DNA methylation is essential for mammalian development and physiology. Here we report that the developmentally regulated H19 lncRNA binds to and inhibits S-adenosylhomocysteine hydrolase (SAHH), the only mammalian enzyme capable of hydrolysing S-adenosylhomocysteine (SAH). SAH is a potent feedback inhibitor of S-adenosylmethionine (SAM)-dependent methyltransferases that methylate diverse cellular components, including DNA, RNA, proteins, lipids and neurotransmitters. We show that H19 knockdown activates SAHH, leading to increased DNMT3B-mediated methylation of an lncRNA-encoding gene Nctc1 within the Igf2-H19-Nctc1 locus. Genome-wide methylation profiling reveals methylation changes at numerous gene loci consistent with SAHH modulation by H19. Our results uncover an unanticipated regulatory circuit involving broad epigenetic alterations by a single abundantly expressed lncRNA that may underlie gene methylation dynamics of development and diseases and suggest that this mode of regulation may extend to other cellular components.

Background: Many pentatricopeptide repeat (PPR) proteins are RNA site specificity factors and include C-terminal DYW deaminase domains. Results: ELI1 and DOT4 are required for editing single sites. The DYW deaminase domain binds two zinc atoms. Conclusion: The C terminus of PLS-type PPR proteins shares molecular characteristics with cytidine deaminase. Significance: This study provides the first evidence that DYW deaminase domains bind zinc.

BackgroundPentatricopeptide repeat (PPR) proteins are required for numerous RNA processing events in plant organelles including C-to-U editing, splicing, stabilization, and cleavage. Fifteen PPR proteins are known to be required for RNA editing at 21 sites in Arabidopsis chloroplasts, and belong to the PLS class of PPR proteins. In this study, we investigate the co-evolution of four PPR genes (CRR4, CRR21, CLB19, and OTP82) and their six editing targets in Brassicaceae species. PPR genes are composed of approximately 10 to 20 tandem repeats and each repeat has two α-helical regions, helix A and helix B, that are separated by short coil regions. Each repeat and structural feature was examined to determine the selective pressures on these regions.ResultsAll of the PPR genes examined are under strong negative selection. Multiple independent losses of editing site targets are observed for both CRR21 and OTP82. In several species lacking the known editing target for CRR21, PPR genes are truncated near the 17th PPR repeat. The coding sequences of the truncated CRR21 genes are maintained under strong negative selection; however, the 3’ UTR sequences beyond the truncation site have substantially diverged. Phylogenetic analyses of four PPR genes show that sequences corresponding to helix A are high compared to helix B sequences. Differential evolutionary selection of helix A versus helix B is observed in both plant and mammalian PPR genes.ConclusionPPR genes and their cognate editing sites are mutually constrained in evolution. Editing sites are frequently lost by replacement of an edited C with a genomic T. After the loss of an editing site, the PPR genes are observed with three outcomes: first, few changes are detected in some cases; second, the PPR gene is present as a pseudogene; and third, the PPR gene is present but truncated in the C-terminal region. The retention of truncated forms of CRR21 that are maintained under strong negative selection even in the absence of an editing site target suggests that unrecognized function(s) might exist for this PPR protein. PPR gene sequences that encode helix A are under strong selection, and could be involved in RNA substrate recognition.

The distribution of 5-methylcytosine (5-mC) in DNA within the eukaryotic genome is known to greatly affect gene regulation and is currently a major topic of research. Studies on DNA methylation have been aided by advancements in bisulfite conversion and Next-Gen sequencing technologies which can provide single-base resolution of 5-mC across the entire genome. Many whole-genome bisulfite sequencing (WGBS) library preparation protocols designed for analysis of 5-mC distribution employ bisulfite to chemically convert unmethylated cytosine bases into uracil following the library preparation step. While these protocols produce reliable results, degradation of DNA is always an inherent issue when dealing with bisulfite conversion, and a large proportion of the adapterized library is often fragmented and can no longer be amplified. This requires high levels of DNA to serve as the input material. However, by rearranging the order of library preparation and bisulfite conversion, we developed a streamlined protocol that necessitates less input material for accurate whole-genome methylation analysis at single-base resolution. This unique workflow accommodates picogram quantities of starting material, making it ideal for analysis of precious samples that are of limited availability (e.g., FFPE). Comparison of sequencing data generated using this new library preparation method and data generated with established Reduced Representation Bisulfite Sequencing demonstrated a correlation of 0.95 in CpG sites with > 10X coverage in human DNA. Also, with only slight modification, this protocol is versatile enough to be used in the preparation of libraries for ChIP-Seq and RNA-Seq analyses. Citation Format: Karolyn Giang, TzuHung Chung, Xueguang Sun, Marc E. Van Eden, Xi Yu Jia. A fast and simple method for whole-genome bisulfite library preparation from ultra-low DNA inputs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2293. doi:10.1158/1538-7445.AM2014-2293