McCallion Lab Family

"FUNCTIONAL GENETICS ILLUMINATING HUMAN DISEASE"

Zach Stine

Graduate Student: Human Genetics (McCallion lab 2006-present)
Email: zstine@yahoo.com
Supervisor: McCallion

My Project:

The focus of my research is to examine the regulatory network underlying a critical gene in enteric nervous system (ENS) development. The Phox2b paired-like homeodomain transcription factor is essential for the development of all aspects of the autonomic nervous system. My goal is to identify Phox2b regulatory sequences, the transcription factors that bind them in regulating Phox2b expression, and the genes regulated by the PHOX2B protein during ENS development. I have begun by identifying conserved non-coding sequences at Phox2b to determine their regulatory potential. At present, I am evaluating the biological relevance of selected sequences using transgenesis in zebrafish as an in vivo assay.

Publications since joining the lab:

McGaughey, D.M., Stine, Z.S., Huynh, J., Vinton, RM., McCallion, A.S.,Symmetrical Distribution Of Non-Conserved Regulatory Sequences At PHOX2B IsReflected At The ENCODE Loci And Illuminates A Possible Genome-Wide Trend. (2008) BMC Genomics (In Press)

Zachary E. Stine, Jimmy L. Huynh, David Gorkin, Todd Purves, Thomas Novak, Amirali H. Salmasi, Anthony Antonellis, Stacie Loftus, William J. Pavan, John P. Gearhart and Andrew S. McCallion. A transgenic mouse strain directs pan-neural crest expression of Cre recombinase under the control of a distal Sox10 enhancer element. Genesis [Epub ahead of print]

Megana Prasad



Graduate Student:  Human Genetics (McCallion lab 2008-present)
Email: megan.prasad@gmail.com
Supervisor: McCallion

My Project:

My interest lies in studying the role of non coding (regulatory) DNA in the development and function of epidermal melanocytes, and how mutations result in pigmentation variation or disease-risk in humans and other vertebrate species. Our primary goal is to uncover DNA sequence-based vocabularies that specify gene expression within developing and mature fate in neural crest-derived melanocytes. We are screening putative regulatory sequence elements (REs) for function in melanocytes using a transgenic zebrafish assay developed in our lab. By looking for motifs that are over-represented in melanocyte REs compared with non-melanocyte REs, we hope to be able to predict the regulatory potential of additional non coding DNA elements in directing melanocytic development.

We are also studying the functional impact of non-coding DNA variants in human pigmentation phenotypes. We are using two approaches to meet this goal: a) testing known generic variants for their effect on pigmentation using in vitro and zebrafish assays, and b) sequencing non-coding regulatory elements of melanocytic genes in patients with pigmentation phenotypes.

By combining these approaches, we hope to begin to probe the impact of non-coding DNA in human pigmentation phenotypes and diseases.

Publications since joining the lab:

Gpnmb is a Melanoblast-Expressed, MITF-Dependent Gene. Loftus SK, Antonellis A, Matera I, Renaud G, Baxter LL, Reid D, Wolfsberg TG, Chen Y, Wang C; NISC Comparative Sequencing Program, Prasad MK, Bessling SL, McCallion AS, Green ED, Bennett DC, Pavan WJ. Pigment Cell Melanoma Res. 2008 Nov 1. PMID: 18983539.

David Gorkin

Graduate Student: Human Genetics (McCallion lab 2008-present)
Recipient - National Science Foundation Graduate Research Fellowship
Email: dgorkin@jhmi.edu
Supervisor: McCallion

My Project:

Enhancer-blocking insulators, or enhancer blockers (EBs), are a class of regulatory elements that have the potential to block the activating effects of an enhancer on a gene. EBs can inhibit an enhancer from upregulating a gene's expression when positioned in between that enhancer and the gene's promoter. There are several well-characterized examples of EBs in vertebrate genomes, including the 5' HS4 elements of the chicken Beta-globin locus and the Imprinted Control Region (ICR) of the murine H19/IGF2 locus. To expand our understanding of EB elements and to begin to assess their role in human genetics, we have set out to identify novel EBs in the human genome. To this end, we have used bioinformatic approaches to identify a number of regions in the human genome that we believe are good candidates to house EBs. I am currently developing an in vivo assay, making use of ToI2-mediated transgenesis in zebrafish, which we will use to functionally evaluate the potential enhancer-blocking activity of our candidate regions.

Publications since joining the lab:

Zachary E. Stine, Jimmy L. Huynh, David Gorkin, Todd Purves, Thomas Novak, Amirali H. Salmasi, Anthony Antonellis, Stacie Loftus, William J. Pavan, John P. Gearhart and Andrew S. McCallion. A transgenic mouse strain directs pan-neural crest expression of Cre recombinase under the control of a distal Sox10 enhancer element. Genesis [Epub ahead of print]

Seneca Bessling

Senior Lab Technician (McCallion lab 2008-present)
Supervisor: McCallion

My Project:

I am the facilities manager of the FInZ (Functional Investigation in Zebrafish) Center, and the senior technician of the McCallion lab. My responsibilities are centered on the running and implementation of zebrafish-based experimental strategies. My research is presently focused on the analysis of a critical interval of the human genome, implicated in facial development and the analysis of the genes therein.

Greg Burzynski

Post-Doctoral Fellow (McCallion lab 2009-present)
Supervisor: McCallion

My Project:

My research areas of interest focus on human neurodegenerative and neurodevelopmental diseases. My doctoral thesis concerned various aspects of complex human genetic disorder - Hirschsprung's disease (HSCR). This pediatric condition manifests with the lack of innervation of the distal gastro-intestinal (GI) tract. My studies involved elucidating the role of non-coding regulatory mutations in the RET gene, as well as searching for new susceptibility regions involved in the disease etiology. Subsequently, my first postdoctoral appointment at Iain Shepherd lab (Emory Univ) allowed me to study enteric nervous system development and to gain experience in the zebrafish animal system model.

In the McCallion lab my research is focused on the central nervous system (CNS) and involves studying the development and transcriptional regulation of catecholaminergic neurons in vivo. While coding sequence mutations have been associated with genetic disorders in around 20,000 protein coding genes, very little information exists on the role of regulatory region variants in the human genetic diseases. Only recently, thanks to the availability of increasing number of sequenced vertebrate genomes, comparative genomics techniques have shown a strong correlation between non-coding conserved regions and a presence of large number of transcriptional enhancers. Moreover, from the very recent development of technologies like ChIP-seq (chromatin immunoprecipitation coupled to parallel genome sequencing) and the discovery of chromatin signatures that mark with high probability active enhancers, it has become apparent that those regulatory regions are active in a very dynamic, cell/tissue type specific manner, which likely holds the key to the complexity of vertebrate organisms. Establishing 'the enhancer code' and the identifications of factors forming the enhaceosome modules in catecholaminergic neurons will shed light on their involvement in neurodegenerative and psychiatric disorders like Parkinson's , Alzheimer disease or schizophrenia. In a long perspective, this study might be extended and adapted to other neuronal populations and facilitate creating a regulatory lexicon for neuronal development.

Publications since joining the lab:

Differential Contributions of Rare and Common, Coding and Noncoding Ret Mutations to Multifactorial Hirschsprung Disease Liability. Emison ES, Garcia-Barcelo M, Grice EA, Lantieri F, Amiel J, Burzynski G, Fernandez RM, Hao L, Kashuk C, West K, Miao X, Tam PK, Griseri P, Ceccherini I, Pelet A, Jannot AS, de Pontual L, Henrion-Caude A, Lyonnet S, Verheij JB, Hofstra RM, Antiñolo G, Borrego S, McCallion AS, Chakravarti A. Am J Hum Genet. 2010 Jul 9;87(1):60-74.PMID: 20598273

Takeshi Matsui

Senior Lab Technician (McCallion lab 2009-present)
Supervisor: McCallion

My Project:

I am and senior lab tech in the McCallion lab.  I work together with Greg (Dr Burzynski) and Professor McCallion on a project which aims to identify neuronal regulatory sequences from non-coding regions of the DNA.  We anticipate that we are able to predict the neuronal gene expression patterns by looking for motif combinations of transcription factor binding sites in sequences regulating the expression of the genes.  My current role in the lab is to evaluate conserved non-coding human sequences for their ability to regulate tissue-specific gene expression.  My goal is to create a data set from which neuronal regulatory sequences can be identified and analyzed.

Samantha Maragh

Graduate student: Human Genetics (McCallion lab 2009-present)
Supervisor: McCallion

My Project:

Rbm24 is a novel gene identified in the lab. Transcriptional profiling on mouse ES cells to identify genes involved in cardiogenesis yielded Rbm24 as a primary candidate.  Rbm24 is expressed in the heart, somites, and central vein of developing mouse embryos. Two orthologs of Rbm24 (rbm24a & rbm24b) have been identified in zebrafish. Zebrafish have allowed an excellent model system to study rbm24a/b in early development. Knockdown of either zebrafish ortholog in zebrafish results in severe cardiac malformation, edema and aberrant somite structure. Edema in the heart indicates circulation abnormalities. I am interested in furthering the understanding of the role of Rbm24 in cardiovascular development. I am currently examining the implications of Rbm24 knockdown on the development of the vascular system.

In addition to paying a role in normal development Rbm24 may have a potential role in cancer.  Meta analysis of tissue and expression array data shows Rbm24 to be upregulated in estrogen responsive breast cancer as well as ovarian, prostate and pancreatic cancers.  I have an interest in cancer detection, particularly early detection of cancer and will be pursuing experiments to better understand the role Rbm24 plays in cancer and cancer progression.  I will be using breast cancer as an initial investigation system. I am currently conducting Rbm24 expression analysis experiments on various breast cancers and generating an Rbm24 antibody for protein localization and tissue microarray studies.

Xylena Reed

Graduate student: Human Genetics (McCallion lab 2010-present)

Email: xreed1@jhmi.edu
Supervisor: McCallion

My Project:

My project is in its infancy but here is the plan.  I want to apply ChIP-Seq and cutting edge transgenic approaches in zebrafish to better understand how gene transcription in the human brain is regulated. I am acquiring the techniques and skills necessary to make this happen, and in doing so will expand on an existing study to analyze regulatory control of the critical neuronal transcription factor PHOX2B.

Ryan Vinton

Senior Lab Technician (McCallion lab 2004-2007)
Email: rvinton1@jhmi.edu

My Project:

As part of the McCallion lab my interests are tied to the studies of gene regulation and disease development.My scientific role within the lab is based heavily on the use of zebrafish transgenesis; I am responsible for the implementation of our technologies in the projects of ourmany in-house and collaborative research projects. Most of this work centers around the role of conserved non-coding sequences in regulating gene expression. I ensure the smooth day to day operation of the lab while training graduate students and post docs.Since joining the lab, I have completed my M.S. in Biotechnology here at JHU and also interned at the Phoebe R. Berman Bioethics Institute.My plans for the future include law school and a career in intellectual property law.

Publications since joining the lab:

McGaughey, D.M., Stine, Z.S., Huynh, J., Vinton, RM., McCallion, A.S.,Symmetrical Distribution Of Non-Conserved Regulatory Sequences At PHOX2B IsReflected At The ENCODE Loci And Illuminates A Possible Genome-Wide Trend. (2008) BMC Genomics (In Press)

Fisher, S., Grice, E.A., Vinton, R., Bessling, S.L., Urasaki, A., Kawakami, K. and McCallion, A.S. (2006) Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish. Nature protocols 1, 1297-1305.

Fisher S, Grice EA, Vinton RM, Bessling SL and McCallion AS. (2006) Conservation of RET Regulatory Function from Human to Zebrafish Without Sequence Similarity. Science. 2006 Mar 23; [Epub ahead of print]

McGaughey, D.M., Vinton, R.M., Huynh, J., Al-Saif, A., Beer, M., and McCallion, A.S. Metrics of sequence constraint overlook regulatory sequences in an exhaustive analysis at phox2b. Genome Research, 12/2007.

Elizabeth Grice

"Elizabeth is now a PRAT Postdoctoral Fellow in the Segre lab at NHGRI/NIH"

Postdoctoral Fellow (McCallion lab 2006 - 2008)
Graduate Student: Human Genetics (McCallion lab 2004 - 2006)
Email: egrice1@jhmi.edu

My Project:

Regulatory mutations at the RET locus are predicted to underlie susceptibility to Hirschsprung disease (HSCR: aganglionic megacolon), a relatively common congenital disorder characterized by incomplete colonization of a variable length of the large intestine by neural crest-derived enteric ganglia. Thus far, mutations in the RET gene have been implicated in as many as 90% of familial HSCR cases, yet one half of the HSCR families demonstrating linkage to RET lack coding sequence mutations. My project focuses on elucidating the regulatory landscape of RET as an entry point to elucidating the role of regulatory RET mutations in disease susceptibility.

My past work focused on systematically examining conserved non-coding sequences in a 220 kb interval encompassing RET, combining in silico prediction methods with in vitro cell-based functional assays, molecular analysis, and regulation of transgenic expression in mouse models. We have shown that most of these conserved elements are capable of enhancer or suppressor activity in vitro, and discrete sequences within regulatory elements bind nuclear proteins. We then focused on an enhancer sequence implicated in HSCR (MCS+9.7) to demonstrate that this element drives reporter gene expression in vivo cell populations consistent with the stage and tissue expression of the endogenous RET protein, including the enteric nervous system, consistent with its proposed role in HSCR.

Because regulatory sequences commonly mediate their effect upon binding transcription factors, we have identified relevant putative transcription factor binding sites (TFBS) localized within MCS+9.7. My recent efforts have focused on determining the biological relevance of identified TFBS in regulatory control through in vitro cDNA transactivation and chromatin immunoprecipitation (ChIP) assays.

Additionally, I am interested in dissecting the biological and disease relevance of MCS+9.7 in mouse models. Toward this end, I have generated a mouse transgenic for a BAC encompassing Ret and containing a GFP-Cre reporter cassette. These mice faithfully recapitulate expression of Ret, and I am performing crosses to attempt to rescue the Ret null phenotype by BAC complementation.

Publications since joining the lab:

Emison ES, Garcia-Barcelo M, Grice EA, Lantieri F, Amiel J, Burzynski G, Fernandez RM, Hao L, Kashuk C, West K, Miao X, Tam PK, Griseri P, Ceccherini I, Pelet A, Jannot AS, de Pontual L, Henrion-Caude A, Lyonnet S, Verheij JB, Hofstra RM, Antiñolo G, Borrego S, McCallion AS, Chakravarti A. Differential Contributions of Rare and Common, Coding and Noncoding Ret Mutations to Multifactorial Hirschsprung Disease Liability. Am J Hum Genet. 2010 Jul 9;87(1):60-74.PMID: 20598273

Fisher, S., Grice, E.A., Vinton, R.., Bessling, S.L., Urasaki, A., Kawakami, K. and McCallion, A.S. (2006) Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish. Nature protocols 1, 1297-1305.

Grice, E.A. and McCallion, A.S. (2007) Genomic dissection of RET signaling in human disease. Signal Transduction: A Systems Biology Approach. Pandey; 1st edition, Humana Press, New Jersey, In Press.

Fisher S, Grice EA, Vinton RM, Bessling SL and McCallion AS. (2006) Conservation of RET Regulatory Function from Human to Zebrafish Without Sequence Similarity. Science. 2006 Mar 23; [Epub ahead of print]

Grice, E.A., Rochelle, E.S., Green, E.D., Chakravarti, A., and McCallion, A.S. (2005). Evaluation of the RET regulatory landscape reveals the biological relevance of a HSCR-implicated enhancer. Human Molecular Genetics, 14 (23).

Kashuk, C.S., Stone, A.E., Grice, E.A., Portnoy, M.E., Green, E.D., Sidow, A., Chakravarti, A. and McCallion, A.S. (2005) Phenotype:Genotype correlation in HSCR and MEN2 facilitated comparative analysis of the RET protein sequence. Proceedings of the National Academy of Science (USA) 102: 8949-8954.

Sproat-Emison, E.E., McCallion, A.S., Kashuk, C.S., Bush, R.T., Grice, E.A., Lin, S., Portnoy, M.E., NISC Comparative Sequencing Program, Cutler, D.J., Green, E.D. and Chakravarti, A. (2005) A common, sex-dependent mutation in a putative RET enhancer underlies Hirschsprung disease susceptibility. Nature, 434: 857-63.

"Russ is now a Postdoctoral Fellow at Univ. Penn"

Graduate student: Human Genetics (Gearhart/McCallion lab 2004-2008)
Email: raddis@jhmi.edu

My Project:

My research is aimed at turning stem cells into dopamine neurons, the cells that are destroyed in Parkinson's Disease. In order to better direct the differentiation of embryonic stem cells into these neurons, I am studying how they are formed in a developing embryo. Two genes known to be important in this developmental process are NR4A2 (aka Nurr1) and PITX3. My goal is to identify the transcription factors that activate these genes so that I may ultimately manipulate these factors in stem cells to direct their differentiation into dopamine neurons. I used comparative genomics to identify evolutionarily conserved sequences in the vicinity of NR4A2 and PITX3. We tested our hypothesis that these sequences are cis-acting regulatory elements by cloning them into a GFP reporter vector and using this vector to make transgenic zebrafish. Those sequences that drove tissue-speficic GFP expression in zebrafish dopamine neurons were selected for further analysis to identify the transcription factors that bind them. These factors will then be used for the directed differentiation of embryonic stem cells into dopamine neurons.

Ron Miller

"Ron is now a Researcher with the The Weinberg Group, Inc."

Postdoctoral Fellow (McCallion lab 2008-2009)
Graduate student: Human Genetics (Gearhart/McCallion lab 2005-2008)
Email: ronaldm@jhmi.edu

My Project:

Ischemic heart disease is a major health problem in the United States affecting ~5 million individuals. In general terms the heart muscle is damaged by a acute episodes when it is starved of oxygenated blood; the damage persists because the heart muscle is largely unable to repair itself. Consequently the ability to restore function to a damaged heart is a major medical challenge. We believe that a better understanding of the mechanisms underlying the genesis of the heart will aid progress towards improved therapeutic interventions for heart disease. One attractive possibility lies in cell-based therapeutics. We propose to use embryonic stem cells as the basis for the generation of new heart muscle to repair the heart. This has been hampered because of the inefficiencies of stem cell differentiation to heart muscle and our poor understanding of the process. By studying the process of differentiation of embryonic stem cell, we aim to understand what genes are necessary for the formation of heart muscle in an effort to make informed decisions about directing cellular differentiation and the selection of cells for transplantation. Using this information we can make the process of cardiac differentiation more efficient and move closer to cell based therapies.

Publications since joining the lab:

Christoforou, N.,* Miller, R.A.,* Hill, C.M., Jie, C.C., McCallion, A.S., and Gearhart. J.D. The characterization of ES-derived cardiac precursor cells demonstrates their multipotentiality and identifies novel cardiac genes. J. Clin. Invest. 2/2008.

David McGaughey

Graduate Student: Human Genetics (McCallion lab 2006-2010)
Email: davidm@jhmi.edu
Supervisor: McCallion

My Project:

The availability of multiple completed vertebrate genomes sequencing, familitates comparisions of the genetic composition of a variety of species. This gives researchers the power to identify sequences that have stayed relatively constand over the course of evolution. This has led to the surprising finding that the vast majorityof the human sequence appears to have little function. In fact, only approximately 5% of the human genome's content is under evolutionary selection. This 5% can be further subdivided into coding (~1.5%) and non-coding (~3.5%) DNA. A tremendous amount of work has been invested into studying coding DNA, which is used to form proteins. This "gene-focused" approach has led to numerous advances in the study of development and disease However, a full understanding of many diseases and developmental pathways cannot be understood without knowledge of how proteins are regulated.

This is where the non-coding DNA comes into play. I'm interested in probing the function of conserved non-coding DNA in development and disease. Through the use of zebrafish trangenesis, I'm currently isolating non-coding elements with a role in the regulation of the gne Phox2b, a transcription factor vital in the development of the nervous system.

Publications since joining the lab:

McGaughey DM, McCallion AS. Efficient discovery of ASCL1 regulatory sequences through transgene pooling.Genomics. Jun;95(6):363-9. Epub 2010 Mar 4.PMID: 20206680

McGaughey, D.M., Vinton, R.M., Huynh, J., Al-Saif, A., Beer, M., and McCallion, A.S. Metrics of sequence constraint overlook regulatory sequences in an exhaustive analysis at phox2b. Genome Research, 12/2007.

Loïc De Pontual, Sophie Thomas, Norann A. Zaghloul, David M. McGaughey, Hélène Dollfus, Clarisse Baumann, Erica E. Davis, Arnold Munnich, Heather Etchevers, Michel Vekemans, Stanislas Lyonnet, Andrew S. McCallion, Tania Attie-Bitach, Nicholas Katsanis, Jeanne Amiel. The Association Of Bardet-Biedl Syndrome And Hirschsprung Disease Highlights The Role Of The Primary Cilium In ENS Development. Proceedings of the National Academy of Science (USA), 2009; 106(33):13921-6.

McGaughey, D.M., Stine, Z.S., Huynh, J., Vinton, RM., McCallion, A.S.,Symmetrical Distribution Of Non-Conserved Regulatory Sequences At PHOX2B IsReflected At The ENCODE Loci And Illuminates A Possible Genome-Wide Trend. (2008) BMC Genomics (In Press)

Efficient discovery of ASCL1 regulatory sequences through transgene pooling. McGaughey DM, McCallion AS. Genomics. 2010 Jun;95(6):363-9. Epub 2010 Mar 4.PMID: 20206680