Q-RRBS: a quantitative reduced representation bisulfite sequencing method for single-cell methylome analyses

Reduced representation bisulfite sequencing (RRBS) is a powerful method of DNA methylome profiling that can be applied to single cells. However, no previous report has described how PCR-based duplication-induced artifacts affect the accuracy of this method when measuring DNA methylation levels. For quantifying the effects of duplication-induced artifacts on methylome profiling when using ultra-trace amounts of starting material, we developed a novel method, namely quantitative RRBS (Q-RRBS), in which PCR-induced duplication is excluded through the use of unique molecular identifiers (UMIs). By performing Q-RRBS on varying amounts of starting material, we determined that duplication-induced artifacts were more severe when small quantities of the starting material were used. However, through using the UMIs, we successfully eliminated these artifacts. In addition, Q-RRBS could accurately detect allele-specific methylation in absence of allele-specific genetic variants. Our results demonstrate that Q-RRBS is an optimal strategy for DNA methylation profiling of single cells or samples containing ultra-trace amounts of cells.     

De novo quantitative bisulfite sequencing using the pyrosequencing technology

Current protocols for DNA methylation analysis are either labor intensive or limited to the measurement of only one or two CpG positions. Pyrosequencing is a real-time sequencing technology that can overcome these limitations and be used as an epigenotype-mapping tool. Initial experiments demonstrated reliable quantification of the degree of DNA methylation when 2-6 CpGs were analyzed. We sought to improve the sequencing protocol so as to analyze as many CpGs as possible in a single sequencing run. By using an improved enzyme mix and adding single-stranded DNA-binding protein to the reaction, we obtained reproducible results for as many as 10 successive CpGs in a single sequencing reaction spanning up to 75 nucleotides. A minimum amount of 10 ng of bisulfite-treated DNA is necessary to obtain good reproducibility and avoid preferential amplification. We applied the assay to the analysis of DNA methylation patterns in four CpG islands in the vicinity of IGF2 and H19 genes. This allowed accurate and quantitative de novo sequencing of the methylation state of each CpG, showing reproducible variations of methylation state in contiguous CpGs, and proved to be a useful adjunct to current technologies.     

Systematic assessment of reduced representation bisulfite sequencing to human blood samples: A promising method for large-sample-scale epigenomic studies

Complementary to the time- and cost-intensive direct bisulfite sequencing, we applied reduced representation bisulfite sequencing (RRBS) to the human peripheral blood mononuclear cells (PBMC) from YH, the Asian individual whose genome and epigenome has been deciphered in the YH project and systematically assessed the genomic coverage, coverage depth and reproducibility of this technology as well as the concordance of DNA methylation levels measured by RRBS and direct bisulfite sequencing for the detected CpG sites. Our result suggests that RRBS can cover more than half of CpG islands and promoter regions with a good coverage depth and the proportion of the CpG sites covered by the biological replicates reaches 80-90%, indicating good reproducibility. Given a smaller data quantity, RRBS enjoys much better coverage depth than direct bisulfite sequencing and the concordance of DNA methylation levels between the two methods is high. It can be concluded that RRBS is a time and cost-effective sequencing method for unbiased DNA methylation profiling of CpG islands and promoter regions in a genome-wide scale and it is the method of choice to assay certain genomic regions for multiple samples in a rapid way.     

Timing and plasticity of shoaling behaviour in the zebrafish, Danio rerio

The zebrafish has become a major model system for biomedical research and is an emerging model for the study of behaviour. While adult zebrafish express a visually mediated shoaling preference, the onset of shoaling behaviour and of this preference is unknown. To assess the onset of these behaviours, we first manipulated the early social environment of larval zebrafish subjects, giving them three model shoaling partners of the same pigment phenotype. We then assayed the subjects' preferences using binary preference tests in which we presented subjects with two shoals, one shoal of fish exhibiting the same pigment pattern phenotype as their models and another shoal with a radically different pigment pattern. To determine whether or not the visually mediated preference could be altered once it was established, we further manipulated the social environment of a number of subjects, rearing them with one model shoal and testing them, then changing their social consorts and retesting them. Our results demonstrate that larval zebrafish shoal early in their development, but do not exhibit a shoaling preference until they are juveniles. Moreover, we find that the shoaling preference is stable, as changing the social environment of fish after they had acquired a preference did not change their preference. These data will facilitate investigations into the mechanisms underlying social behaviour in this vertebrate model system.     

MNNG-induced mutations in the adult gill and hepatopancreas and in embryos of rpsL transgenic zebrafish

To evaluate the feasibility of a mutagenicity assay using adult rpsL transgenic zebrafish, 4- to 8-month-old females were exposed to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (0, 15 or 30 mg/L in a water bath for 2 h). At 2 weeks after exposure, MNNG showed a concentration-dependent significant increase in mutant frequency (MF) of 8 x 10(-5), 18 x 10(-5), and 51 x 10(-5), respectively, in the gill. DNA sequencing revealed that 60-74% of the induced mutations were G:C to A:T transitions, consistent with the known mutagenic effects of MNNG. A marginal but significant increase in MF was observed in the hepatopancreas only in the group exposed to 30 mg/L, with the induction of some G:C to A:T transitions. A time-course of the appearance of mutations was determined in fish treated with 15 mg/L MNNG. In both, the gill and hepatopancreas, a higher MF was observed at 3 weeks than at 2 weeks, suggesting that an expression time of at least 3 weeks is preferable for the assay. When embryos (29 h post-fertilization) were exposed to MNNG (0, 50, and 150 mg/L) for 1 h, MFs increased significantly with an increase in the concentration of MNNG (5 x 10(-5), 40 x 10(-5), and 144 x 10(-5), respectively) at 3 days after exposure. G:C to A:T transitions were the predominant mutations, and these occurred at the same sites in the rpsL gene as in adult tissues. Thus, MNNG induces typical mutations in the gill and hepatopancreas of adult fish, and in embryos, suggesting that the rpsL zebrafish is a useful tool for monitoring genotoxicity caused by water-borne mutagens.     

Assessing the Impact of Advected African Dust on Air Quality and Health in the Eastern United States

Large quantities of African dust are carried into the southeastern United States each summer with concentrations typically in the range of 10 to 100?µg m^?3. Because approximately one-third to one-half of the dust mass is in the size range under 2.5?µm diameter, the advection of African dust has implications for the EPA's newly implemented standard for PM_2.5 particulate matter and for the assessment of human health effects. It will be difficult to assess the impact of African dust events on air quality because they occur during the summer (maximum in July) when photochemical pollution events are frequent and intense in the eastern United States. Indeed, the presence of dust could affect the evolution of photochemical dust events. In order to assess the role of African dust in air quality in the United States, it will be necessary to develop a set of diagnostic indicators; it appears that dust mineralogy and elemental composition might be useful for this purpose. Various satellite products can be used to characterize the spatial coverage of dust events and, when coupled with meteorological tools, to predict impact regions.     

结合激光雷达分析上海地区一次连续浮尘天气过程

通过分析2010年3月至20日至3月22日上海地区一次典型的连续浮尘天气过程,利用激光雷达数据资料反演得到的气溶胶消光系数图和垂直廓线图,结合地面气象数据和气溶胶观测资料以及卫星遥感资料,初步得出了此次连续浮尘天气形成的原因。外源性输入和垂直风场分布是导致此次浮尘天气发生的重要原因,大气层结变化决定着浮尘天气发生的强度,当存在接地逆温时,浮尘天气维持,而太阳辐射强度变化决定着边界层高度的变化。第一次浮尘过程以大颗粒污染为主,且在消光作用中散射性气溶胶的贡献大于吸收性气溶胶,而第二次回流浮尘过程则PM2.5比重上升,吸收性气溶胶对消光系数的贡献与散射性气溶胶大体相当。     

Nucleosome positioning signals in the DNA sequence of the human and mouse H19 imprinting control regions

We have investigated the sequences of the mouse and human H19 imprinting control regions (ICRs) to see whether they contain nucleosome positioning information pertinent to their function as a methylation-regulated chromatin boundary. Positioning signals were identified by an in vitro approach that employs reconstituted chromatin to comprehensively describe the contribution of the DNA to the most basic, underlying level of chromatin structure. Signals in the DNA sequence of both ICRs directed nucleosomes to flank and encompass the short conserved sequences that constitute the binding sites for the zinc finger protein CTCF, an essential mediator of insulator activity. The repeat structure of the human ICR presented a conserved array of strong positioning signals that would preferentially flank these CTCF binding sites with positioned nucleosomes, a chromatin structure that would tend to maintain their accessibility. Conversely, all four CTCF binding sites in the mouse sequence were located close to the centre of positioning signals that were stronger than those in their flanks; these binding sites might therefore be expected to be more readily incorporated into positioned nucleosomes. We found that CpG methylation did not effect widespread repositioning of nucleosomes on either ICR, indicating that allelic methylation patterns were unlikely to establish allele-specific chromatin structures for H19 by operating directly upon the underlying DNA-histone interactions; instead, epigenetic modulation of ICR chromatin structure is likely to be mediated principally at higher levels of control. DNA methylation did, however, both promote and inhibit nucleosome positioning at several sites in both ICRs and substantially negated one of the strongest nucleosome positioning signals in the human sequence, observations that underline the fact that this epigenetic modification can, nevertheless, directly and decisively modulate core histone-DNA interactions within the nucleosome.     

Wnt1 and wnt10b function redundantly at the zebrafish midbrain-hindbrain boundary

Wnt signals have been shown to be involved in multiple steps of vertebrate neural patterning, yet the relative contributions of individual Wnts to the process of brain regionalization is poorly understood. Wnt1 has been shown in the mouse to be required for the formation of the midbrain and the anterior hindbrain, but this function of wnt1 has not been explored in other model systems. Further, wnt1 is part of a Wnt cluster conserved in all vertebrates comprising wnt1 and wnt10b, yet the function of wnt10b during embryogenesis has not been explored. Here, we report that in zebrafish wnt10b is expressed in a pattern overlapping extensively with that of wnt1. We have generated a deficiency allele for these closely linked loci and performed morpholino antisense oligo knockdown to show that wnt1 and wnt10b provide partially redundant functions in the formation of the midbrain-hindbrain boundary (MHB). When both loci are deleted, the expression of pax2.1, en2, and her5 is lost in the ventral portion of the MHB beginning at the 8-somite stage. However, wnt1 and wnt10b are not required for the maintenance of fgf8, en3, wnt8b, or wnt3a expression. Embryos homozygous for the wnt1-wnt10b deficiency display a mild MHB phenotype, but are sensitized to reductions in either Pax2.1 or Fgf8; that is, in combination with mutant alleles of either of these loci, the morphological MHB is lost. Thus, wnt1 and wnt10b are required to maintain threshold levels of Pax2.1 and Fgf8 at the MHB.     

Novel cis-regulatory function in ICR-mediated imprinted repression of H19

Expression of coregulated imprinted genes, H19 and Igf2, is monoallelic and parent-of-origin-dependent. Like most imprinted genes, H19 and Igf2 are regulated by a differentially methylated imprinting control region (ICR). CTCF binding sites and DNA methylation at the ICR have previously been identified as key cis-acting elements required for proper H19/Igf2 imprinting. Here, we use mouse models to elucidate further the mechanism of ICR-mediated gene regulation. We specifically address the question of whether sequences outside of CTCF sites at the ICR are required for paternal H19 repression. To this end, we generated two types of mutant ICRs in the mouse: (i) deletion of intervening sequence between CTCF sites (H19^ICR?IVS), which changes size and CpG content at the ICR; and (ii) CpG depletion outside of CTCF sites (H19^ICR-8nrCG), which only changes CpG content at the ICR. Individually, both mutant alleles (H19^ICR?IVS and H19^ICR-8nrCG) show loss of imprinted repression of paternal H19. Interestingly, this loss of repression does not coincide with a detectable change in methylation at the H19 ICR or promoter. Thus, neither intact CTCF sites nor hypermethylation at the ICR is sufficient for maintaining the fully repressed state of the paternal H19 allele. Our findings demonstrate, for the first time in vivo, that sequence outside of CTCF sites at the ICR is required in cis for ICR-mediated imprinted repression at the H19/Igf2 locus. In addition, these results strongly implicate a novel role of ICR size and CpG density in paternal H19 repression.     

Elevated Fmr1 mRNA levels and reduced protein expression in a mouse model with an unmethylated Fragile X full mutation

The human FMR1 gene contains a CGG repeat in its 5' untranslated region. The repeat length in the normal population is polymorphic (5-55 CGG repeats). Lengths beyond 200 CGGs (full mutation) result in the absence of the FMR1 gene product, FMRP, through abnormal methylation and gene silencing. This causes Fragile X syndrome, the most common inherited form of mental retardation. Elderly carriers of the premutation, defined as a repeat length between 55 and 200 CGGs, can develop a progressive neurodegenerative syndrome: Fragile X-associated tremor/ataxia syndrome (FXTAS). In FXTAS, FMR1 mRNA levels are elevated and it has been hypothesised that FXTAS is caused by a pathogenic RNA gain-of-function mechanism. We have developed a knock in mouse model carrying an expanded CGG repeat (98 repeats), which shows repeat instability and displays biochemical, phenotypic and neuropathological characteristics of FXTAS. Here, we report further repeat instability, up to 230 CGGs. An expansion bias was observed, with the largest expansion being 43 CGG units and the largest contraction 80 CGG repeats. In humans, this length would be considered a full mutation and would be expected to result in gene silencing. Mice carrying long repeats ( approximately 230 CGGs) display elevated mRNA levels and decreased FMRP levels, but absence of abnormal methylation, suggesting that modelling the Fragile X full mutation in mice requires additional repeats or other genetic manipulation.     

Gangliosides modulate the activity of the plasma membrane Ca(2+)-ATPase from porcine brain synaptosomes

We systematically examined the effects of gangliosides on the plasma membrane Ca(2+)-ATPase (PMCA) from porcine brain synaptosomes. Our results showed that GD1b (two sialic acid residues) stimulated the activity, GM1 (one sialic acid residue) slightly reduced the activity, while asialo-GM1 (no sialic acid residue) markedly inhibited it, suggesting that sialic acid residues of gangliosides are important in the modulation of the PMCA. We also examined the oligosaccharide effects by using GM1, GM2, and GM3 whose only difference was in the length of their oligosaccharide chain. GM1, GM2, and GM3 reduced the enzyme activities, whereas GM2 and GM3 were potent inhibitors. Gangliosides affect both affinity for Ca(2+) and the Vmax of enzyme. It was observed that GD1b and GM2 increased the affinity of the enzyme for Ca(2+). GD1b, GM2 affected the Vmax with an increase of GD1b, but decreases of GM2. The study of the affinity for ATP and the Vmax of enzyme in the presence of gangliosides showed that GD1b and GM2 had little effect on the ATP binding to the enzyme, but the Vmax was apparently changed. Moreover, the effects of gangliosides are additive to that of calmodulin, suggesting that the modulation of PMCA by gangliosides should be through a different mechanism. The conformational changes induced by gangliosides were probed by fluorescence quenching. We found that fluorescent quenchers (I(-) and Cs(+)) with opposite charges had different accessibility to the IAEDANS binding to the PMCA in the presence of gangliosides. An apparent red shift (25nm) with increased maximum of fluorescence spectrum was also observed in the presence of GD1b.     

Global DNA methylation in a population with aflatoxin B1 exposure

We previously reported that global DNA hypomethylation, measured as Sat2 methylation in white blood cells (WBC), and aflatoxin B₁ (AFB₁) exposure were associated with increased hepatocellular carcinoma risk. In this study, we assessed the association between AFB₁ exposure and global DNA methylation. We measured LINE-1 and Sat2 methylation in WBC DNA samples from 1140 cancer free participants of the Cancer Screening Program (CSP) cohort. Blood and urine samples were used to determine the level of AFB₁-albumin (AFB₁-Alb) adducts and urinary AFB₁ metabolites. In continuous models, we found reverse associations of urinary AFB₁ with LINE-1 and Sat2 methylation. The odds ratio (OR) per 1 unit decrease were 1.12 (95%CI = 1.03–1.22) for LINE-1 and 1.48 (95%CI = 1.10–2.00) for Sat2 methylation. When compared with subjects in the highest quartile of LINE-1, we found that individuals in the 2nd and 3rd quartiles were less likely to have detectable AFB₁-Alb adducts, with ORs (95%CI) of 0.61 (0.40–0.93), 0.61 (0.40-.94), and 1.09 (0.69–1.72), respectively. The OR for detectable AFB₁-Alb was 1.81 (95%CI = 1.15–2.85) for subjects in the lowest quartile of Sat2 methylation. The OR for detection of urinary AFB₁ for those with LINE-1 methylation in the lowest quartile compared with those in the highest quartile was 1.87 (95%CI = 1.15–3.04). The corresponding OR was 1.75 (95%CI = 1.08–2.82) for subjects in the lowest quartile of Sat2 methylation. The association between AFB₁ exposure and global DNA methylation may have implications for the epigenetic effect of AFB₁ on hepatocellular carcinoma development and also suggests that changes in DNA methylation may represent an epigenetic biomarker of dietary AFB₁ exposure.     

Triple helical structure and stabilization of collagen-like molecules with 4(R)-hydroxyproline in the Xaa position

In this study, we examine the relationships between the structure and stability of five related collagen-like molecules that have hydroxyproline residues occupying positions not observed in vertebrate collagen. Two of the molecules contain valine or threonine and form stable triple helices in water. Three of the molecules contain allo-threonine (an enantiomer of threonine), serine, or alanine, and are not stable. Using molecular dynamics simulation methods, we examine possible explanations for the stability difference, including considering the possibility that differences in solvent shielding of the essential interchain hydrogen bonds may result in differences in stability. By comparing the structures of threonine- and allo-threonine-containing molecules in six polar and nonpolar solvation conditions, we find that solvent shielding is not an adequate explanation for the stability difference. A closer examination of the peptides shows that the structures of the unstable molecules are looser, having weaker intermolecular hydrogen bonds. The weakened hydrogen bonds result from extended Yaa residue Psi-angles that prevent optimal geometry. The Phi-Psi-maps of the relevant residues suggest that each residue's most favorable Psi-angle determines the corresponding collagen-like molecule's stability. Additionally, we propose that these molecules illustrate a more general feature of triple-helical structures: interchain hydrogen bonds are always longer and weaker than ideal, so they are sensitive to relatively small changes in molecular structure. This sensitivity to small changes may explain why large stability differences often result from seemingly small changes in residue sequence.