McCallion Lab Family

MCCALLION LAB FAMILY

"Developing Functional Genetic Approaches for Individualized Medicine"

David McGaughey

Graduate student:  Human Genetics (McCallion lab 2006-present)
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, 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.

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.

Ron Miller

Graduate student: Human Genetics (Gearhart/McCallion lab 2005-present)
Email: ronaldm@jhmi.edu
Supervisors: McCallion/Gearhart

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

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

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.

Megana Prasad


Graduate Student, Human Genetics (McCallion 2005-present)
Email: megan.prasad@gmail.com
Supervisors: McCallion

My Project:

My interests lie in the use of functional genetic strategies to uncover the genetic mechanisms of critical developmental and disease processes. I am presently contributing to a range of efforts as I begin to formulate the questions that will comprise my thesis work.

PAST LABORATORY MEMBERS

Elizabeth Grice

Post-doctoral fellow (McCallion lab 2006 - 2007)
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:

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.

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:

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.