Linus T.-Y. Tsai

Charting a dynamic DNA methylation landscape of the human genome

DNA methylation is a defining feature of mammalian cellular identity and is essential for normal development. Most cell types, except germ cells and pre-implantation embryos, display relatively stable DNA methylation patterns, with 70–80% of all CpGs being methylated. Despite recent advances, we still have a limited understanding of when, where and how many CpGs participate in genomic regulation. Here we report the in-depth analysis of 42 whole-genome bisulphite sequencing 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, most of which are distal to transcription start sites. These dynamic CpGs co-localize with gene regulatory elements, particularly enhancers and transcription-factor-binding sites, which allow identification of key lineage-specific regulators. In addition, differentially methylated regions (DMRs) often contain single nucleotide polymorphisms associated with cell-type-related diseases as determined by genome-wide association studies. The results also highlight the general inefficiency of whole-genome bisulphite sequencing, as 70–80% of the sequencing reads across these data sets provided little or no relevant information about CpG methylation. To demonstrate further 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 guide new, more effective reduced representation approaches to capture the most informative fraction of CpGs, as well as further pinpoint putative regulatory elements.

Dopamine modulates acute responses to cocaine, nicotine and ethanol in Drosophila

BACKGROUND Drugs of abuse have a common property in mammals, which is their ability to facilitate the release of the neurotransmitter and neuromodulator dopamine in specific brain regions involved in reward and motivation. This increase in synaptic dopamine levels is believed to act as a positive reinforcer and to mediate some of the acute responses to drugs. The mechanisms by which dopamine regulates acute drug responses and addiction remain unknown. RESULTS We present evidence that dopamine plays a role in the responses of Drosophila to cocaine, nicotine or ethanol. We used a startle-induced negative geotaxis assay and a locomotor tracking system to measure the effect of psychostimulants on fly behavior. Using these assays, we show that acute responses to cocaine and nicotine are blunted by pharmacologically induced reductions in dopamine levels. Cocaine and nicotine showed a high degree of synergy in their effects, which is consistent with an action through convergent pathways. In addition, we found that dopamine is involved in the acute locomotor-activating effect, but not the sedating effect, of ethanol. CONCLUSIONS We show that in Drosophila, as in mammals, dopaminergic pathways play a role in modulating specific behavioral responses to cocaine, nicotine or ethanol. We therefore suggest that Drosophila can be used as a genetically tractable model system in which to study the mechanisms underlying behavioral responses to multiple drugs of abuse.

moody encodes two GPCRs that regulate cocaine behaviors and blood-brain barrier permeability in Drosophila

We identified moody in a genetic screen for Drosophila mutants with altered cocaine sensitivity. Hypomorphic mutations in moody cause an increased sensitivity to cocaine and nicotine exposure. In contrast, sensitivity to the acute intoxicating effects of ethanol is reduced. The moody locus encodes two novel GPCRs, Moody-alpha and Moody-beta. While identical in their membrane-spanning domains, the two Moody proteins differ in their long carboxy-terminal domains, which are generated by use of alternative reading frames. Both Moody forms are required for normal cocaine sensitivity, suggesting that they carry out distinct but complementary functions. Moody-alpha and Moody-beta are coexpressed in surface glia that surround the nervous system, where they are actively required to maintain the integrity of the blood-brain barrier in the adult fly. We propose that a Moody-mediated signaling pathway functions in glia to regulate nervous system insulation and drug-related behaviors.

MUTATIONS AFFECTING THE PATTERN OF THE PNS IN DROSOPHILA REVEAL NOVEL ASPECTS OF NEURONAL DEVELOPMENT

Through a systematic genetic screen, we have identified 55 mutations that affect the development of the PNS of Drosophila embryos. These mutations specify 13 novel and 5 previously characterized genes and define new phenotypes for 2 other known genes. Five classes of mutant phenotypes were identified in the screen: gain of neurons, loss of neurons, abnormal position of chordotonal neurons, aberrant neuronal trajectories, and abnormal morphology of neurons. Phenotypic analyses of mutations identified in this study revealed three novel aspects of PNS development. First, we have identified a novel gene that may be required to define glial versus neuronal cell identity. Second, our data indicate that neuronal migration plays an important role in pattern formation in the embryonic PNS. Third, we have identified mutations that cause a lack of sensory organs, but unlike mutations in proneural genes, do not affect the formation of sensory organ precursors. These genes may be required for key aspects of neuronal differentiation. Our studies suggest that approximately 70 essential genes are required for proper PNS development in Drosophila embryos.

Distinct Behavioral Responses to Ethanol Are Regulated by Alternate RhoGAP18B Isoforms

In most organisms, low ethanol doses induce increased activity, while high doses are sedating. To investigate the underlying mechanisms, we isolated Drosophila mutants with altered ethanol responsiveness. Mutations in white rabbit (whir), disrupting RhoGAP18B, are strongly resistant to the sedating effects of ethanol. This resistance can be suppressed by reducing the levels of Rho1 or Rac, implicating these GTPases in the behavioral response to ethanol. Indeed, expression of constitutively active forms of Rho1 or Rac1 in adult flies results in ethanol resistance similar to that observed in whir mutants. The whir locus produces several transcripts, RA-RD, which are predicted to encode three distinct RhoGAPs that share only the GAP domain. The RC transcript mediates the sedating effects of ethanol, while the RA transcript regulates its stimulant effects. Thus, distinct RhoGAPs, encoded by the same gene, regulate different manifestations of acute ethanol intoxication.

A molecular census of arcuate hypothalamus and median eminence cell types

The hypothalamic arcuate–median eminence complex (Arc-ME) controls energy balance, fertility and growth through molecularly distinct cell types, many of which remain unknown. To catalog cell types in an unbiased way, we profiled gene expression in 20,921 individual cells in and around the adult mouse Arc-ME using Drop-seq. We identify 50 transcriptionally distinct Arc-ME cell populations, including a rare tanycyte population at the Arc-ME diffusion barrier, a new leptin-sensing neuron population, multiple agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) subtypes, and an orexigenic somatostatin neuron population. We extended Drop-seq to detect dynamic expression changes across relevant physiological perturbations, revealing cell type–specific responses to energy status, including distinct responses in AgRP and POMC neuron subtypes. Finally, integrating our data with human genome-wide association study data implicates two previously unknown neuron populations in the genetic control of obesity. This resource will accelerate biological discovery by providing insights into molecular and cell type diversity from which function can be inferred.

Novel Mechanism of Positive versus Negative Regulation by Thyroid Hormone Receptor β1 (TRβ1) Identified by Genome-wide Profiling of Binding Sites in Mouse Liver

Background: Examining the TRβ1 cistrome in mouse liver is critical to understanding thyroid hormone signaling. Results: Novel mechanisms of positive versus negative regulation by biotinylated TRβ1 were identified. Conclusion: TRβ1 regulates transcription by changes in relative binding and use of preferred binding motifs. Significance: This study demonstrates that a mechanism other than differential co-regulator recruitment is involved in transcriptional regulation by TRβ1 Triiodothyronine (T3) regulates key metabolic processes in the liver through the thyroid hormone receptor, TRβ1. However, the number of known target genes directly regulated by TRβ1 is limited, and the mechanisms by which positive and especially negative transcriptional regulation occur are not well understood. To characterize the TRβ1 cistrome in vivo, we expressed a biotinylated TRβ1 in hypo- and hyperthyroid mouse livers, used ChIP-seq to identify genomic TRβ1 targets, and correlated these data with gene expression changes. As with other nuclear receptors, the majority of TRβ1 binding sites were not in proximal promoters but in the gene body of known genes. Remarkably, T3 can dictate changes in TRβ1 binding, with strong correlation to T3-induced gene expression changes, suggesting that differential TRβ1 binding regulates transcriptional outcome. Additionally, DR-4 and DR-0 motifs were significantly enriched at binding sites where T3 induced an increase or decrease in TRβ1 binding, respectively, leading to either positive or negative regulation by T3. Taken together, the results of this study provide new insights into the mechanisms of transcriptional regulation by TRβ1 in vivo.

Drosophila tao Controls Mushroom Body Development and Ethanol-Stimulated Behavior through par-1

In both mammalian and insect models of ethanol-induced behavior, low doses of ethanol stimulate locomotion. However, the mechanisms of the stimulant effects of ethanol on the CNS are mostly unknown. We have identified tao, encoding a serine-threonine kinase of the Ste20 family, as a gene necessary for ethanol-induced locomotor hyperactivity in Drosophila. Mutations in tao also affect behavioral responses to cocaine and nicotine, making flies resistant to the effects of both drugs. We show that tao function is required during the development of the adult nervous system and that tao mutations cause defects in the development of central brain structures, including the mushroom body. Silencing of a subset of mushroom body neurons is sufficient to reduce ethanol-induced hyperactivity, revealing the mushroom body as an important locus mediating the stimulant effects of ethanol. We also show that mutations in par-1 suppress both the mushroom body morphology and behavioral phenotypes of tao mutations and that the phosphorylation state of the microtubule-binding protein Tau can be altered by RNA interference knockdown of tao, suggesting that tao and par-1 act in a pathway to control microtubule dynamics during neural development.

Drosophila, a genetic model system to study cocaine-related behaviors: a review with focus on LIM-only proteins.

In the last decade, the fruit fly Drosophila melanogaster, highly accessible to genetic, behavioral and molecular analyses, has been introduced as a novel model organism to help decipher the complex genetic, neurochemical, and neuroanatomical underpinnings of behaviors induced by drugs of abuse. Here we review these data, focusing specifically on cocaine-related behaviors. Several of cocaines most characteristic properties have been recapitulated in Drosophila. First, cocaine induces motor behaviors in flies that are remarkably similar to those observed in mammals. Second, repeated cocaine administration induces behavioral sensitization a form of behavioral plasticity believed to underlie certain aspects of addiction. Third, a key role for dopaminergic systems in mediating cocaines effects has been demonstrated through both pharmacological and genetic methods. Finally, and most importantly, unbiased genetic screens, feasible because of the simplicity and scale with which flies can be manipulated in the laboratory, have identified several novel genes and pathways whose role in cocaine behaviors had not been anticipated. Many of these genes and pathways have been validated in mammalian models of drug addiction. We focus in this review on the role of LIM-only proteins in cocaine-induced behaviors.

Nuclear Mechanisms of Insulin Resistance.

Insulin resistance is a sine qua non of type 2 diabetes and is associated with many other clinical conditions. Decades of research into mechanisms underlying insulin resistance have mostly focused on problems in insulin signal transduction and other mitochondrial and cytosolic pathways. By contrast, relatively little attention has been focused on transcriptional and epigenetic contributors to insulin resistance, despite strong evidence that such nuclear mechanisms play a major role in the etiopathogenesis of this condition. In this review, we summarize the evidence for nuclear mechanisms of insulin resistance, focusing on three transcription factors with a major impact on insulin action in liver, muscle, and fat.