesis that altered R-loop formation leads to aberrant DNA methylation patterns.

Public Health Relevance

Project Narrative: This proposal provides a novel framework for understanding CGI promoter function and for addressing how deregulated protection of CGIs often leads to human diseases, including cancer, imprinting, and most importantly, auto-immune disorders such as Aicardi-Goutieres Syndrome.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM094299-04
Application #
8512740
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Carter, Anthony D
Project Start
2010-08-01
Project End
2015-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
4
Fiscal Year
2013
Total Cost
$268,079
Indirect Cost
$86,562
Name
University of California Davis
Department
Biochemistry
Type
Schools of Medicine
DUNS #
047120084
City
Davis
State
CA
Country
United States
Zip Code
95618
Chédin, Frédéric (2016) Nascent Connections: R-Loops and Chromatin Patterning. Trends Genet 32:828-838
Sanz, Lionel A; Hartono, Stella R; Lim, Yoong Wearn et al. (2016) Prevalent, Dynamic, and Conserved R-Loop Structures Associate with Specific Epigenomic Signatures in Mammals. Mol Cell 63:167-78
Stork, Caroline Townsend; Bocek, Michael; Crossley, Madzia P et al. (2016) Co-transcriptional R-loops are the main cause of estrogen-induced DNA damage. Elife 5:
Lim, Yoong Wearn; Sanz, Lionel A; Xu, Xiaoqin et al. (2015) Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi-Goutières syndrome. Elife 4:
Hartono, Stella R; Korf, Ian F; Chédin, Frédéric (2015) GC skew is a conserved property of unmethylated CpG island promoters across vertebrates. Nucleic Acids Res 43:9729-41
Yang, Yanzhong; McBride, Kevin M; Hensley, Sean et al. (2014) Arginine methylation facilitates the recruitment of TOP3B to chromatin to prevent R loop accumulation. Mol Cell 53:484-97
Loomis, Erick W; Sanz, Lionel A; Chédin, Frédéric et al. (2014) Transcription-associated R-loop formation across the human FMR1 CGG-repeat region. PLoS Genet 10:e1004294
Powell, Weston T; Coulson, Rochelle L; Gonzales, Michael L et al. (2013) R-loop formation at Snord116 mediates topotecan inhibition of Ube3a-antisense and allele-specific chromatin decondensation. Proc Natl Acad Sci U S A 110:13938-43
Fagan, Rebecca L; Wu, Meng; Chédin, Frédéric et al. (2013) An ultrasensitive high throughput screen for DNA methyltransferase 1-targeted molecular probes. PLoS One 8:e78752
Ginno, Paul A; Lim, Yoong Wearn; Lott, Paul L et al. (2013) GC skew at the 5' and 3' ends of human genes links R-loop formation to epigenetic regulation and transcription termination. Genome Res 23:1590-600

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