Functionally conserved enhancers with divergent sequences in distant vertebrates

Conserved transcription factor binding motifs in the five zebrafish/mouse syntenic enhancers. Identical n-mers (n ≥ 7) identified in the zebrafish, mouse, and human sequences of the five syntenic CNS were examined for the presence of transcription factor binding motifs; only motifs with E-value E ≤ 0.1 are shown. (XLSX 15 kb

The rice XA21 ectodomain fused to the Arabidopsis EFR cytoplasmic domain confers resistance to Xanthomonas oryzae pv. oryzae

Rice (Oryza sativa) plants expressing the XA21 cell-surface receptor kinase are resistant to Xanthomonas oryzae pv. oryzae (Xoo) infection. We previously demonstrated that expressing a chimeric protein containing the ELONGATION FACTOR Tu RECEPTOR (EFR) ectodomain and the XA21 endodomain (EFR: XA21) in rice does not confer robust resistance to Xoo. To test if the XA21 ectodomain is required for Xoo resistance, we produced transgenic rice lines expressing a chimeric protein consisting of the XA21 ectodomain and EFR endodomain (XA21:EFR) and inoculated these lines with Xoo. We also tested if the XA21:EFR rice plants respond to a synthetic sulfated 21 amino acid derivative (RaxX21-sY) of the activator of XA21-mediated immunity, RaxX. We found that five independently transformed XA21:EFR rice lines displayed resistance to Xoo as measured by lesion length analysis, and showed that five lines share characteristic markers of the XA21 defense response (generation of reactive oxygen species and defense response gene expression) after treatment with RaxX21-sY. Our results indicate that expression of the XA21:EFR chimeric receptor in rice confers resistance to Xoo. These results suggest that the endodomain of the EFR and XA21 immune receptors are interchangeable and the XA21 ectodomain is the key determinant conferring robust resistance to Xoo.This project was funded through NIH grant GM59962 and the NSF PGRP grant IOS1237975. Benjamin Schwessinger was supported by a Human Frontiers Science Program long-term postdoctoral fellowship (LT000674/2012) and a Discovery Early Career Research Award (DE150101897)

Integrating Diverse Datasets Improves Developmental Enhancer Prediction

Gene-regulatory enhancers have been identified using various approaches, including evolutionary conservation, regulatory protein binding, chromatin modifications, and DNA sequence motifs. To integrate these different approaches, we developed EnhancerFinder, a two-step method for distinguishing developmental enhancers from the genomic background and then predicting their tissue specificity. EnhancerFinder uses a multiple kernel learning approach to integrate DNA sequence motifs, evolutionary patterns, and diverse functional genomics datasets from a variety of cell types. In contrast with prediction approaches that define enhancers based on histone marks or p300 sites from a single cell line, we trained EnhancerFinder on hundreds of experimentally verified human developmental enhancers from the VISTA Enhancer Browser. We comprehensively evaluated EnhancerFinder using cross validation and found that our integrative method improves the identification of enhancers over approaches that consider a single type of data, such as sequence motifs, evolutionary conservation, or the binding of enhancer-associated proteins. We find that VISTA enhancers active in embryonic heart are easier to identify than enhancers active in several other embryonic tissues, likely due to their uniquely high GC content. We applied EnhancerFinder to the entire human genome and predicted 84,301 developmental enhancers and their tissue specificity. These predictions provide specific functional annotations for large amounts of human non-coding DNA, and are significantly enriched near genes with annotated roles in their predicted tissues and lead SNPs from genome-wide association studies. We demonstrate the utility of EnhancerFinder predictions through in vivo validation of novel embryonic gene regulatory enhancers from three developmental transcription factor loci. Our genome-wide developmental enhancer predictions are freely available as a UCSC Genome Browser track, which we hope will enable researchers to further investigate questions in developmental biology. © 2014 Erwin et al

The Function and Regulation of AUTS2

The autism susceptibility candidate 2 (AUTS2) gene is associated with multiple neurological diseases, including autism, and has been implicated as an important gene in human-specific evolution. In this thesis, I begin (chapter 1) with an introduction reviewing the literature regarding AUTS2, including its discovery, expression, association with autism and other neurological and non-neurological traits, implication in human evolution, function, regulation, and genetic pathways. Part of the research included in this introduction was performed by me and co-authors, and described in detail in the following chapters. In chapter 2, I investigate the expression, function, and regulation of auts2. auts2 is expressed primarily in the zebrafish brain. Knockdown of this gene in zebrafish leads to a smaller head size, neuronal reduction and decreased mobility. I identified twenty-three functional zebrafish enhancers, ten of which are active in the brain. Mouse enhancer assays characterized three brain enhancers that overlap an ASD-associated deletion and four enhancers that reside in regions implicated in human evolution, two of which are active in the brain. Combined, I show that AUTS2 is important for neurodevelopment and expose candidate enhancer sequences in which nucleotide variation could lead to neurological disease and human-specific traits.In chapter 3, I investigate the regulatory role and targets of Auts2. Using ChIP-seq and RNA-seq on mouse embryonic day 16.5 forebrains, we I elucidated the gene regulatory networks of Auts2. It was found that the majority of promoters bound by Auts2 belong to genes highly expressed in the developing forebrain, suggesting that Auts2 is involved in transcriptional activation. Auts2 non-promoter bound regions significantly overlap developing brain-associated enhancer marks and are located near genes involved in neurodevelopment. Auts2 marked sequences are enriched for binding site motifs of neurodevelopmental transcription factors, including Pitx3 and TCF3. I characterized ten non-coding Auts2 marked sites near critical ASD-related genes for enhancer activity in zebrafish, four of which showed positive enhancer activity. Additionally, I characterized two of the positive brain enhancers near NRXN1 and ATP2B2 in mice. The results implicate Auts2 as an active regulator of important neurodevelopmental genes and pathways and identify novel genomic regions which could be associated with ASD and other neurodevelopmental diseases. In summary, this thesis investigates the function of AUTS2. I conclude that this gene is critical for the proper development of neurons, and may act as a cofactor to positively regulate genes expressed in the forebrain involved in neurodevelopment

The role of AUTS2 in neurodevelopment and human evolution

The autism susceptibility candidate 2 (AUTS2) gene is associated with multiple neurological diseases, including autism, and has been implicated as an important gene in human-specific evolution. Recent functional analysis of this gene has revealed a potential role in neuronal development. Here, we review the literature regarding AUTS2, including its discovery, expression, association with autism and other neurological and non-neurological traits, implication in human evolution, function, regulation, and genetic pathways. Through progress in clinical genomic analysis, the medical importance of this gene is becoming more apparent, as highlighted in this review, but more work needs to be done to discover the precise function and the genetic pathways associated with AUTS2

Functional characterization of SIM1-associated enhancers.

Haploinsufficiency of the single-minded homology 1 (SIM1) gene in humans and mice leads to severe obesity, suggesting that altered expression of SIM1, by way of regulatory elements such as enhancers, could predispose individuals to obesity. Here, we identified transcriptional enhancers that could regulate SIM1, using comparative genomics coupled with zebrafish and mouse transgenic enhancer assays. Owing to the dual role of Sim1 in hypothalamic development and in adult energy homeostasis, the enhancer activity of these sequences was annotated from embryonic to adult age. Of the seventeen tested sequences, two SIM1 candidate enhancers (SCE2 and SCE8) were found to have brain-enhancer activity in zebrafish. Both SCE2 and SCE8 also exhibited embryonic brain-enhancer expression in mice, and time course analysis of SCE2 activity showed overlapping expression with Sim1 from embryonic to adult age, notably in the hypothalamus in adult mice. Using a deletion series, we identified the critical region in SCE2 that is needed for enhancer activity in the developing brain. Sequencing this region in obese and lean cohorts revealed a higher prevalence of single nucleotide polymorphisms (SNPs) that were unique to obese individuals, with one variant reducing developmental-enhancer activity in zebrafish. In summary, we have characterized two brain enhancers in the SIM1 locus and identified a set of obesity-specific SNPs within one of them, which may predispose individuals to obesity

Function and Regulation of <em>AUTS2</em>, a Gene Implicated in Autism and Human Evolution

<div><p>Nucleotide changes in the <em>AUTS2</em> locus, some of which affect only noncoding regions, are associated with autism and other neurological disorders, including attention deficit hyperactivity disorder, epilepsy, dyslexia, motor delay, language delay, visual impairment, microcephaly, and alcohol consumption. In addition, <em>AUTS2</em> contains the most significantly accelerated genomic region differentiating humans from Neanderthals, which is primarily composed of noncoding variants. However, the function and regulation of this gene remain largely unknown. To characterize <em>auts2</em> function, we knocked it down in zebrafish, leading to a smaller head size, neuronal reduction, and decreased mobility. To characterize <em>AUTS2</em> regulatory elements, we tested sequences for enhancer activity in zebrafish and mice. We identified 23 functional zebrafish enhancers, 10 of which were active in the brain. Our mouse enhancer assays characterized three mouse brain enhancers that overlap an ASD–associated deletion and four mouse enhancers that reside in regions implicated in human evolution, two of which are active in the brain. Combined, our results show that <em>AUTS2</em> is important for neurodevelopment and expose candidate enhancer sequences in which nucleotide variation could lead to neurological disease and human-specific traits.</p> </div

Schematic of the <i>AUTS2</i> genomic region.

<p>Human accelerated sequences are shown as blue lines above the gene <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003221#pgen.1003221-Green1" target="_blank">[17]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003221#pgen.1003221-Prabhakar1" target="_blank">[19]</a>. Structural variants <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003221#pgen.1003221-Sultana1" target="_blank">[4]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003221#pgen.1003221-Talkowski1" target="_blank">[12]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003221#pgen.1003221-Elia1" target="_blank">[14]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003221#pgen.1003221-Mefford1" target="_blank">[15]</a> are represented as colored lines (red: deletion, orange: inversion, green: duplication, purple: translocation). The rs6943555 SNP associated with alcohol consumption <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003221#pgen.1003221-Schumann1" target="_blank">[16]</a> is shown as a magenta star. Arrows in bars signify that the structural variant extends past the gene in that direction. Exons are depicted as light blue rectangles, as defined by the RefSeq genes track in the UCSC Genome Browser <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003221#pgen.1003221-Pruitt1" target="_blank">[52]</a>. Numbers to the left of the lines correspond to a reference number. Human Accelerated Conserved Non-coding Sequence (HACNS), Human Accelerated Region (HAR), developmental delay (DD), intellectual disability (ID), dysmorphic features (DF), seizure disorder (SD), multiple congenital anomalies (MCA), language disability (LD).</p