Uncovering the roles of DNA hemi-methylation in transcriptional regulation using MspJI-assisted hemi-methylation sequencing.

Hemi-methylated cytosine dyads widely occur on mammalian genomic DNA, and can be stably inherited across cell divisions, serving as potential epigenetic marks. Previous identification of hemi-methylation relied on harsh bisulfite treatment, leading to extensive DNA degradation and loss of methylation information. Here we introduce Mhemi-seq, a bisulfite-free strategy, to efficiently resolve methylation status of cytosine dyads into unmethylation, strand-specific hemi-methylation, or full-methylation. Mhemi-seq reproduces methylomes from bisulfite-based sequencing (BS-seq & hpBS-seq), including the asymmetric hemi-methylation enrichment flanking CTCF motifs. By avoiding base conversion, Mhemi-seq resolves allele-specific methylation and associated imprinted gene expression more efficiently than BS-seq. Furthermore, we reveal an inhibitory role of hemi-methylation in gene expression and transcription factor (TF)-DNA binding, and some displays a similar extent of inhibition as full-methylation. Finally, we uncover new hemi-methylation patterns within Alu retrotransposon elements. Collectively, Mhemi-seq can accelerate the identification of DNA hemi-methylation and facilitate its integration into the chromatin environment for future studies.

A continuum of zinc finger transcription factor retention on native chromatin underlies dynamic genome organization.

Transcription factor (TF) residence on chromatin translates into quantitative transcriptional or structural outcomes on genome. Commonly used formaldehyde crosslinking fixes TF-DNA interactions cumulatively and compromises the measured occupancy level. Here we mapped the occupancy level of global or individual zinc finger TFs like CTCF and MAZ, in the form of highly resolved footprints, on native chromatin. By incorporating reinforcing perturbation conditions, we established S-score, a quantitative metric to proxy the continuum of CTCF or MAZ retention across different motifs on native chromatin. The native chromatin-retained CTCF sites harbor sequence features within CTCF motifs better explained by S-score than the metrics obtained from other crosslinking or native assays. CTCF retention on native chromatin correlates with local SUMOylation level, and anti-correlates with transcriptional activity. The S-score successfully delineates the otherwise-masked differential stability of chromatin structures mediated by CTCF, or by MAZ independent of CTCF. Overall, our study established a paradigm continuum of TF retention across binding sites on native chromatin, explaining the dynamic genome organization.

Towards a predictive model of chromatin 3D organization.

Architectural proteins mediate interactions between distant regions in the genome to bring together different regulatory elements while establishing a specific three-dimensional organization of the genetic material. Depletion of specific architectural proteins leads to miss regulation of gene expression and alterations in nuclear organization. The specificity of interactions mediated by architectural proteins depends on the nature, number, and orientation of their binding site at individual genomic locations. Knowledge of the mechanisms and rules governing interactions among architectural proteins may provide a code to predict the 3D organization of the genome.

Resolution of the DNA methylation state of single CpG dyads using in silico strand annealing and WGBS data.

Whole-genome bisulfite sequencing (WGBS) has been widely used to quantify cytosine DNA methylation frequency in an expanding array of cell and tissue types. Because of the denaturing conditions used, this method ultimately leads to the measurement of methylation frequencies at single cytosines. Hence, the methylation frequency of CpG dyads (two complementary CG dinucleotides) can be only indirectly inferred by overlaying the methylation frequency of two cytosines measured independently. Furthermore, hemi-methylated CpGs (hemiCpGs) have not been previously analyzed in WGBS studies. We recently developed in silico strand annealing (iSA), a bioinformatics method applicable to WGBS data, to resolve the methylation status of CpG dyads into unmethylated, hemi-methylated, and methylated. HemiCpGs account for 4-20% of the DNA methylome in different cell types, and some can be inherited across cell divisions, suggesting a role as a stable epigenetic mark. Therefore, it is important to resolve hemiCpGs from fully methylated CpGs in WGBS studies. This protocol describes step-by-step commands to accomplish this task, including dividing alignments by strand, pairing alignments between strands, and extracting single-fragment methylation calls. The versatility of iSA enables its application downstream of other WGBS-related methods such as nasBS-seq (nascent DNA bisulfite sequencing), ChIP-BS-seq (ChIP followed by bisulfite sequencing), TAB-seq, oxBS-seq, and fCAB-seq. iSA is also tunable for analyzing the methylation status of cytosines in any sequence context. We exemplify this flexibility by uncovering the single-fragment non-CpG methylome. This protocol provides enough details for users with little experience in bioinformatic analysis and takes 2-7 h.

Genome-Wide Mapping of Protein-DNA Interactions on Nascent Chromatin.

Chromatin immunoprecipitation (ChIP) is the most widely used method to analyze protein-DNA interactions in vivo. Coupled with next generation sequencing, ChIP-seq experiments map protein-DNA interactions in a genome-wide fashion. Here we describe a novel method called nasChIP-seq for mapping genome-wide occupancy of posttranslationally modified histones or transcription factors on newly replicated DNA.

Nascent DNA methylome mapping reveals inheritance of hemimethylation at CTCF/cohesin sites.

The faithful inheritance of the epigenome is critical for cells to maintain gene expression programs and cellular identity across cell divisions. We mapped strand-specific DNA methylation after replication forks and show maintenance of the vast majority of the DNA methylome within 20 minutes of replication and inheritance of some hemimethylated CpG dinucleotides (hemiCpGs). Mapping the nascent DNA methylome targeted by each of the three DNA methyltransferases (DNMTs) reveals interactions between DNMTs and substrate daughter cytosines en route to maintenance methylation or hemimethylation. Finally, we show the inheritance of hemiCpGs at short regions flanking CCCTC-binding factor (CTCF)/cohesin binding sites in pluripotent cells. Elimination of hemimethylation causes reduced frequency of chromatin interactions emanating from these sites, suggesting a role for hemimethylation as a stable epigenetic mark regulating CTCF-mediated chromatin interactions.