Publications by authors named "Xylena Reed"

17 Publications

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Transcriptional signatures in iPSC-derived neurons are reproducible across labs when differentiation protocols are closely matched.

Stem Cell Res 2021 Oct 3;56:102558. Epub 2021 Oct 3.

Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892, USA.

Reproducibility of expression patterns in iPSC-derived cells from different labs is an important first step in ensuring replication of biochemical or functional assays that are performed in different labs. Here we show that reproducible gene expression patterns from iPSCs and iPSC-derived neurons matured and collected at two separate laboratory locations can be achieved by closely matching protocols and reagents. While there are significant differences in gene expression between iPSCs and differentiated neurons, as well as between different donor lines of the same cell type, transcriptional changes that vary with laboratory sites are relatively small. These results suggest that making great efforts to match protocols, reagents and technical methods between labs may improve the reproducibility of iPSC-derived cell models.
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http://dx.doi.org/10.1016/j.scr.2021.102558DOI Listing
October 2021

Coding and Noncoding Variation in LRRK2 and Parkinson's Disease Risk.

Mov Disord 2021 Sep 20. Epub 2021 Sep 20.

Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, Maryland, USA.

Background: The leucine-rich repeat kinase 2 (LRRK2) gene harbors both rare highly damaging missense variants (eg, p.G2019S) and common noncoding variants (eg, rs76904798) with lower effect sizes that are associated with Parkinson's disease (PD) risk.

Objectives: This study aimed to investigate in a large meta-analysis whether the LRRK2 Genome-Wide Association Study (GWAS) signal represented by rs76904798 is independently associated with PD risk from LRRK2 coding variation and whether complex linkage disequilibrium structures with p.G2019S and the 5' noncoding haplotype account for the association of LRRK2 coding variants.

Methods: We performed a meta-analysis using imputed genotypes from 17,838 patients, 13,404 proxy patients, and 173,639 healthy controls of European ancestry. We excluded carriers of p.G2019S and/or rs76904798 to clarify the role of LRRK2 coding variation in mediating disease risk and excluded carriers of relatively rare LRRK2 coding variants to assess the independence of rs76904798. We also investigated the co-inheritance of LRRK2 coding variants with p.G2019S, rs76904798, and p.N2081D.

Results: LRRK2 rs76904798 remained significantly associated with PD after excluding the carriers of relatively rare LRRK2 coding variants. LRRK2 p.R1514Q and p.N2081D were frequently co-inherited with rs76904798, and the allele distribution of p.S1647T significantly changed among patients after removing rs76904798 carriers.

Conclusions: These data suggest that the LRRK2 coding variants previously related to PD (p.N551K, p.R1398H, p.M1646T, and p.N2081D) do not drive the 5' noncoding GWAS signal. These data, however, do not preclude the independent association of the haplotype p.N551K-p.R1398H and p.M1646T with altered disease risk. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society. This article has been contributed to by US Government employees and their work is in the public domain in the USA.
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http://dx.doi.org/10.1002/mds.28787DOI Listing
September 2021

THAP1 modulates oligodendrocyte maturation by regulating ECM degradation in lysosomes.

Proc Natl Acad Sci U S A 2021 Aug;118(31)

Peter O'Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390;

Mechanisms controlling myelination during central nervous system (CNS) maturation play a pivotal role in the development and refinement of CNS circuits. The transcription factor THAP1 is essential for timing the inception of myelination during CNS maturation through a cell-autonomous role in the oligodendrocyte lineage. Here, we demonstrate that THAP1 modulates the extracellular matrix (ECM) composition by regulating glycosaminoglycan (GAG) catabolism within oligodendrocyte progenitor cells (OPCs). OPCs accumulate and secrete excess GAGs, inhibiting their maturation through an autoinhibitory mechanism. THAP1 controls GAG metabolism by binding to and regulating the gene encoding β-glucuronidase, a GAG-catabolic lysosomal enzyme. Applying GAG-degrading enzymes or overexpressing β-glucuronidase rescues OL maturation deficits in vitro and in vivo. Our studies establish lysosomal GAG catabolism within OPCs as a critical mechanism regulating oligodendrocyte development.
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http://dx.doi.org/10.1073/pnas.2100862118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8346877PMC
August 2021

Generation of fourteen isogenic cell lines for Parkinson's disease-associated leucine-rich repeat kinase (LRRK2).

Stem Cell Res 2021 05 15;53:102354. Epub 2021 Apr 15.

Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892, USA.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with inherited forms of Parkinson's disease (PD), causing disease by a gain of kinase function. Here, we describe a series of isogenic iPSC lines with any of five pathogenic mutations (N1437H, R1441C, Y1699C, G2019S and I2020T); two hypothesis testing mutations (GTP binding null, T1348N, and kinase dead, K1906M) and two LRRK2 knockouts. This resource could be used to assess effects of mutations on the function of endogenous LRRK2 and/or to study LRRK2 interactors and substrates in iPSC-derived cellular models.
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http://dx.doi.org/10.1016/j.scr.2021.102354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8314873PMC
May 2021

Advancing Personalized Medicine in Common Forms of Parkinson's Disease through Genetics: Current Therapeutics and the Future of Individualized Management.

J Pers Med 2021 Mar 1;11(3). Epub 2021 Mar 1.

Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, 35 Convent Drive, Room 1A-211, Bethesda, MD 20892-3707, USA.

Parkinson's disease (PD) is a condition with heterogeneous clinical manifestations that vary in age at onset, rate of progression, disease course, severity, motor and non-motor symptoms, and a variable response to antiparkinsonian drugs. It is considered that there are multiple PD etiological subtypes, some of which could be predicted by genetics. The characterization and prediction of these distinct molecular entities provides a growing opportunity to use individualized management and personalized therapies. Dissecting the genetic architecture of PD is a critical step in identifying therapeutic targets, and genetics represents a step forward to sub-categorize and predict PD risk and progression. A better understanding and separation of genetic subtypes has immediate implications in clinical trial design by unraveling the different flavors of clinical presentation and development. Personalized medicine is a nascent area of research and represents a paramount challenge in the treatment and cure of PD. This manuscript summarizes the current state of precision medicine in the PD field and discusses how genetics has become the engine to gain insights into disease during our constant effort to develop potential etiological based interventions.
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http://dx.doi.org/10.3390/jpm11030169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7998972PMC
March 2021

The Parkinson's Disease Genome-Wide Association Study Locus Browser.

Mov Disord 2020 11 31;35(11):2056-2067. Epub 2020 Aug 31.

Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, Maryland, USA.

Background: Parkinson's disease (PD) is a neurodegenerative disease with an often complex component identifiable by genome-wide association studies. The most recent large-scale PD genome-wide association studies have identified more than 90 independent risk variants for PD risk and progression across more than 80 genomic regions. One major challenge in current genomics is the identification of the causal gene(s) and variant(s) at each genome-wide association study locus. The objective of the current study was to create a tool that would display data for relevant PD risk loci and provide guidance with the prioritization of causal genes and potential mechanisms at each locus.

Methods: We included all significant genome-wide signals from multiple recent PD genome-wide association studies including themost recent PD risk genome-wide association study, age-at-onset genome-wide association study, progression genome-wide association study, and Asian population PD risk genome-wide association study. We gathered data for all genes 1 Mb up and downstream of each variant to allow users to assess which gene(s) are most associated with the variant of interest based on a set of self-ranked criteria. Multiple databases were queried for each gene to collect additional causal data.

Results: We created a PD genome-wide association study browser tool (https://pdgenetics.shinyapps.io/GWASBrowser/) to assist the PD research community with the prioritization of genes for follow-up functional studies to identify potential therapeutic targets.

Conclusions: Our PD genome-wide association study browser tool provides users with a useful method of identifying potential causal genes at all known PD risk loci from large-scale PD genome-wide association studies. We plan to update this tool with new relevant data as sample sizes increase and new PD risk loci are discovered. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. This article has been contributed to by US Government employees and their work is in the public domain in the USA.
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http://dx.doi.org/10.1002/mds.28197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7754106PMC
November 2020

Genetic modifiers of risk and age at onset in GBA associated Parkinson's disease and Lewy body dementia.

Brain 2020 01;143(1):234-248

Institute of Clinical Medicine, Department of Neurology, University of Oulu, Oulu, Finland.

Parkinson's disease is a genetically complex disorder. Multiple genes have been shown to contribute to the risk of Parkinson's disease, and currently 90 independent risk variants have been identified by genome-wide association studies. Thus far, a number of genes (including SNCA, LRRK2, and GBA) have been shown to contain variability across a spectrum of frequency and effect, from rare, highly penetrant variants to common risk alleles with small effect sizes. Variants in GBA, encoding the enzyme glucocerebrosidase, are associated with Lewy body diseases such as Parkinson's disease and Lewy body dementia. These variants, which reduce or abolish enzymatic activity, confer a spectrum of disease risk, from 1.4- to >10-fold. An outstanding question in the field is what other genetic factors that influence GBA-associated risk for disease, and whether these overlap with known Parkinson's disease risk variants. Using multiple, large case-control datasets, totalling 217 165 individuals (22 757 Parkinson's disease cases, 13 431 Parkinson's disease proxy cases, 622 Lewy body dementia cases and 180 355 controls), we identified 1691 Parkinson's disease cases, 81 Lewy body dementia cases, 711 proxy cases and 7624 controls with a GBA variant (p.E326K, p.T369M or p.N370S). We performed a genome-wide association study and analysed the most recent Parkinson's disease-associated genetic risk score to detect genetic influences on GBA risk and age at onset. We attempted to replicate our findings in two independent datasets, including the personal genetics company 23andMe, Inc. and whole-genome sequencing data. Our analysis showed that the overall Parkinson's disease genetic risk score modifies risk for disease and decreases age at onset in carriers of GBA variants. Notably, this effect was consistent across all tested GBA risk variants. Dissecting this signal demonstrated that variants in close proximity to SNCA and CTSB (encoding cathepsin B) are the most significant contributors. Risk variants in the CTSB locus were identified to decrease mRNA expression of CTSB. Additional analyses suggest a possible genetic interaction between GBA and CTSB and GBA p.N370S induced pluripotent cell-derived neurons were shown to have decreased cathepsin B expression compared to controls. These data provide a genetic basis for modification of GBA-associated Parkinson's disease risk and age at onset, although the total contribution of common genetics variants is not large. We further demonstrate that common variability at genes implicated in lysosomal function exerts the largest effect on GBA associated risk for disease. Further, these results have implications for selection of GBA carriers for therapeutic interventions.
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http://dx.doi.org/10.1093/brain/awz350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935749PMC
January 2020

Parkinson's disease-associated mutations in the GTPase domain of LRRK2 impair its nucleotide-dependent conformational dynamics.

J Biol Chem 2019 04 22;294(15):5907-5913. Epub 2019 Feb 22.

From the Departments of Biochemistry and Molecular Biology, Indianapolis, Indiana 46202; The Stark Neurosciences Institute, Indiana University School of Medicine, Indianapolis, Indiana 46202; Department of Neurology, Indiana University School of Medicine, Indianapolis, Indiana 46202. Electronic address:

Mutation in leucine-rich repeat kinase 2 (LRRK2) is a common cause of familial Parkinson's disease (PD). Recently, we showed that a disease-associated mutation R1441H rendered the GTPase domain of LRRK2 catalytically less active and thereby trapping it in a more persistently "on" conformation. However, the mechanism involved and characteristics of this on conformation remained unknown. Here, we report that the Ras of complex protein (ROC) domain of LRRK2 exists in a dynamic dimer-monomer equilibrium that is oppositely driven by GDP and GTP binding. We also observed that the PD-associated mutations at residue 1441 impair this dynamic and shift the conformation of ROC to a GTP-bound-like monomeric conformation. Moreover, we show that residue Arg-1441 is critical for regulating the conformational dynamics of ROC. In summary, our results reveal that the PD-associated substitutions at Arg-1441 of LRRK2 alter monomer-dimer dynamics and thereby trap its GTPase domain in an activated state.
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http://dx.doi.org/10.1074/jbc.RA119.007631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463707PMC
April 2019

Parkinson-Associated SNCA Enhancer Variants Revealed by Open Chromatin in Mouse Dopamine Neurons.

Am J Hum Genet 2018 12 29;103(6):874-892. Epub 2018 Nov 29.

McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA. Electronic address:

The progressive loss of midbrain (MB) dopaminergic (DA) neurons defines the motor features of Parkinson disease (PD), and modulation of risk by common variants in PD has been well established through genome-wide association studies (GWASs). We acquired open chromatin signatures of purified embryonic mouse MB DA neurons because we anticipated that a fraction of PD-associated genetic variation might mediate the variants' effects within this neuronal population. Correlation with >2,300 putative enhancers assayed in mice revealed enrichment for MB cis-regulatory elements (CREs), and these data were reinforced by transgenic analyses of six additional sequences in zebrafish and mice. One CRE, within intron 4 of the familial PD gene SNCA, directed reporter expression in catecholaminergic neurons from transgenic mice and zebrafish. Sequencing of this CRE in 986 individuals with PD and 992 controls revealed two common variants associated with elevated PD risk. To assess potential mechanisms of action, we screened >16,000 proteins for DNA binding capacity and identified a subset whose binding is impacted by these enhancer variants. Additional genotyping across the SNCA locus identified a single PD-associated haplotype, containing the minor alleles of both of the aforementioned PD-risk variants. Our work posits a model for how common variation at SNCA might modulate PD risk and highlights the value of cell-context-dependent guided searches for functional non-coding variation.
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http://dx.doi.org/10.1016/j.ajhg.2018.10.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6288322PMC
December 2018

The role of monogenic genes in idiopathic Parkinson's disease.

Neurobiol Dis 2019 04 15;124:230-239. Epub 2018 Nov 15.

Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD, USA. Electronic address:

In the past two decades, mutations in multiple genes have been linked to autosomal dominant or recessive forms of monogenic Parkinson's disease (PD). Collectively, these monogenic (often familial) cases account for less than 5% of all PD, the majority being apparently sporadic cases. More recently, large-scale genome-wide association studies have identified over 40 loci that increase risk of PD. Importantly, there is overlap between monogenic and sporadic PD genes, particularly for the loci that contain the genes SNCA and LRRK2, which are mutated in monogenic dominant PD. There have also been reports of idiopathic PD cases with heterozygous variants in autosomal recessive genes suggesting that these mutations may increase risk of PD. These observations suggest that monogenic and idiopathic PD may have shared pathogenic mechanisms. Here, we focus mainly on the role of monogenic PD genes that represent pleomorphic risk loci for idiopathic PD. We also discuss the functional mechanisms that may play a role in increasing risk of disease in both monogenic and idiopathic forms.
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http://dx.doi.org/10.1016/j.nbd.2018.11.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363864PMC
April 2019

Frequency of Loss of Function Variants in LRRK2 in Parkinson Disease.

JAMA Neurol 2018 11;75(11):1416-1422

Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, Maryland.

Importance: Pathogenic variants in LRRK2 are a relatively common genetic cause of Parkinson disease (PD). Currently, the molecular mechanism underlying disease is unknown, and gain and loss of function (LOF) models of pathogenesis have been postulated. LRRK2 variants are reported to result in enhanced phosphorylation of substrates and increased cell death. However, the double knockout of Lrrk2 and its homologue Lrrk1 results in neurodegeneration in a mouse model, suggesting that disease may occur by LOF. Because LRRK2 inhibitors are currently in development as potential disease-modifying treatments in PD, it is critical to determine whether LOF variants in LRRK2 increase or decrease the risk of PD.

Objective: To determine whether LRRK1 and LRRK2 LOF variants contribute to the risk of developing PD.

Design, Setting, And Participants: To determine the prevailing mechanism of LRRK2-mediated disease in human populations, next-generation sequencing data from a large case-control cohort (>23 000 individuals) was analyzed for LOF variants in LRRK1 and LRRK2. Data were generated at 5 different sites and 5 different data sets, including cases with clinically diagnosed PD and neurologically normal control individuals. Data were collected from 2012 through 2017.

Main Outcomes And Measures: Frequencies of LRRK1 and LRRK2 LOF variants present in the general population and compared between cases and controls.

Results: Among 11 095 cases with PD and 12 615 controls, LRRK1 LOF variants were identified in 0.205% of cases and 0.139% of controls (odds ratio, 1.48; SE, 0.571; 95% CI, 0.45-4.44; P = .49) and LRRK2 LOF variants were found in 0.117% of cases and 0.087% of controls (odds ratio, 1.48; SE, 0.431; 95% CI, 0.63-3.50; P = .36). All association tests suggested lack of association between LRRK1 or LRRK2 variants and PD. Further analysis of lymphoblastoid cell lines from several heterozygous LOF variant carriers found that, as expected, LRRK2 protein levels are reduced by approximately half compared with wild-type alleles.

Conclusions And Relevance: Together these findings indicate that haploinsufficiency of LRRK1 or LRRK2 is neither a cause of nor protective against PD. Furthermore, these results suggest that kinase inhibition or allele-specific targeting of mutant LRRK2 remain viable therapeutic strategies in PD.
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http://dx.doi.org/10.1001/jamaneurol.2018.1885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6248108PMC
November 2018

Genetics in Parkinson disease: Mendelian versus non-Mendelian inheritance.

J Neurochem 2016 10 18;139 Suppl 1:59-74. Epub 2016 Apr 18.

Laboratory of Neurogenetics, National Institute on Aging, Bethesda, Maryland, USA.

Parkinson's disease is a common, progressive neurodegenerative disorder, affecting 3% of those older than 75 years of age. Clinically, Parkinson's disease (PD) is associated with resting tremor, postural instability, rigidity, bradykinesia, and a good response to levodopa therapy. Over the last 15 years, numerous studies have confirmed that genetic factors contribute to the complex pathogenesis of PD. Highly penetrant mutations producing rare, monogenic forms of the disease have been discovered in singular genes such as SNCA, Parkin, DJ-1, PINK 1, LRRK2, and VPS35. Unique variants with incomplete penetrance in LRRK2 and GBA have been shown to be strong risk factors for PD in certain populations. Additionally, over 20 common variants with small effect sizes are now recognized to modulate the risk for PD. Investigating Mendelian forms of PD has provided precious insight into the pathophysiology that underlies the more common idiopathic form of disease; however, no treatment methodologies have developed. Furthermore, for identified common risk alleles, the functional basis underlying risk principally remains unknown. The challenge over the next decade will be to strengthen the findings delivered through genetic discovery by assessing the direct, biological consequences of risk variants in tandem with additional high-content, integrated datasets. This review discusses monogenic risk factors and mechanisms of Mendelian inheritance of Parkinson disease. Highly penetrant mutations in SNCA, Parkin, DJ-1, PINK 1, LRRK2 and VPS35 produce rare, monogenic forms of the disease, while unique variants within LRRK2 and GBA show incomplete penetrance and are strong risk factors for PD. Additionally, over 20 common variants with small effect sizes modulate disease risk. The challenge over the next decade is to strengthen genetic findings by assessing direct, biological consequences of risk variants in tandem with high-content, integrated datasets. This article is part of a special issue on Parkinson disease.
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http://dx.doi.org/10.1111/jnc.13593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5155439PMC
October 2016

Integration of genomic and functional approaches reveals enhancers at LMX1A and LMX1B.

Mol Genet Genomics 2013 Nov 13;288(11):579-89. Epub 2013 Aug 13.

McKusick-Nathans Institute of Genetic Medicine, Department of Molecular and Comparative Pathobiology, The Johns Hopkins University School of Medicine, 733 N. Broadway, BRB Suite 407, Baltimore, MD, 21205, USA.

LMX1A and LMX1B encode two closely related members of the LIM homeobox family of transcription factors. These genes play significant, and frequently overlapping, roles in the development of many structures in the nervous system, including the cerebellum, hindbrain, spinal cord roof plate, sensory systems and dopaminergic midbrain neurons. Little is known about the cis-acting regulatory elements (REs) that dictate their temporal and spatial expression or about the regulatory landscape surrounding them. The availability of comparative sequence data and the advent of genomic technologies such as ChIP-seq have revolutionized our capacity to identify regulatory sequences like enhancers. Despite this wealth of data, the vast majority of loci lack any significant in vivo functional exploration of their non-coding regions. We have completed a significant functional screen of conserved non-coding sequences (putative REs) scattered across these critical human loci, assaying the temporal and spatial control using zebrafish transgenesis. We first identify and describe the LMX1A paralogs lmx1a and lmx1a-like, comparing their expression during embryogenesis with that in mammals, along with lmx1ba and lmx1bb genes. Consistent with their prominent neuronal expression, 47/71 sequences selected within and flanking LMX1A and LMX1B exert spatial control of reporter expression in the central nervous system (CNS) of mosaic zebrafish embryos. Upon germline transmission, we identify CNS reporter expression in multiple independent founders for 22 constructs (LMX1A, n = 17; LMX1B, n = 5). The identified enhancers display significant overlap in their spatial control and represent only a fraction of the conserved non-coding sequences at these critical genes. Our data reveal the abundance of regulatory instruction located near these developmentally important genes.
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http://dx.doi.org/10.1007/s00438-013-0771-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3812808PMC
November 2013

Integration of ChIP-seq and machine learning reveals enhancers and a predictive regulatory sequence vocabulary in melanocytes.

Genome Res 2012 Nov 27;22(11):2290-301. Epub 2012 Sep 27.

McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

We take a comprehensive approach to the study of regulatory control of gene expression in melanocytes that proceeds from large-scale enhancer discovery facilitated by ChIP-seq; to rigorous validation in silico, in vitro, and in vivo; and finally to the use of machine learning to elucidate a regulatory vocabulary with genome-wide predictive power. We identify 2489 putative melanocyte enhancer loci in the mouse genome by ChIP-seq for EP300 and H3K4me1. We demonstrate that these putative enhancers are evolutionarily constrained, enriched for sequence motifs predicted to bind key melanocyte transcription factors, located near genes relevant to melanocyte biology, and capable of driving reporter gene expression in melanocytes in culture (86%; 43/50) and in transgenic zebrafish (70%; 7/10). Next, using the sequences of these putative enhancers as a training set for a supervised machine learning algorithm, we develop a vocabulary of 6-mers predictive of melanocyte enhancer function. Lastly, we demonstrate that this vocabulary has genome-wide predictive power in both the mouse and human genomes. This study provides deep insight into the regulation of gene expression in melanocytes and demonstrates a powerful approach to the investigation of regulatory sequences that can be applied to other cell types.
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http://dx.doi.org/10.1101/gr.139360.112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3483558PMC
November 2012

Systematic elucidation and in vivo validation of sequences enriched in hindbrain transcriptional control.

Genome Res 2012 Nov 3;22(11):2278-89. Epub 2012 Jul 3.

McKusick-Nathans Institute of Genetic Medicine, Department of Molecular and Comparative Pathobiology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Illuminating the primary sequence encryption of enhancers is central to understanding the regulatory architecture of genomes. We have developed a machine learning approach to decipher motif patterns of hindbrain enhancers and identify 40,000 sequences in the human genome that we predict display regulatory control that includes the hindbrain. Consistent with their roles in hindbrain patterning, MEIS1, NKX6-1, as well as HOX and POU family binding motifs contributed strongly to this enhancer model. Predicted hindbrain enhancers are overrepresented at genes expressed in hindbrain and associated with nervous system development, and primarily reside in the areas of open chromatin. In addition, 77 (0.2%) of these predictions are identified as hindbrain enhancers on the VISTA Enhancer Browser, and 26,000 (60%) overlap enhancer marks (H3K4me1 or H3K27ac). To validate these putative hindbrain enhancers, we selected 55 elements distributed throughout our predictions and six low scoring controls for evaluation in a zebrafish transgenic assay. When assayed in mosaic transgenic embryos, 51/55 elements directed expression in the central nervous system. Furthermore, 30/34 (88%) predicted enhancers analyzed in stable zebrafish transgenic lines directed expression in the larval zebrafish hindbrain. Subsequent analysis of sequence fragments selected based upon motif clustering further confirmed the critical role of the motifs contributing to the classifier. Our results demonstrate the existence of a primary sequence code characteristic to hindbrain enhancers. This code can be accurately extracted using machine-learning approaches and applied successfully for de novo identification of hindbrain enhancers. This study represents a critical step toward the dissection of regulatory control in specific neuronal subtypes.
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http://dx.doi.org/10.1101/gr.139717.112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3483557PMC
November 2012

SOX10 directly modulates ERBB3 transcription via an intronic neural crest enhancer.

BMC Dev Biol 2011 Jun 14;11:40. Epub 2011 Jun 14.

McKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

Background: The ERBB3 gene is essential for the proper development of the neural crest (NC) and its derivative populations such as Schwann cells. As with all cell fate decisions, transcriptional regulatory control plays a significant role in the progressive restriction and specification of NC derived lineages during development. However, little is known about the sequences mediating transcriptional regulation of ERBB3 or the factors that bind them.

Results: In this study we identified three transcriptional enhancers at the ERBB3 locus and evaluated their regulatory potential in vitro in NC-derived cell types and in vivo in transgenic zebrafish. One enhancer, termed ERBB3_MCS6, which lies within the first intron of ERBB3, directs the highest reporter expression in vitro and also demonstrates epigenetic marks consistent with enhancer activity. We identify a consensus SOX10 binding site within ERBB3_MCS6 and demonstrate, in vitro, its necessity and sufficiency for the activity of this enhancer. Additionally, we demonstrate that transcription from the endogenous Erbb3 locus is dependent on Sox10. Further we demonstrate in vitro that Sox10 physically interacts with that ERBB3_MCS6. Consistent with its in vitro activity, we also show that ERBB3_MCS6 drives reporter expression in NC cells and a subset of its derivative lineages in vivo in zebrafish in a manner consistent with erbb3b expression. We also demonstrate, using morpholino analysis, that Sox10 is necessary for ERBB3_MCS6 expression in vivo in zebrafish.

Conclusions: Taken collectively, our data suggest that ERBB3 may be directly regulated by SOX10, and that this control may in part be facilitated by ERBB3_MCS6.
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http://dx.doi.org/10.1186/1471-213X-11-40DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3124416PMC
June 2011

Decreased proliferation kinetics of mouse myoblasts overexpressing FRG1.

PLoS One 2011 16;6(5):e19780. Epub 2011 May 16.

Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.

Although recent publications have linked the molecular events driving facioscapulohumeral muscular dystrophy (FSHD) to expression of the double homeobox transcription factor DUX4, overexpression of FRG1 has been proposed as one alternative causal agent as mice overexpressing FRG1 present with muscular dystrophy. Here, we characterize proliferative defects in two independent myoblast lines overexpressing FRG1. Myoblasts isolated from thigh muscle of FRG1 transgenic mice, an affected dystrophic muscle, exhibit delayed proliferation as measured by decreased clone size, whereas myoblasts isolated from the unaffected diaphragm muscle proliferated normally. To confirm the observation that overexpression of FRG1 could impair myoblast proliferation, we examined C2C12 myoblasts with inducible overexpression of FRG1, finding increased doubling time and G1-phase cells in mass culture after induction of FRG1 and decreased levels of pRb phosphorylation. We propose that depressed myoblast proliferation may contribute to the pathology of mice overexpressing FRG1 and may play a part in FSHD.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019780PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3095625PMC
October 2011
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