1. Volume 45, Issue 6, Pages 814-825 (March 2012)
R-Loop Formation Is a Distinctive Characteristic of Unmethylated Human CpG Island Promoters 
Paul A. Ginno, Paul L. Lott, Holly C. Christensen, Ian Korf, Frédéric Chédin 
Molecular Cell 
Volume 45, Issue 6, Pages (March 2012)
DOI: /j.molcel
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2012 45, 814-825DOI: (10.1016/j.molcel.2012.01.017)
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 1
Co-oriented Positive GC Skew Is a Common Property of Strong Human CGI Promoters
(A) Transcription through regions of GC skew such that a G-rich RNA is generated can lead to R loop formation (top). In contrast, transcription through the same region such that a C-rich RNA is produced does not lead to R loop formation (bottom). The G-rich and C-rich strands are color-coded in red and blue, respectively; open lollipops represent unmethylated CpG sites. Note that transcription through regions devoid of GC skew does not give rise to R loop formation either.
(B) Percent of human genes (RefSeq) showing overlap with GC skew at their 5′ (−500 to +1500 relative to the beginning of the gene) or 3′ (−500 to +1500 relative to the end of the gene) extremities, as determined by the SkewR algorithm. The overlap was calculated for each chromosome and the average and standard deviation for the genome are shown.
(C) Metagene analysis of the 7,820 genes showing positive GC skew co-oriented with transcription. All genes were oriented from left to right (as denoted by the arrow above) and aligned at their TSSs. The graph shows the aggregate GC%, CpG obs/exp ratio, and GC skew values calculated for a 50 nucleotide sliding window. The gray shaded area highlights the portion of the region corresponding to a CpG island (GC% > 50% and CpG o/e > 0.60).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

4. Figure 2
Formation of Genomic R Loops at the Endogenous SNRPN CGI Promoter
(A) Schematic representation of the 4.5 kb region surrounding the SNRPN CGI (green). The major transcription start site and first untranslated exon are indicated by a broken arrow and blue box, respectively. The CpG o/e ratio, GC percent, and GC skew are indicated in UCSC Genome Browser dense track format; strong SkewR blocks are indicated by black boxes. The analyzed region is indicated by dashed lines and expanded in the panel below.
(B) The GC skew over the analyzed region is plotted using a 100 nucleotide sliding window. The solid line represents the average genomic GC skew with the standard deviation shown as dotted lines.
(C) This panel depicts the distribution of ssDNA footprints over the analyzed region in a stack format. This was generated from the analysis of 21 individual DNA molecules recovered from H1 ESCs, Ntera2 cells, blood, and brain. Vertical tick marks indicate when a given cytosine on the nontemplate DNA strand was sequenced as thymine, indicative of a single-stranded conformation. Green tick marks indicate converted cytosines in CpG dinucleotides. The position of all cytosines along the region is indicated on the line at the bottom of the stack (all Cs). The gray-shaded area highlights the span of the longest ssDNA footprints. The primers used to generate the sequenced amplicons are indicated at the bottom (red primers corresponding to “converted” primers matching bisulfite-modified DNA).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

5. Figure 3 R Loop Formation at the Endogenous APOE Promoter
All symbols are as described in Figure 2. DNA was recovered from human H1 ESCs and Ntera2 cells and ssDNA footprints determined from 12 independent DNA molecules.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

6. Figure 4
Formation of Genomic R Loops at the Endogenous Mouse Airn CGI Promoter
(A) Schematic description of the 4.5 kb region surrounding the Airn promoter.
(B) GC skew over the analyzed region.
(C) Analysis of R loop-derived ssDNA footprints. Symbols are as in Figure 2, except each line corresponds to one individual DNA molecule. DNA was analyzed from mESCs differentiated along a neural path by addition of retinoic acid (−LIF+RA, high Airn expression) with (+H) or without (−H) RNase H pretreatment prior to bisulfite footprinting. Amplicons were also recovered from undifferentiated mESCs (+LIF, little to no Airn expression). Brackets indicate regions that underwent short deletions due to instability of the DNA sequence in E. coli.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

7. Figure 5 Widespread R Loop Formation at Human Promoters
(A) H1 hESCs were stained with the S9.6 antibody (top left) and counterstained with DAPI (top right). A merge of both channels is shown (bottom left). Nucleolar and mitochondrial staining are indicated by arrows, whereas the boxed inset shows a magnified view of the nucleoplasm. The bottom right panel shows the cellular distribution of the HA-tagged human RNASEH1(ΔMLS) protein (red) upon transfection in HEK293 cells. Cells were counterstained with DAPI (blue).
(B) The aggregate GC skew for all newly identified R loop forming promoters is graphed in red. All genes were aligned at their TSS and the GC skew computed using a 50 nt sliding window over the −500/+1500 region. The overall GC skew predicted for all 7,820 highly skewed promoters (Figure 1C) is shown in green for comparison.
(C and D) Examples of DRIP-seq data. Each panel shows a schematic description of the region analyzed with TSSs indicated by broken arrows (minor TSSs are shown by dashed lines), exons by blue boxes, CpG islands by green boxes. The SkewR tract shows the position of GC skew blocks with red indicating G-rich blocks and blue C-rich blocks. The RE tract indicates cut sites for the five restriction enzymes used to fragment the genome. Below are two tracts representing DRIP-seq read density in the absence (top) or presence (bottom) of Ribonuclease pretreatment. The red box indicates restriction fragments that coincide with RNase H-sensitive DRIP-seq peaks and therefore harbor R loop forming regions. The coordinates of each region analyzed (Hg19) are given. The two regions shown here also correspond to DRIVE-seq peaks (data not shown).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

8. Figure 6
R Loop Formation Potential Is Predictive of the Unmethylated Status of CGI Promoters
(A) Percent of promoter CGIs (n = 13,636) and gene body CGIs (n = 4,598) with GC skew overlap.
(B) Percent of unmethylated (n = 1,785) and methylated (n = 1,594) CGIs showing GC skew overlap (methylation data from the hESC data set; Straussman et al., 2009). Unmethylated CGIs showed a methylation score <−0.8, whereas methylated CGIs had a score >1.3 in both hESC cell lines). In both panels, the overlap was calculated for each chromosome, and the average and standard deviation for the genome are shown.
(C) Metagene analysis of a subset of 4,528 promoter CGIs lacking strong GC skew. Symbols and analysis are as described for Figure 1C.
(D) Aggregate CpG methylation levels around the TSS of genes corresponding to strong CGIs characterized by high GC skew (n = 7,820; red), weak CGIs characterized by intermediate GC skew (n = 4,526; orange), and “CpG-poor” promoters characterized by little to no skew (n = 7,570; green), respectively. The DNA methylation data were from Laurent et al. (2010).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

9. Figure 7 R Loop-Mediated Protection from DNA Methylation
(A and B) The ability of DNMT3B1 to methylate the SNRPN (A) and Airn (B) CGIs in the presence or absence of the stimulatory factor DNMT3L is shown. CGIs were cloned in episomes in an R loop forming or non-R loop forming orientation as graphically indicated above. The episomes were harvested 7 days posttransfection and methylation was analyzed after cleavage by the methyl-sensitive HpaII enzyme, gel electrophoresis, and Southern blotting with SNRPN and Airn CGI probes, respectively. Regions showing clear lack of methylation are highlighted by brackets and an asterisk.
(C) Identical to (A), except the constitutive CMV promoter driving transcription through the SNRPN region was deleted.
(D) For each sample, the graph depicts the fold reduction in methylation comparing the R loop to the non-R loop forming orientation, as determined by band densitometry. Values were calculated from three independent experiments and are shown with means and standard error.
(E) The average methylation levels (in %) measured by bisulfite sequencing on the G-rich strand of the SNRPN insert is presented for both orientations. The number of independent molecules sequenced in each case is indicated together with the standard error for each sample.
(F) Model for the function of R loops in the protection against de novo DNA methylation and epigenetic silencing at GC skewed CGIs.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions