Research Expertise

Research Interests: Elucidate genetic, epigenetic, and biochemical mechanisms by which mammals develop their immune systems while suppressing autoimmunity and genomic aberrations that cause leukemia or lymphoma.

Key Words: Genome topology, chromatin modifications, transcriptional regulation, RAG1/RAG2 (RAG) endonuclease, DNA double strand breaks (DSBs), DNA damage responses, post-transcriptional control of protein expression, antigen receptor gene diversification, lymphocyte development, genomic stability, translocations, lymphoma, leukemia, severe combined immunodeficiency, autoimmunity

Research Details: The ability to generate immune systems that discriminate between self and foreign antigens is vital for the life, health, and reproduction of mammals. Starting in utero, mammals develop several distinct lymphocyte lineages that segregate into innate and adaptive arms of immune systems. Innate lymphocytes express invariant germline-encoded antigen receptors, while adaptive lymphocytes express diverse antigen receptors assembled from recombination of germline variable (V), diversity (D), and joining (J) gene segments. The RAG1/RAG2 (RAG) endonuclease-mediated V(D)J recombination of immunoglobulin (Ig) and T cell receptor (TCR) genes in immature B and T lymphocytes, respectively, is essential for the development and function of adaptive immunity. Yet, this process also confers fatal risk as evidenced by generation of auto-reactive receptors and lymphoid malignancies with oncogenic translocations involving Ig or TCR loci. Developing B and T cells share developmental strategies that include mono-allelic gene recombination and feedback mechanisms, which link antigen receptor protein expression from functional rearrangements to further developmental progression or negative selection of auto-reactive cells. However, the genetic, epigenetic, and biochemical mechanisms governing these facets of B and T cell differentiation remain largely unknown. RAG DNA double strand breaks (DSBs) generated during V(D)J recombination engage conserved cellular DNA damage responses to inhibit the formation and oncogenic potential of aberrant genomic rearrangements. In the past few years, we have discovered that RAG DSBs activate tissue-specific DNA damage responses and transcend hazardous intermediates during antigen receptor gene assembly. In the latter context, RAG cleavage within the genomes of lymphocyte progenitors and immature lymphocytes regulates the expression of ubiquitous and lymphocyte-specific gene transcripts to control the differentiation and function of both adaptive and innate lymphocyte lineages. These unexpected discoveries raise important novel questions that have broad-ranging implications for basic immunology research and the screening, diagnosis, and treatment of human immunological disease.

Current Projects:

Elucidate How Three-Dimensional Genome Topology Regulates Transcription and Recombination
Gene expression relies on dynamic interplay among cis elements, chromatin domains, and genome architecture. The latter is of intense interest as ~10% of human diseases may arise from defects in genome topology that impact gene transcription. Genomes divide into conserved, megabase (Mb)-sized topologically associated domains (TADs) that are further subdivided into cell type-specific loops between promoter and enhancers (regulatory loops) or between CTCF binding sites (structural loops). In addition, chromatin architecture can be shaped by tissue-specific boundary elements (BEs) that divide active and inactive regions of transcription. These two types of domains tend to associate spatially, perhaps via homotypic chromatin interactions. Foundational questions remain about mechanisms of genome architecture reorganization and its impact on gene expression during cellular differentiation. Answers to these questions have profound implications because disease-associated variants in the human genome can disrupt CTCF elements or BEs, enabling aberrant communication between enhancers and alternative promoters that normally partition into separate architectural domains. In collaboration with the Oltz lab at Washington University, we have approached mechanistic relationships between genome topology and gene regulation by focusing on the Tcrb antigen receptor locus for several reasons, including: (i) it is a physiological model of manageable complexity (ii) its architecture and transcription are dynamically regulated during T cell development, (iii) it divides into alternating chromatin domains, (iv) changes in topology and transcription are critical for Tcrb assembly by long-range recombination, and (v) its recombination center (RC) has a simple regulatory landscape with one enhancer that communicates with two promoters to initiate all aspects of Tcrb assembly. Our recent collaborations have provided important clues into the dynamics of Tcrb structure at a low level of resolution, but fundamental insights into mechanisms that sculpt the observed architectural changes are still lacking. These and other data support our hypothesis that developmental switches between inactive and active Tcrb conformations are orchestrated by tissue- and stage-specific changes in the binding of CTCF to cornerstone elements and by the transcription status of individual gene segments, which cooperate to compartmentalize Tcrb into distinct structural domains and promote homotypic interactions that facilitate long-range Tcrb gene assembly. To test foundational aspects of our model, we seek to elucidate detailed topologies of active versus inactive Tcrb loci through cutting-edge chromosome conformation capture methodologies, assess whether transcription status and homotypic chromatin interactions shape Tcrb conformations, and determine if by which CTCF elements direct Tcrb topology. We monitor multiple physiological readouts - genome topology, gene transcription, chromatin structure, and V(D)J recombination - to gain unprecedented molecular insights into relationships among genome architecture, gene expression.

Determine the Physiological Role of Allelic Exclusion
V(D)J recombination of Tcrb, Igh, and Igk loci is regulated so that functional genes are assembled and expressed on one allele in most lymphocytes. This antigen receptor allelic exclusion is achieved by mono-allelic initiation and feedback inhibition of V recombination. The physiological role of allelic exclusion remains a major unresolved question in basic immunology. The greatest obstacle to finding an answer has been lack of any proven mechanism for mono-allelic V recombination. This has blocked development of experimental means to simultaneously increase the frequencies of bi-allelic assembly and expression of antigen receptor genes, without otherwise affecting V(D)J recombination or lymphocyte development. Tcrb and Igh recombination signal sequences (RSSs) target RAG activity to promote V rearrangements to DJ complexes and block V rearrangements to J segments. It has been proposed that Vβ (Trbv) and VH (Ighv) RSSs enforce allelic exclusion by directing mono-allelic initiation and feedback inhibition of V-to-DJ recombination. To test roles of RSSs in allelic exclusion, we have made mice harboring replacement of the weak Trbv2 or Trvb31 RSS with a stronger 3’Dβ RSS that recombines with Dβ and Jβ RSSs. These mice exhibit profoundly increased recombination of their RSS-replaced Vβ gene segment. Mice with these Vβs on opposite alleles (Trbv2R/Trbv31R mice) exhibit a 30-fold higher than normal fraction of T lymphocytes with bi-allelic TCRβ expression. We hypothesize that weak V RSSs and incompatible V and J RSSs enforce Tcrb and Igh allelic exclusion by decreasing the probability of assembling functional genes on both alleles before feedback inhibition permanently halts V recombination. We further hypothesize that these stochastic mechanisms inhibit oncogenic Tcrb and Igh translocations by limiting the incidence of RAG DSBs at these loci while proteins from in-frame V(D)J rearrangements signal proliferation. To test fundamental points of our model, we seek to: (i) determine how Tcrb RSSs limit V rearrangements and enforce allelic exclusion, (ii) assess whether these mechanisms control Igh recombination and allelic exclusion, (iii) elucidate contributions of V and J RSSs to mono-allelic initiation and feedback inhibition of V recombination, and (iv) investigate potential roles of RSSs in suppressing oncogenic Tcrb and Igh translocations. This project will generate unprecedented advances that answer a long-standing and much-investigated question in basic immunology. In the long-term, the knowledge, mouse models, and approaches acquired from this study also will empower us to assess the prevailing model that allelic exclusion suppresses auto-immunity by facilitating negative selection of self-reactive lymphocytes.

Elucidate Mechanisms and Functions of DSB Feedback Inhibition of V(D)J Recombination
While most studies of antigen receptor gene assembly have focused on how RAG cleavage is initiated, RAG activity also must be restrained to limit DSBs and resultant genomic instability. It has been recognized for decades that the assembly of Tcrb, Igh, and Igk genes occurs on one allele at a time. Two facets of this fundamental control mechanism are asynchronous initiation of V rearrangements between alleles and Ig/TCR-mediated feedback inhibition to permanently halt further V recombination after a functional Ig/TCR gene is assembled and expressed. In addition, it was proposed in 1980 that V recombination must activate more immediate signals to transiently block further V recombination, providing time for V(D)J rearrangements to be repaired, expressed, and signal feedback inhibition. Yet, evidence for this long-predicted transient aspect of regulation has been lacking. We discovered that RAG DSBs induced during Vk-to-Jk recombination signal through the Ataxia Telangiectasia mutated (ATM) protein kinase to transiently inhibit further Vk rearrangements. We showed that ATM down-regulates levels of RAG1 and RAG2 mRNA and RAG1 protein in response to RAG DSBs, and helps enforce mono-allelic Ig expression. In collaboration with the Sleckman Lab at Cornell, we found that RAG DSBs also signal via ATM to up-regulate expression of the SpiC transcriptional repressor, which in turn lowers recombination potential of Igk loci. DSBs induced by ionizing radiation, etoposide, or bleomycin suppress Rag1 and Rag2 mRNA levels in primary pre-B cells, pro-B cells, and pro-T cells, indicating that inhibition of Rag1 and Rag2 expression is a prevalent DSB response among immature lymphocytes. DSBs induced in pre-B cells signal rapid transcriptional repression of Rag1 and Rag2, causing down-regulation of both Rag1 and Rag2 mRNA but only Rag1 protein. This response requires ATM and the NF-κB essential modulator protein, implicating a role for ATM-mediated activation of canonical NF-κB transcription factors. DSBs induced in pre-B cells by etoposide or bleomycin inhibit recombination of Ig loci and a chromosomally integrated substrate. These data indicate that immature lymphocytes exploit a common DDR signaling pathway to limit DSBs at multiple genomic locations in developmental stages wherein mono-allelic antigen receptor locus recombination is enforced. We hypothesize that RAG and other types of DSBs transiently suppress V rearrangements through multiple independent but complementary mechanisms to safeguard from oncogenic Ig and TCR translocations and help ensure mono-specificity of antigen-binding B and T cells. We hypothesize that these mechanisms create a failsafe regulatory strategy that includes suppression of: (i) RAG expression by DSB response pathways that are ATM-dependent but restricted to developing lymphocytes, (ii) recombination potential in trans at the second non-cleaved Ig/TCR allele, and 3) sequential recombination in cis on the cleaved Ig/TCR allele. We seek to dissect underlying mechanisms for these ATM-dependent restrictions of V(D)J recombination and determine their independent contributions to enforcement of mono-allelic antigen receptor gene assembly and expression, suppression of Ig/TCR translocations, and shaping Ig/TCR repertoire. The knowledge acquired from this research will define molecules and pathways that protect from lymphoid malignancies and production of lymphocytes with multiple antigen specificities that cause autoimmunity. In the long-term, such knowledge should foster the development of novel prognostics, diagnostics, and therapeutics for specific human immunological disorders.

Determine Mechanisms and Roles of RAG1 Post-Transcriptional Control of Gene Expression
The RAG proteins each can be separated into “core” domains that are vital for V(D)J recombination and “non-core” regions that regulate the induction and repair of RAG DSBs. The discovery that mutation of the RAG1 N-terminal non-core region causes Omenn Syndrome, an immunodeficiency associated with autoimmunity, led to the notion that RAG1 non-core regions might control V(D)J recombination and lymphocyte development independent of regulating RAG endonuclease activity. Since then, the RAG1 N-terminus has been discovered to encode an ubiquitin ligase and associate with other proteins, including another ubiquitin ligase and a kinase. However, little is known today about potential roles of the RAG1 N-terminus beyond regulating RAG endonuclease function. We have recently found that the RAG1 N-terminus promotes Igλ+ B cell development by stimulating Pim2 protein expression from Pim2 transcripts induced in response to RAG DSBs. We hypothesize that the RAG1 N-terminus regulates lymphocyte development through post-transcriptional control of gene expression by mechanisms that transcend its known roles in modulating RAG endonuclease activity. These data raise fundamental questions including: (i) how does RAG1 post-transcriptionally increase Pim2 expression, and (ii) to what extent does RAG1 post-transcriptionally control gene expression in response to and independent of RAG DSBs. We seek to elucidate mechanisms by which the RAG1 N-terminus post-transcriptionally stimulates Pim2 expression in response to RAG DSBs, and determine the extent to which this part of RAG1 controls gene expression downstream and independent of RAG DSBs. This work will illuminate a critical physiological function of RAG1 that transcends its canonical roles in forming and regulating the RAG endonuclease. The knowledge acquired from this work will prompt new lines of research on RAG biology, V(D)J recombination, and lymphocyte development and function. This research should yield novel insights into pathological mechanisms that cause immunodeficiency, autoimmunity, and lymphoid cancers. The culmination of work sparked from this exploratory project could be improved therapies for human immunological disorders.

Elucidate Mechanisms and Functions of Tissue-, Lineage-, and Developmental-Stage Specific DNA Damage Responses of Immature Lymphocytes
Mammalian cells are thought to protect themselves and their host organisms from DSBs via universal mechanisms that restrain cellular proliferation until DNA is repaired. The Cyclin D3 protein drives G1-to-S cell cycle progression and is required for rapid proliferation and expansion of immature T and B cells following RAG-dependent assembly of functional Tcrb or Igh genes, respectively. We have discovered that DSBs repress transcription of Rag1 and Rag2 in pre-B cells, pro-B cells, and pro-T cells, but not in pre-T cells, indicating that immature T cells engage developmental stage-specific mechanisms to down-regulate RAG expression in response to DSBs. We also have found that DSBs down-regulate Cyclin D3 protein levels in immature, but not mature, B and T cells. Each of these responses requires the ATM kinase. Cyclin D3 protein loss in immature T cells coincides with decreased association of Cyclin D3 mRNA with the HuR RNA binding protein that ATM regulates. HuR inactivation reduces basal Cyclin D3 protein levels without affecting Cyclin D3 mRNA levels, indicating that immature T cells repress Cyclin D3 expression through ATM-dependent inhibition of Cyclin D3 mRNA translation. In contrast, ATM-dependent transcriptional repression of the Cyclin D3 gene inhibits Cyclin D3 protein levels in pre-B cells. Retrovirus-driven Cyclin D3 expression is resistant to transcriptional repression by DSBs; this prevents pre-B cells from reducing Cyclin D3 protein levels and from inhibiting DNA synthesis to the normal extent following DSBs. Our data indicate that immature B and T lymphocytes use lymphocyte lineage- and developmental stage-specific mechanisms to inhibit RAG and Cyclin D3 protein expression and thereby help suppress proliferation of cells with RAG DSBs. We seek to elucidate the mechanisms and roles of these cellular context-dependent DSB response mechanisms in restraining proliferation, maintaining genomic integrity, and suppressing malignant transformation of lymphocytes.

Current Lab Members:
Dr. Rahul Arya – CHOP Research Associate
Katharina Hayer – CHOP Computational Biologist
Morgann Reed – CHOP Research Technician
Glendon Wu – Penn IGG Student

Former Lab Trainees:
Dr. Lori Ehrlich – Clinical Fellow		Julie Horowitz – Penn IGG Student
Dr. Angella Fusello – Postdoctoral Fellow		Marta Rowh – Penn IGG Student
Brenna Brady - Penn IGG Student		Levi Rupp – Penn CAMB/GTV Student
Andrea Carpenter – Penn IGG Student		Velibor Savic – Penn CAMB/GTV Student
Amy DeMicco – Penn CAMB/CB Student		Natalie Steinel – Penn IGG Student
Megan Fisher – Penn IGG Student		Bu Yin – Penn CAMB/CB Student

Education

BA (Biology), The Johns Hopkins University, 1992
PhD (Biology), Duke University, 1997

Specialty Certification

Postgraduate Training

Research Fellow, Genetics, Harvard Medical School, 1997-2002
Research Fellow, Molecular Immunology, Irvington Institute for Immunological Research, 1997-2000
Research Fellow, Medicine, Boston Children's Hospital, 1997-2002
Research Fellow, Molecular Immunology, CBR Institute for Biomedical Research, 1997-2000
Research Associate, Molecular Immunology, Howard Hughes Medical Institute, 2000-03
Research Fellow, Cancer Genetics, Lymphoma Research Foundation, 2003-05

Awards and Honors

Alpha Epsilon Delta Honor Society, The Johns Hopkins University Chapter, 1990
Summer Fellow, Howard Hughes Medical Institute, 1991
Phi Beta Kappa Honor Society, The Johns Hopkins University Chapter, 1991
Golden Key Honor Society, The Johns Hopkins University Chapter, 1992
Pre-Doctoral Fellow Award, National Science Foundation, 1992-95
Departmental Honors (Biology), The Johns Hopkins University, 1992
University Honors, The Johns Hopkins University, 1992
Fellow Award, Irvington Institute for Immunological Research, 1997-2000
Fellow Award, Lymphoma Research Foundation, 2003-05
Foerderer-Murray Award, 2005-06
Pew Scholar in the Biomedical Sciences, 2005-09
American Cancer Society Institutional Research Award, 2005-08
W.W. Smith Charitable Trust Award, 2005-07
Lassin Family Cancer Research Award at the Abramson Cancer Center of the University of Pennsylvania School of Medicine, 2008-09
Michael S. Brown New Investigator Research Award, University of Pennsylvania School of Medicine, 2009
Pathology and Laboratory Medicine Chair Award of Children's Hospital of Philadelphia, 2010
Leukemia and Lymphoma Society Scholar Award, 2010-15
Simon Flexner Award (for teaching and mentoring), Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, 2017

Memberships and Professional Organizations

American Association for the Advancement of Science, 1992-present

Immunology Graduate Group (IGG), University of Pennsylvania, 2004-present
--- IGG Admissions Committee, 2005-14
--- IGG Student Affairs Committee: Member, 2005-08; Ad Hoc Member, 2009-13
--- IGG Curriculum Committee, 2010-14
--- IGG Executive Committee, 2010-14
--- IGG Curriculum Review Panel, 2011

Cell and Molecular Biology (CAMB) Graduate Group, University of Pennsylvania, 2004-present
--- CAMB Events Committee, 2005-06
--- Cancer Biology Program: Curriculum Committee, 2007-08; Executive Committee, 2008; Chair of Executive Committee, 2014-18; Preliminary Exams Committee, 2009; Advising Committee 2009-14 (Chair, 2012-14); Program Chair, 2014-18
--- Executive Committee, Cell and Molecular Biology Training Grant, 2012-present
--- CAMB Executive Committee, 2014-present
--- CAMB Curriculum Committee, 2018-present
--- Vice Chair, 2018-present

Abramson Cancer Center, Penn Medicine, 2005-present
--- Tumor Biology, 2005-10
--- Review Committee for American Cancer Society Pilot & Institutional Grants, 2009-17
--- Pediatric Oncology Research, 2010-present

Abramson Family Cancer Research Institute, 2005-present
--- Assistant Investigator, 2005-11
--- Associate Investigator, 2011-present

American Association of Immunologists, 2005-present

Cerimon Pharmaceuticals, Inc., Scientific Advisory Board, 2006-08

Irvington Institute for Immunological Research, Fundraising and Selection Committee for the

Frederick W. Alt Award, 2006

National Cancer Institute, Avon-NCI Progress for Patients Grants Program Reviewer, 2006

CHOP Institutional Animal Care and Use Committee, 2007-16 (Chair, 2011-13)

CHOP Institutional Core Advisory Committee, 2007-16

American Association for Cancer Research, 2008-present

CHOP Rodent Users Committee, Chair, 2008

UK Cancer Research Project, Project Program Reviewer, 2008

Wellcome Trust, Reviewer for the Research Career Development Fellowship Program, 2008

Children's Hospital of Philadelphia Research Institute, Center for Childhood Cancer Research, 2009-present (Biobank Scientific Review Council, 2012-present)

National Institutes of Health:
--- Cellular and Molecular Immunology B Study Section, Section of Scientific Review (Ad Hoc Reviewer, 2009, 2016; Standing Member, 2018-present)
--- Special Emphasis Panel/Scientific Review Group 2009/10 ZRG1 OBT-A (58) R Meeting, 2009
--- Study Section ZRG1 IMM-D (02) M - Member Conflict: Myeloid cell Development and Immunoglobulin Repertoire, 2010
--- National Institute of Arthritis and Musculoskeletal and Skin Diseases, Division of Intramural Research, Board of Scientific Counselors, 2014, 2021
--- Special Emphasis Panel/Scientific Review Group 2014/05 ZRG1 IMM-M (80) S Meeting, 2014

New York Academy of Sciences, 2009-present

Austrian Academy of Sciences, Austrian Programme for Advanced Research and Technology, Reviewer of Postdoctoral Fellowships, 2010

The Henry Kunkel Society, 2010-12

Institute for Immunology, Perelman School of Medicine at the University of Pennsylvania, 2010-present (Pilot Grant Program Review Panel, 2010, 2014)

Israel Science Foundation Grant Program Reviewer, 2010

Michael S. Brown New Investigator Research Award Selection Committee, Perelman School of Medicine at the University of Pennsylvania, 2010-15

H1 Connect (fka Faculty Opinions, Faculty of 1000), Immunology Experts, Leukocyte Development Section, 2012-present

Penn Biomedical Advisory Committee, 2014-present

Radiation Safety Committee of the University of Pennsylvania, 2014-present

Biomedical Graduate Studies (BGS) at the University of Pennsylvania, Curriculum Committee, 2018-present

CHOP Department of Veterinary Resources, Vice Chair of Faculty Advisory Committee, 2022-24

Web Links

Selected Publications