My long-term goal is to become an independent investigator at a major American research institution, developing cutting-edge research at the interface between immunology and systems biology. To achieve this goal my immediate objectives are: (1) to produce innovative, high quality research that will result in high impact publications that are independent of my mentor?s lines of research, (2) to acquire a new set of skills in bioinformatics, complex data analyses, immunology, and genome editing approaches to complement my existing background. Obtaining the prestigious NIH pathway to independence award would bolster my career opportunities and provide me with the means to develop and mature my own research program, plus acquire the additional training I need to succeed as an independent researcher. The proposed research encompasses my major scientific interests: integrating immune regulation with systems biology approaches to study gene expression. Cytokines are cell-to-cell signaling proteins that play fundamental roles in delineating immune responses. Cytokine production is highly regulated at the transcriptional level, and failure to express them appropriately often results in several pathologies such as autoimmune diseases, cancer and susceptibility to infections. After more than three decades of research, many aspects of cytokine expression are still not well understood, and transcriptional regulation of cytokine gene expression has not been explored on a systems level. My research will delineate the first gene-centered cytokine gene regulatory network (GRN) by comprehensively identifying the transcription factors (TFs) that regulate cytokine genes. To do so, I will first identify the TFs that can bind to the promoters and other known regulatory regions for all human cytokines by directly testing for protein-DNA interactions (PDIs) using high-throughput yeast one-hybrid (eY1H) assays, recently developed in the Walhout lab. I will model the PDIs detected into a GRN and will integrate this GRN with publicly available expression data to define which TFs positively or negatively regulate cytokine expression. These analyses, together with functional validation assays in knockout cell lines, will identify novel cytokine regulators linking them to their functional role and relevance in disease. Noncoding mutations are at the core of many human genetic diseases, and constitute 90% of all mutations identified in genome-wide association studies. For instance, mutations in cytokine regulatory regions have been associated with cancer, higher susceptibility to infections, chronic arthritis and systemic lupus erythematosus. These mutations can result in loss or gain of transcription factor binding sites. Assays to test for PDI changes are labor intensive and limited to a few dozen TFs. Thus, I propose to use a novel eY1H pipeline to systematically interrogate for PDI changes that result as a consequence of disease-associated mutations in cytokine regulatory regions. Findings from this experimental approach will be further validated in immune cell lines and will provide mechanistic insights and therapeutic targets for many immune related diseases. The cross-disciplinary program I propose requires that I obtain additional training in multiple areas of research that will complement my background, and will provide me with state-of-the-art tools for my future research as an independent investigator. I have identified Dr. Walhout and Dr. Fitzgerald as ideal mentors to carry out my proposed work, together with Dr. Wolfe and Dr. Myers as collaborators, with highly complementary expertise. Working in collaboration with these world-renowned researchers in a stimulating environment such as that of UMMS, together with the training activities my mentoring committee and I have planned for the mentored phase of the award, will enable me to: (1) become proficient in state-of-the-art immunology techniques and learn up-to-date immunology concepts, (2) acquire bioinformatics and complex data analysis skills, and (3) learn genome editing approaches, all necessary for the interpretation and validation of the proposed work. This scientific expertise will be complemented with career development seminars and activities to acquire the mentoring, leadership, grantsmanship and writing skills I will need to succeed as an independent investigator. Securing this award would place me in an ideal position to capitalize on my experience to date in the Walhout laboratory, while allowing me to define my own research niche. This award will lead to a better understanding of how immune genes are regulated in normal and pathologic conditions, which will have a broad impact on public health.
Cytokines are signaling proteins that play fundamental roles in delineating immune responses to infections. Cytokine expression is a highly regulated process that, when affected, can lead to diseases such as immune deficiencies, autoimmunity and cancer. This project will determine how the expression of cytokine genes is regulated and will identify potential therapeutic targets for multiple immune related diseases. Further, it will provide mechanistic insights into how mutations in regulatory regions result in altered gene expression leading to disease.
| Sewell, Jared A; Fuxman Bass, Juan I (2018) Options and Considerations When Using a Yeast One-Hybrid System. Methods Mol Biol 1794:119-130 |
| Gan, Kok A; Carrasco Pro, Sebastian; Sewell, Jared A et al. (2018) Identification of Single Nucleotide Non-coding Driver Mutations in Cancer. Front Genet 9:16 |
| Carrasco Pro, Sebastian; Dafonte Imedio, Alvaro; Santoso, Clarissa Stephanie et al. (2018) Global landscape of mouse and human cytokine transcriptional regulation. Nucleic Acids Res 46:9321-9337 |
| Sewell, Jared A; Fuxman Bass, Juan I (2017) Cellular network perturbations by disease-associated variants. Curr Opin Syst Biol 3:60-66 |
