Faculty

Craig H. Bassing, Ph.D.

faculty photo
Associate Professor of Pathology and Laboratory Medicine
Department: Pathology and Laboratory Medicine
Graduate Group Affiliations

Contact information
Children's Hospital of Philadelphia
4054 Colket Translational Research Building
3501 Civic Center Blvd.
Philadelphia, PA 19104
Office: 267-426-0311
Fax: 267-426-2791
Education:
B.A. (Biology)
The Johns Hopkins University, 1992.
Ph.D. (Biology)
Duke University, 1997.
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Description of Research Expertise

Research Interests: Elucidate genetic, epigenetic, and signaling mechanisms that govern the development and function of lymphocytes, thereby establishing effective immunity while suppressing autoimmunity and genomic aberrations that cause leukemia or lymphoma.

Key Words: genome topology, transcriptional control, chromatin biology, genomic stability, lymphocyte antigen receptor gene diversification, lymphocyte development and selection, DNA damage response signaling, immunodeficiency, autoimmunity, lymphoid cancers

Research Overview: Our laboratory studies genetic, epigenetic, and signaling mechanisms that orchestrate gene expression and recombination programs in lymphocytes. We seek to elucidate on molecular levels how developing B and T cells alter their genomes to create the antigen receptor gene diversity necessary to recognize a veritable universe of external pathogens and internal damage. We also aim to determine ubiquitous and cell type-specific signaling mechanisms by which immature B and T cells manage and exploit DNA breakage, respectively, to suppress oncogenic translocations and direct gene expression changes that govern the selection of lymphocytes based on antigen receptor specificity. Insights from our studies will guide novel strategies to establish targeted genetic, epigenetic, or biochemical therapeutics for lymphoid malignancies, immunodeficiencies, auto-immunities, and chronic inflammatory diseases.

Current Projects:

1. Lymphocyte Lineage- and Developmental Stage-Specific Regulation of Gene Expression and Recombination. Proper gene expression and recombination relies on dynamic interplay among cis-acting genomic elements, chromatin domains, and three-dimensional genome architecture. The latter is of intense interest as ~10% of human diseases may arise from defects in chromosome topology that impact chromatin domains and gene transcription. We have approached mechanistic relationships between genome topology and gene regulation by focusing on the T cell receptor beta (Tcrb) antigen receptor locus for several reasons: (i) it is a physiological model of manageable complexity (ii) its architecture and transcription are dynamically regulated throughout T cell development, (iii) it divides into alternating active and repressive chromatin domains, (iv) changes in topology and transcription are critical for Tcrb assembly through long-range recombination, and (v) it has one enhancer that communicates with many promoters to regulate all aspects of Tcrb gene expression and assembly. We are testing our hypothesis that developmental switches between inactive and active Tcrb conformations are orchestrated by two independent mechanisms: 1) the transcription status of gene segments nucleating homotypic chromatin interactions that drive large-scale locus compaction, and 2) CTCF/Cohesin protein-mediated chromosome looping that focusses contacts within compacted loci. To test our hypothesis and elucidate mechanisms that govern Tcrb expression and recombination, we manipulate cis-elements and/or trans-factors and monitor multiple physiological readouts - genome topology, gene transcription, chromatin structure, V(D)J recombination, DNA repair, Tcrb gene repertoire, and T cell development and function. This line of work is yielding unprecedented molecular insights into relationships among genome architecture, chromatin biology, gene expression, and recombination, as well as how these mechanisms or their dysfunction impact normal or abnormal T cell biology, respectively.

2. Regulation of Mono-Allelic Antigen Receptor Gene Expression and Recombination. The mono-allelic expression of genes is essential for normal biology, preventing disease through X chromosome inactivation and genetic imprinting in all cells and permitting recognition of specific odors or antigens through allelic exclusion of receptor genes in olfactory neurons or lymphocytes, respectively. While common epigenetic mechanisms appear to direct these processes by silencing transcription on an allele, additional mechanisms might control antigen receptor allelic exclusion because this process also involves mono-allelic DNA recombination. Despite discovery of antigen receptor allelic exclusion in 1965, elucidating mechanisms that orchestrate this process and determining reasons for this gene expression pattern remain elusive. We have approached these issues from the perspectives that: i) mechanisms that direct RAG endonuclease activity and cellular responses to RAG DSBs might cooperate with ubiquitous epigenetic mechanisms to enforce allelic exclusion, and ii) suppression of oncogenic lesions through mono-allelic induction of RAG DSBs might be as essential as facilitating immunity through mono-specific antigen specificity of lymphocytes. Indeed, we have demonstrated that intrinsic poor-quality of RAG-targeting DNA elements enforce Tcrb allelic exclusion by stochastically limiting the bi-allelic assembly of Tcrb genes before signals from resulting proteins engage epigenetic mechanisms to permanently silence further recombination. Moreover, we have shown that RAG DSBs induced on one allele signal through the ATM protein kinase to transiently block recombination on the other allele, providing additional time for selection of protein produced from the first allele. This DSB-induced feedback inhibition of V(D)J recombination correlates with transcriptional repression of RAG expression and suppression of Ig and TCR translocations and resulting lymphomas. We seek to elucidate precise molecular mechanisms by which poor-quality RAG-targeting sequences and DSB-induced feedback inhibition govern allelic exclusion. Our work has provided us the unique ability to create mice that recombine and/or express specific Ig/TCR genes from both alleles and monitor multiple physiological readouts – lymphocyte selection by antigen specificity, Ig/TCR translocations, efficiency of immune responses, autoimmunity, and lymphoid malignancies. This work is producing unexpected insights into cooperation among genetic, epigenetic, and DSB signaling mechanisms to enforce allelic exclusion and reasons for this mono-allelic control of antigen receptor gene recombination and expression.

3. RAG1 Ubiquitin Ligase Regulation of Antigen Receptor Gene Assembly and Selection. Protein ubiquitylation serves fundamental roles in most aspects of biology including control of gene transcription, chromatin, DNA repair, and intra-cellular signaling. The RAG1 protein has a ubiquitin ligase domain that is dispensable for RAG endonuclease directed cleavage, recombination, and repair of DNA. RAG1 mutations in humans that inactivate this ubiquitin ligase cause immunodeficiency and autoimmunity through undetermined means. We have discovered that inactivation of RAG1 ubiquitin ligase activity in mice lowers the efficiency of antigen receptor gene assembly and impairs both negative selection of highly self-reactive lymphocytes and the ability of RAG cleavage to signal changes in expression of proteins that control antigen receptor signaling and antigen presentation. We hypothesize that the RAG1 ubiquitin ligase modulates chromatin to promote RAG cleavage of Ig/TCR loci and amplifies signals from RAG DSBs to fully activate a genetic program that regulates antigen receptor selection. We are employing genetic approaches to isolate each of these steps of lymphocyte development to elucidate molecular mechanisms by which the RAG1 ubiquitin ligase promotes antigen receptor gene assembly and proper selection of protein products of these genes.

Current Lab Members:
Becca Glynn – Penn CAMB/CB PhD Student
Brittney Allyn - Penn IGG PhD Student
Katharina Hayer – CHOP Bioinformatics Scientist II & Drexel Computational PhD Student
Erica Culberson - CHOP Research Technician
Chao Di - CHOP Bioinformatics Scientist III


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

Selected Publications

Beilinson, H.A., Glynn, R.A., Yadavailli, A.D., Xiao, J., Corbett, E., Saribasak, H., Arya, R., Miot, C., Bhattacharyya, A., Jones, J.M., Pongubala, J., Bassing, C.H., and Schatz, D.G. : The RAG1 N-terminal region regulates the efficiency and pathways of synapsis for V(D)J recombination. Journal of Experimental Medicine. Oct 4;218(10):e20210250. doi: 10.1084/jem.20210250. (eds.). 218(10): :e20210250, Oct 2021.

Dauphers, D.J., Wu, G.S., Bassing, C.H., and Krangel, M.S.: Molecular Analysis of Mouse T Cell Receptor α and β Gene Rearrangements Methods in Molecular Biology In Press, 2021.

Wu, G.S. and Bassing, C.H.: Inefficient V(D)J Recombination Underlies Monogenic T Cell Receptor β Expression. Proceedings of the National Academy of Sciences, USA 117: 18172-18174, 2020.

Collins, P., Purman, C., Porter, S., Nganga, V., Siani, A., Hayer, K., Gurewitz, G., Sleckman, B.P., Bednarski, J., Bassing, C.H., Oltz, E.M.: DNA Double-Strand Breaks Induce H2Ax Phosphorylation Domains in a Contact-Dependent Manner. Nature Communications 11: 3158, 2020.

Wu, G. S., Yang-Iott, K. S., Klink, M.A., Hayer, K.E., Lee, K.D., and Bassing, C.H.: Poor Quality Vb Recombination Signal Sequences Stochastically Enforce TCRb Allelic Exclusion. Journal of Experimental Medicine 217: e20200412, 2020.

Allyn, B.M., Lee, K.D., and Bassing, C.H.: Genome Topology Control of Antigen Receptor Gene Assembly. Journal of Immunology 204: 2617-2626, 2020.

Lee, K.D., and Bassing, C.H.: Two Successive Inversional Vb Rearrangements on a Single Tcrb Allele can Contribute to the TCRb Repertoire. Journal of Immunology 204: 78-86, 2020.

Burn, Thomas, N., Lee, Kyutae, D., Dawany, N., Robertson, Tanner, F., Fisher, Megan, R., Bassing, Craig H., and Behrens, Edward M. : A Spontaneous Rag1 Nonsense Mutations Unveils Naturally Occurring N-truncated RAG1 Isoforms ImmunoHorizons 4: 119-128, 2020.

Glynn, R.A. and Bassing, C.H. : From RAG2 to T Cell Riches and Future Fortunes Journal of Immunology 202: 1315-1316, 2019.

Wu, G.S. and Bassing, C.H.: BTG2-PRMT1 protein complexes antagonize pre-B cell proliferation to promote B cell development. Cellular and Molecular Immunology 15: 808-811, 2018.

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Last updated: 09/16/2021
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