Complete Genome Phasing of Family Quartet by Combination of Genetic, Physical and Population-Based Phasing Analysis

<div><p>Phased genome maps are important to understand genetic and epigenetic regulation and disease mechanisms, particularly parental imprinting defects. Phasing is also critical to assess the functional consequences of genetic variants, and to allow precise definition of haplotype blocks which is useful to understand gene-flow and genotype-phenotype association at the population level. Transmission phasing by analysis of a family quartet allows the phasing of 95% of all variants as the uniformly heterozygous positions cannot be phased. Here, we report a phasing method based on a combination of transmission analysis, physical phasing by pair-end sequencing of libraries of staggered sizes and population-based analysis. Sequencing of a healthy Caucasians quartet at 120x coverage and combination of physical and transmission phasing yielded the phased genotypes of about 99.8% of the SNPs, indels and structural variants present in the quartet, a phasing rate significantly higher than what can be achieved using any single phasing method. A false positive SNP error rate below 10*E-7 per genome and per base was obtained using a combination of filters. We provide a complete list of SNPs, indels and structural variants, an analysis of haplotype block sizes, and an analysis of the false positive and negative variant calling error rates. Improved genome phasing and family sequencing will increase the power of genome-wide sequencing as a clinical diagnosis tool and has myriad basic science applications.</p></div

DNMT3C is a putative DNA methyltransferase with similarity to DNMT3B.

<p><b>(A)</b> Schematics of DNMT3C and DNMT3B showing the location of conserved domains and the <i>rahu</i> mutation (asterisk). <b>(B)</b> Cladogram of Clustal Omega aligned human and mouse DNMT3 family sequences rooted with HhaI. <b>(C)</b> Motif IX in Clustal Omega aligned sequences showing the location of the <i>rahu</i> mutation (asterisk). DNMT3L proteins do not contain Motif IX. Amino acids identical to those in DNMT3C are shaded gray. Amino acid positions refer to DNMT3C. <b>(D)</b> Homology-based model of DNMT3C carboxy-terminal domain (cytosine methyltransferase domain and the preceding four amino acid residues) with E692 depicted in red. <b>(E)</b> Crystal structure of the DNMT3A carboxy-terminal domain dimer (PDB ID:2QRV), with monomers depicted in two shades of blue. The DNMT3A amino acid equivalent to the glutamic acid that is mutated in <i>Dnmt3c</i>^<i>rahu</i> mutants (DNMT3A E861) is shown in red.</p

Retrotransposons belonging to the L1, ERVK, and ERV1 families are hypomethylated in <i>Dnmt3c</i>^<i>rahu</i> mutants.

<p><b>(A)</b> Proportions of differentially methylated CpGs within genomic elements, as determined by WGBS. WGBS was performed on whole-testis DNA samples from six 12-<i>dpp</i> animals (two <i>Dnmt3c</i>^<i>rahu</i> mutant and two wild-type mice from one litter, and one <i>Dnmt3c</i>^<i>rahu</i> mutant and one wild-type mice from a second litter). CpGs with >25% differential methylation (up or down) and with methylKit Sliding Linear Model-adjusted p-value < 0.01 were considered differentially methylated. “Remaining intergenic” refers to genomic regions that do not overlap with LINE, LTR, SINE, genic, or CpG island annotations. <b>(B)</b> Meta-element plot showing the average difference in methylation levels between individual <i>Dnmt3c</i>^<i>rahu</i> mutants and their wild-type littermates (mice u, v, u′, v′ from one litter; w, w′ from a second litter), across LINE, LTR, and SINE retrotransposons (including 5,000 bp of flanking sequence on each side) with minimum 95% coverage of the consensus sequence. <b>(C)</b> Heatmap showing the mean methylation levels of significantly changed retrotransposon families in individual <i>Dnmt3c</i>^<i>rahu</i> mutants and their wild-type littermates (p < 0.01, two-sided Student’s t-test). Labels on rows indicate the retrotransposon family and differentially expressed retrotransposon families (families with greater than two-fold increase in median expression in <i>Dnmt3c</i>^<i>rahu</i> mutants; rows in bold in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006964#pgen.1006964.g006" target="_blank">Fig 6B</a></b>) are in bold. Three differentially expressed retrotransposon families with changed methylation levels using a less stringent cutoff (p < 0.05, two-sided Student’s t-test) are included and are indicated with an asterisk. The median methylation difference is provided in the bar graph at left.</p

Profiling Genome-Wide Chromatin Methylation with Engineered Posttranslation Apparatus within Living Cells

Protein methyltransferases (PMTs) have emerged as important epigenetic regulators in myriad biological processes in both normal physiology and disease conditions. However, elucidating PMT-regulated epigenetic processes has been hampered by ambiguous knowledge about <i>in vivo</i> activities of individual PMTs particularly because of their overlapping but nonredundant functions. To address limitations of conventional approaches in mapping chromatin modification of specific PMTs, we have engineered the chromatin-modifying apparatus and formulated a novel technology, termed clickable chromatin enrichment with parallel DNA sequencing (CliEn-seq), to probe genome-wide chromatin modification within living cells. The three-step approach of CliEn-seq involves <i>in vivo</i> synthesis of <i>S</i>-adenosyl-l-methionine (SAM) analogues from cell-permeable methionine analogues by engineered SAM synthetase (methionine adenosyltransferase or MAT), <i>in situ</i> chromatin modification by engineered PMTs, subsequent enrichment and sequencing of the uniquely modified chromatins. Given critical roles of the chromatin-modifying enzymes in epigenetics and structural similarity among many PMTs, we envision that the CliEn-seq technology is generally applicable in deciphering chromatin methylation events of individual PMTs in diverse biological settings

<i>Dnmt3c</i>^<i>rahu</i> mutants up-regulate retrotransposons belonging to L1 and ERVK families.

<p><b>(A)</b> Volcano plot of differential RNA-seq values for various classes of retrotransposons. RNA-seq was performed on testis RNA samples from six 14-<i>dpp</i> animals from a single litter: three <i>Dnmt3c</i>^<i>rahu</i> mutants and three heterozygotes (same mice analyzed in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006964#pgen.1006964.g005" target="_blank">Fig 5A</a></b>). Q-value is the Benjamini-Hochberg-adjusted p-value from DESeq2. Retrotransposons with expression fold change of >2 (up or down) and q < 0.01 are depicted as large, colored circles. <b>(B)</b> Heatmap showing the z-score of differentially expressed retrotransposon families (with expression fold change >2 and q < 0.01) in individual <i>Dnmt3c</i>^<i>rahu</i> mutants (x, y, z) and their heterozygous littermates (x′, y′, z′). Labels on rows indicate the retrotransposon family, followed by superfamily, followed by class, and then in parentheses the log₂ fold change of median expression in mutant versus heterozygote. Rows with greater than two-fold change in median expression (up or down) are in bold. The log₂ fold changes are also provided in the bar graph at left (greater than two-fold change shown as black bars). <b>(C)</b> Correlation between differentially expressed retrotransposon families in 14-<i>dpp</i> <i>Dnmt3c</i>^<i>rahu</i> mutants and 20-<i>dpp</i> <i>Dnmt3l</i> mutants. The regression line is shown and <i>r</i> is the Pearson correlation coefficient.</p

Males from the ENU-induced mutant line <i>rahu</i> display meiotic defects.

<p><b>(A)</b> Breeding scheme used to isolate third-generation males with recessive defects in meiosis. Un-filled shapes represent animals that are wild-type for a mutation of interest, half-filled shapes are heterozygous carriers, and filled shapes are homozygotes. <b>(B)</b> Representative images showing the SYCP3 and γH2AX immunofluorescence patterns during meiotic prophase stages in squashed spermatocyte preparations from wild type and <i>rahu</i> mutants. Examples of cells with abnormal staining (nucleus-wide γH2AX along with longer tracks of SYCP3) are also shown. <b>(C)</b> Distribution of meiotic prophase stages in four G3 mutants obtained from the <i>rahu</i> line (a, b, c, d) and their phenotypically wild-type littermates (a′, b′, c′, d′). The number of SYCP3-positive spermatocytes counted from each animal is indicated.</p

<i>rahu</i> and CRISPR/Cas9-targeted frameshift alleles of <i>Gm14490</i> cause meiotic arrest and fail to complement each other.

<p><b>(A)</b> The ratios of testes weight to body weight for 5- to 9-week-old mice carrying the <i>rahu</i> and CRISPR/Cas9-targeted alleles (<i>em</i>). Half-filled and fully filled circles represent heterozygous and homozygous genotypes, respectively. <b>(B)</b> Representative PAS-stained testis sections from adult mice of the indicated genotypes. Arrows indicate post-meiotic germ cells (spermatids) and arrowheads point to spermatocytes with an apoptotic morphology (condensed and/or fragmented). <b>(C)</b> Representative TUNEL-stained testis sections from adult mice of the indicated genotypes. Arrowheads point to TUNEL-positive cells (stained dark brown). <b>(D and E)</b> Representative PAS-stained testis sections from adult mice of the indicated genotypes. Arrows indicate post-meiotic germ cells.</p

<i>Dnmt3c</i>^<i>rahu</i> mutants exhibit phenotypes consistent with a role in DNA methylation and transposon repression in the male germline.

<p><b>(A)</b> Quantitative RT-PCR analysis of whole-testis RNA samples from six littermates (represented as individual data points) aged 14 <i>dpp</i>. Asterisk represents p<0.05 and double-asterisk represents p<0.01 in one-sided Student’s t-test. <b>(B)</b> Immunofluorescence of retrotransposon-encoded proteins L1 ORF1p and IAP Gag in testis sections from adults and from juveniles at 14 <i>dpp</i>. Matched exposures are shown comparing heterozygotes with <i>Dnmt3c</i>^<i>rahu</i> mutants. Arrows indicate spermatogonia and arrowheads point to spermatocytes. <b>(C)</b> Southern blot analysis of DNA extracted from either the testes or the tails of three <i>Dnmt3c</i>^<i>rahu</i> mutants or a wild-type littermate at 15 <i>dpp</i>. DNA was digested with either the methylation-sensitive restriction enzyme HpaII, or its methylation-insensitive isoschizomer MspI. Arrowheads mark the positions expected for fully digested bands.</p

High-Resolution Mapping of H1 Linker Histone Variants in Embryonic Stem Cells

<div><p>H1 linker histones facilitate higher-order chromatin folding and are essential for mammalian development. To achieve high-resolution mapping of H1 variants H1d and H1c in embryonic stem cells (ESCs), we have established a knock-in system and shown that the N-terminally tagged H1 proteins are functionally interchangeable to their endogenous counterparts <i>in vivo</i>. H1d and H1c are depleted from GC- and gene-rich regions and active promoters, inversely correlated with H3K4me3, but positively correlated with H3K9me3 and associated with characteristic sequence features. Surprisingly, both H1d and H1c are significantly enriched at major satellites, which display increased nucleosome spacing compared with bulk chromatin. While also depleted at active promoters and enriched at major satellites, overexpressed H1⁰ displays differential binding patterns in specific repetitive sequences compared with H1d and H1c. Depletion of H1c, H1d, and H1e causes pericentric chromocenter clustering and de-repression of major satellites. These results integrate the localization of an understudied type of chromatin proteins, namely the H1 variants, into the epigenome map of mouse ESCs, and we identify significant changes at pericentric heterochromatin upon depletion of this epigenetic mark.</p> </div

Increased nucleosome repeat length at major satellite repeats in ESCs.

<p>(A) Nucleosome repeat length analyses of bulk chromatin (left), major satellite sequences (middle) and minor satellite sequences (right) in WT ESCs. DNA isolated from ESC nuclei digested with MNase at different time points were analyzed by ethidium bromide (EB) –stained gel (left), transferred to membrane which was sequentially probed with major satellites (middle) and minor satellites (right) using Southern blotting. The positions of di-nucleosomes with 10-minute MNase digestion are marked by *. The dashed line indicates di-nucleosome position of major satellites, which is higher than that of bulk chromatin and minor satellites. (B) The NRLs were calculated from the images presented in (A) by extrapolating the corresponding curves to time “0” as described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003417#pgen.1003417-Gilbert2" target="_blank">[72]</a>.</p