Charting a dynamic DNA methylation landscape of the human genome

DNA methylation is a defining feature of mammalian cellular identity and essential for normal development1,2. Most cell types, except germ cells and pre-implantation embryos3–5, display relatively stable DNA methylation patterns with 70–80% of all CpGs being methylated6. Despite recent advances we still have a too limited understanding of when, where and how many CpGs participate in genomic regulation. Here we report the in depth analysis of 42 whole genome bisulfite sequencing (WGBS) data sets across 30 diverse human cell and tissue types. We observe dynamic regulation for only 21.8% of autosomal CpGs within a normal developmental context, a majority of which are distal to transcription start sites. These dynamic CpGs co-localize with gene regulatory elements, particularly enhancers and transcription factor binding sites (TFBS), which allow identification of key lineage specific regulators. In addition, differentially methylated regions (DMRs) often harbor SNPs associated with cell type related diseases as determined by GWAS. The results also highlight the general inefficiency of WGBS as 70–80% of the sequencing reads across these data sets provided little or no relevant information regarding CpG methylation. To further demonstrate the utility of our DMR set, we use it to classify unknown samples and identify representative signature regions that recapitulate major DNA methylation dynamics. In summary, although in theory every CpG can change its methylation state, our results suggest that only a fraction does so as part of coordinated regulatory programs. Therefore our selected DMRs can serve as a starting point to help guide novel, more effective reduced representation approaches to capture the most informative fraction of CpGs as well as further pinpoint putative regulatory elements

Lmo mutants reveal a novel role for circadian pacemaker neurons in cocaine-induced behaviors.

Drosophila has been developed recently as a model system to investigate the molecular and neural mechanisms underlying responses to drugs of abuse. Genetic screens for mutants with altered drug-induced behaviors thus provide an unbiased approach to define novel molecules involved in the process. We identified mutations in the Drosophila LIM-only (LMO) gene, encoding a regulator of LIM-homeodomain proteins, in a genetic screen for mutants with altered cocaine sensitivity. Reduced Lmo function increases behavioral responses to cocaine, while Lmo overexpression causes the opposite effect, reduced cocaine responsiveness. Expression of Lmo in the principal Drosophila circadian pacemaker cells, the PDF-expressing ventral lateral neurons (LN(v)s), is sufficient to confer normal cocaine sensitivity. Consistent with a role for Lmo in LN(v)function,Lmomutants also show defects in circadian rhythms of behavior. However, the role for LN(v)s in modulating cocaine responses is separable from their role as pacemaker neurons: ablation or functional silencing of the LN(v)s reduces cocaine sensitivity, while loss of the principal circadian neurotransmitter PDF has no effect. Together, these results reveal a novel role for Lmo in modulating acute cocaine sensitivity and circadian locomotor rhythmicity, and add to growing evidence that these behaviors are regulated by shared molecular mechanisms. The finding that the degree of cocaine responsiveness is controlled by the Drosophila pacemaker neurons provides a neuroanatomical basis for this overlap. We propose that Lmo controls the responsiveness of LN(v)s to cocaine, which in turn regulate the flies' behavioral sensitivity to the drug

Simultaneous Transcriptional and Epigenomic Profiling from Specific Cell Types within Heterogeneous Tissues In Vivo

Epigenomic mechanisms direct distinct gene expression programs for different cell types. Various in vivo tissues have been subjected to epigenomic analysis; however, these studies have been limited by cellular heterogeneity, resulting in composite gene expression and epigenomic profiles. Here, we introduce “NuTRAP,” a transgenic mouse that allows simultaneous isolation of cell-type-specific translating mRNA and chromatin from complex tissues. Using NuTRAP, we successfully characterize gene expression and epigenomic states of various adipocyte populations in vivo, revealing significant differences compared to either whole adipose tissue or in vitro adipocyte cell lines. We find that chromatin immunoprecipitation sequencing (ChIP-seq) using NuTRAP is highly efficient, scalable, and robust with even limited cell input. We further demonstrate the general utility of NuTRAP by analyzing hepatocyte-specific epigenomic states. The NuTRAP mouse is a resource that provides a powerful system for cell-type-specific gene expression and epigenomic profiling

Cocaine Responses Are Not a Circadian Output and <i>pdf</i> Mutants Show Wild-Type Cocaine Sensitivity

<div><p>(A) Cocaine responses do not vary with the circadian clock. Control <i>(EP1631)</i> flies were raised under LD conditions and assayed for cocaine phenotypes in the crackometer at the indicated Zeitgeber (ZT) times. One-way ANOVA revealed no significant effect of time of day (<i>F</i> = 0.53, <i>p</i> = 0.82, <i>n</i> = 32).</p> <p>(B) Flies lacking the neuropeptide PDF <i>(pdf⁰¹)</i> display normal cocaine sensitivity. <i>pdf⁰¹</i> homozygotes <i>(pdf⁰¹/pdf⁰¹)</i> and <i>pdf⁰¹</i> hemizygotes <i>(pdf⁰¹/Df)</i> showed wild-type responses to cocaine in the crackometer. Individual pairwise comparisons using Student's <i>t</i>-tests revealed no significant differences between control (+/+ and +/<i>Df</i>) and <i>pdf</i> mutant genotypes (<i>p</i> = 0.69, <i>p</i> = 0.97, <i>n</i> = 6–8 experiments)</p></div

Molecular Structure of the <i>Lmo</i> Locus

<div><p>(A) A genomic map of the <i>Lmo</i> locus. Three different first exons can be utilized, forming the basis for three alternative transcripts. Exon RA-1 is separated from the alternative start sites RB-1 and RC-1 by a large (∼30 kb) intron. <i>EP1306, EP1383,</i> and <i>pdrm</i> carry insertions 25, 73, and 91 bp, respectively, upstream of the exon RA-1 transcriptional start site. Arrows within the EP elements refer to the orientation of the insertion and the expected direction of inducible expression via UAS sites contained within the EP element. <i>Bx</i> alleles are insertions of natural transposons into the 3′ UTR of the <i>Lmo</i> gene that have been shown to stabilize <i>Lmo</i> transcript (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020408#pbio-0020408-Shoresh1" target="_blank">Shoresh et al. 1998</a>). Protein-coding exons are shaded.</p> <p>(B) Expression of the <i>Lmo</i> RA transcript is enriched in <i>Drosophila</i> heads, and is reduced in the <i>EP1306</i> mutant. RNA was isolated from heads and bodies; after cDNA synthesis, quantitative RT-PCR was performed using primers specific to the RA transcript of <i>Lmo</i> in addition to primers to a reference transcript, the ribosomal protein <i>rp49</i>. Relative abundance is expressed as fold increase over control <i>(EP1631)</i> body mRNA. No detectable amplification was seen in RNase-treated controls (data not shown). Error bars represent standard error of the mean. Asterisk denotes significant difference from control (Student's paired <i>t</i>-test assuming equal variance; <i>p <</i> 0.001, <i>n</i> = 3).</p></div

<i>Lmo</i> Loss-of-Function Mutants Show Increased Sensitivity to Cocaine

<div><p>(A) Cocaine phenotypes of various <i>Lmo</i> mutants. Male flies hemizygous for the indicated <i>Lmo</i> alleles (and their appropriate genetic controls) were exposed to 150 μg of cocaine and tested in the crackometer as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020408#s4" target="_blank">Materials and Methods</a>. Compared to their control (Ctl-1), <i>EP1383</i> (<i>p <</i> 0.02) and <i>EP1306</i> (<i>p <</i> 0.001) flies show significantly increased sensitivity to cocaine. Similarly, compared to their respective controls, <i>pdrm</i> and <i>hdp</i> flies are significantly more sensitive to cocaine (<i>p <</i> 0.001). Asterisks denote significant differences from controls (Student's paired <i>t</i>-test assuming equal variance); <i>n</i> = 20 experiments.</p> <p>(B) Cocaine dose–response. <i>EP1306</i> flies (filled squares) and <i>pdrm</i> flies (filled circles) and their respective controls were exposed to the indicated doses of cocaine. At each dose, the responses of <i>EP1306</i> and <i>pdrm</i> flies are significantly higher than their controls (<i>p <</i> 0.001, <i>n</i> = 16–20 experiments).</p> <p>(C) <i>EP1306</i> flies show alterations in cocaine-induced locomotor patterns of activity. Flies were exposed to 0, 75, or 100 μg of cocaine, as indicated, for 1 min. Representative traces shown correspond to 30 s of recorded activity of about ten flies starting 1 min after the end of cocaine exposure (<i>n</i> ≥ 4). Top panels show response of control flies to indicated amounts of cocaine; bottom panels show activity of <i>EP1306</i> flies after cocaine administration. Ctl-1 is <i>EP1631,</i> Ctl-2 is P[GAL4] line <i>8.142,</i> and Ctl-3 is <i>w¹¹¹⁸</i>.</p></div

A Model for LN_v and LMO Regulation of Cocaine Sensitivity

<div><p>(A) In wild type, LN_vs modulate locomotor responses via electrical activity and synaptic transmission. We propose a model in which cocaine acts to directly increase LN_v activity. Upon cocaine administration, synaptic DA concentrations are increased (via cocaine's inhibition of the plasma membrane DA transporter). Activation of presumed DA receptors on the LN_v (dark arrowheads) stimulates electrical activity and subsequent synaptic output. This activity contributes to the behavioral response of the fly to cocaine.</p> <p>(B) LN_v ablations eliminate LN_v contribution to the cocaine response, reducing cocaine sensitivity.</p> <p>(C) In our model, <i>Lmo</i> loss-of-function mutants <i>(Lmo^LOF),</i> which have increased cocaine sensitivity, have increased activity/output during the cocaine response. This increased activity may be mediated by increases in receptor content on the LN_v or by recruitment of other LN_vs that normally do not participate in the cocaine response.</p> <p>(D) <i>Lmo</i> gain-of-function mutants <i>(Lmo^GOF)</i> mutants have reduced LN_v output and reduced cocaine sensitivity. This could also result from a reduction in receptor density.</p></div

<i>Lmo</i> Gain-of-Function <i>Bx</i> Alleles Show Reduced Sensitivity to Cocaine

<div><p>(A) Cocaine phenotypes of <i>Bx</i> mutants. Male flies hemizygous for <i>Bx</i> alleles <i>Bx¹</i> or <i>Bx^J</i> show significant reductions in sensitivity to cocaine compared to control (Ctl) flies (<i>p <</i> 0.001, <i>n</i> = 12 experiments). Asterisks denote significant differences from control (Student's paired <i>t</i>-test assuming equal variance).</p> <p>(B) Dose–response. <i>Bx^J</i> flies (filled circles) show reduced sensitivity compared to Ctl flies (open circles) at all doses tested (<i>p <</i> 0.001, <i>n</i> = 16–20 for all doses except for 250 μg, where <i>p</i> = 0.0015, <i>n</i> = 8). Two additional <i>Bx</i> alleles (<i>Bx² and Bx³</i>) had similar phenotypes to <i>Bx¹</i> (not shown).</p> <p>(C) <i>Bx^J</i> flies show alterations in cocaine-induced locomotor patterns of activity. Flies were exposed to 0, 100, or 125 μg of cocaine, as indicated, for 1 min. Representative traces shown are 30 s of recorded activity of about ten flies starting 30 or 60 s after the end of cocaine exposure (<i>n</i> ≥ 3). Top panels show response of control flies to indicated amounts of cocaine; bottom panels show activity of <i>Bx^J</i> flies after cocaine administration. Ctl flies are <i>w¹¹¹⁸</i>.</p></div