Thomas Gonatopoulos-Pournatzis, Ph.D.
Thomas Gonatopoulos-Pournatzis studies the regulatory pathways and functional roles of alternative splicing and other pre-mRNA processing events in mammalian cells. Towards this, he has developed several CRISPR-based screening platforms which are coupled to high-throughput phenotyping and enable systematic exploration of the regulatory and functional complexity of alternative splicing. Dr. Gonatopoulos-Pournatzis’ team combines these functional genomics tools with molecular and biochemical approaches as well as animal models to identify alternative exons and other genetic segments that underlie phenotypes related to normal physiology and disease states. The long-term goal of his research is to contribute to the functional annotation of all exons in the human genome and to identify novel targets for precision medicine applications.
1) alternative splicing, 2) RNA biology, 3) gene regulation, 4) genome engineering, 5) CRISPR-Cas, 6) functional genomics
Center for Cancer Research
National Cancer Institute
Building 560, Room 21-81A
Frederick, MD, 21702-1201
Advances in next-generation sequencing have revealed that cancer and neurological disorders are often associated with distinct transcriptomic signatures, including widespread alterations in alternative splicing, a process that vastly expands the coding potential of the genome. Loss- or gain-of-function mutations in splicing factors impact entire networks of coordinated alternative exons, which play critical roles in cell fate decisions and impact processes such as cell cycle control, neurogenesis and synaptic transmission. However, given that the functions of the overwhelming majority of alternative exons remain unexplored, Thomas Gonatopoulos-Pournatzis’ research program aims to identify individual events that control these fundamental biological processes, reveal how their disruption contributes to disease- or disorder-related phenotypes, and explore the regulatory networks that control these events.
1. Exon-resolution functional genomics
To advance the systematic mapping of genetic interactions and interrogation of the functions of sizeable genomic segments, we have developed a CRISPR-based screening system for combinatorial genetic perturbations, named CHyMErA. CHyMErA employs co-expression of Cas9 and Cas12a nucleases and libraries of hybrid Cas9-Cas12a guide RNAs that can be employed for multi-targeted genome editing. We have applied CHyMErA in human cells to uncover novel genetic interactions between paralog gene pairs. Importantly, we have also used CHyMErA to systematically target thousands of alternative splicing events, identifying dozens of exons underlying cell fitness. CHyMErA thus represents an effective screening approach for genetic interaction mapping and the functional analysis of sizeable genomic regions, such as alternative exons.
Functional Exomics: Future application of CHyMErA screens coupled to high-throughput phenotyping will enable the efficient functional characterization of alternative exons in the human genome and the identification of novel targets for precision medicine applications.
Technology Development: A major goal of the Gonatopoulos-Pournatzis team is to continue developing cutting-edge technologies that will enable the systematic and efficient functional characterization of transcript isoform diversity and genetic interactions.
2. Identification of regulatory networks that control alternative splicing
We previously developed a CRISPR-based screening platform that allows the systematic identification of regulatory factors that control alternative splicing. CRISPR-Cas9 loss-of-function genome wide screens in neural cells expressing dual-fluorescent splicing reporters uncovered hundreds of splicing regulators and have shed light on how very short neuronal exons are recognized and spliced. The aforementioned strategy represents a highly effective approach for the comprehensive definition of splicing regulatory pathways. Future studies using this tool will systematically delineate regulatory pathways that control the splicing of alternative exons with critical roles in mammalian cells.
3. Uncover microexons that impact animal behavior and cognitive functioning
A program of neuronal-specific, highly conserved microexons is frequently disrupted in the brains of patients with autism spectrum disorder (ASD). We have started to identify individual microexons that contribute to ASD-associated phenotypes by characterizing a set of neuronal microexons in the translation initiation factors eIF4G1 and eIF4G3. The eIF4G microexons selectively up-regulate synaptic proteins that control neuronal activity and plasticity, and mice lacking the Eif4g1 microexon display social behavior as well as learning and memory deficits. Future studies are focused on uncovering additional microexons with such critical roles for animal behavior and cognitive functioning.
The Gonatopoulos-Pournatzis group is looking for enthusiastic lab members who wish to explore the complexity of the mammalian transcriptome and we have several openings for Postdoctoral Fellows and Postbaccalaureate Fellows. If you are interested in our work, please send your C.V., a statement of research interests and the names and contact details of 2-3 references to firstname.lastname@example.org.
View Dr. Gonatopoulos-Pournatzis' Google Scholar page.
Selected Key Publications
Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform.Nat Biotechnol. Epub ahead of print (Mar. 16), 2020. [ Journal Article ]
Autism-Misregulated eIF4G Microexons Control Synaptic Translation and Higher Order Cognitive Functions.Mol Cell. 77(6): 1176-1192, 2020. [ Journal Article ]
Genome-wide CRISPR-Cas9 Interrogation of Splicing Networks Reveals a Mechanism for Recognition of Autism-Misregulated Neuronal Microexons.Mol Cell. 72(3): 510-524, 2018. [ Journal Article ]
- Current Opinion in Genetics & Development. 65: 22-33, 2020. [ Journal Article ]
- Mol Cell. 44(4): 585-96, 2011. [ Journal Article ]
Dr. Gonatopoulos-Pournatzis obtained his Bachelor’s degree in Biology from the National & Kapodistrian University of Athens, Greece in 2007 before moving to King’s College London in England to earn his Master’s degree in 2008. In 2013, Dr. Gonatopoulos-Pournatzis obtained his Ph.D. from the University of Dundee in Scotland where he worked in Prof. Victoria Cowling’s laboratory studying mechanisms underlying mRNA cap methylation and gene expression. For his postdoctoral research, he joined Prof. Ben Blencowe’s laboratory at the University of Toronto in Canada to study the regulation and function of splicing regulatory networks in neurons with the support of postdoctoral fellowships from the European Molecular Biology Organization (EMBO), Canadian Institutes of Health Research (CIHR) and Ontario Institute of Regenerative Medicine (OIRM). Dr. Gonatopoulos-Pournatzis has received multiple awards and recognitions, including the Baxter Prize (2012), the Donnelly Centre Research Excellence Award (2018) and his is also a recipient of the 2020 NIH Distinguished Scholar Program. In 2020, Dr. Gonatopoulos-Pournatzis joined the RNA Biology Laboratory at the National Cancer Institute to establish the Functional Transcriptomics Section, which integrates functional genomics and RNA biology. His lab focuses on developing and applying CRISPR tools to systematically uncover transcript variants that play critical roles in normal physiology and disease state.