The focus of the lab is to understand regulation of transcription and co-transcriptional processing in human cells using a combination of molecular cell biology techniques, and high-throughput sequencing.
Transcriptional integrity is critical for all cellular functions, as mRNA transcripts are the basis for all protein products. RNA polymerase II (RNAPII) is responsible for transcription of the vast majority of protein-coding genes as well as a number of non-coding RNAs. Correct co-transcriptional processing of the nascent RNA transcript is dependent on the C-terminal domain (CTD) of RNAPII. In humans, the CTD consists of 52 heptapeptide repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. The CTD is dynamically phosphorylated and serves as a binding platform of RNAPII-associated factors with certain phosphorylation states enriched in the beginning of the gene and others around termination sites. RNA-binding proteins are a key group of proteins responsible for co-transcriptional processing. Many of them recognize specific phosphorylation states of the RNAPII CTD and associate with the transcription machinery at certain stages during the transcription cycle to dictate correct maturation of nascent RNA transcripts.
We aim to understand fundamental biological mechanisms underlying transcriptional regulation and processing of nascent RNA in mammalian cells and how misregulation of these processes contribute to human disease such as cancer. We are especially interested in understanding how proteins interacting with the CTD of RNAPII impact co-transcriptional RNA processing events and how these processes are regulated.
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Regulation of transition between transcription elongation and termination
Whereas much work in past years has focused on transcriptional activation and initiation, it is becoming clear that post-initiation events are equally crucial for transcriptional integrity. In particular, the transition from elongation to termination has emerged as a significant point of regulation. Transcriptional termination ensures correct maturation of the final RNA transcript, but what is less appreciated is that regulation of transcriptional termination also influences expression of different isoforms with different end-sites. Transcription termination consists of two coupled events: co-transcriptional transcript cleavage and polyadenylation of the pre-mRNA, followed by RNAPII disassociation (i.e. transcriptional termination). We use a combination of molecular biology techniques (including generation of CRISPR modified human cell lines) together with custom next-generation sequencing techniques including RNA-seq, TTchem-seq, mNET-seq, ChIP-seq, CLIP etc. to study mRNA transcript cleavage and transcription termination in human knockout cell lines or in response to stress (see Gregersen et al. 2019 Cell and Gregersen et al. 2020 Nature Protocols).
Molecular mechanism of RNAPII CTD-interacting proteins
Our previous work has uncovered a role for two Serine/Arginine (SR)-related and CTD-associated factors (SCAFs) in suppression of premature mRNA transcript cleavage (Gregersen et al. 2019 Cell). Both proteins couple RNAPII CTD binding and their RNA-binding activity to prevent early mRNA transcript cleavage and ensure the production of full-length mRNA transcripts and proteins. Our current research is focused on elucidating the detailed molecular mechanisms of SCAF proteins as well as other RNAPII CTD associated factors with unknown roles in transcription regulation using a combination of cell biology, molecular biology, and systems biology.
- Pappas G, Munk SHN, Watanabe K, Thomas Q, Gál Z, Gram HH, Lee M, Gómez-Cabello D, Kanellis DC, Olivares-Chauvet P, Larsen DH, Gregersen LH, Maya-Mendoza A, Galanos P, Bartek J. MDC1 maintains active elongation complexes of RNA polymerase II. Cell Rep 2023 Jan, 42(1):111979
- Gregersen LH, Mitter R and Svejstrup JQ. Elongation factor-specific capture of RNA polymerase II complexes. Cell Rep Methods 2022 Dec, 2(12):100368
- Gregersen LH, Mitter R, Svejstrup JQ. Using TTchem-seq for profiling nascent transcription and measuring transcript elongation. Nat Protoc. 2020 Feb;15(2):604-627.
- Zatreanu D, Han Z, Mitter R, Tumini E, Williams H, Gregersen LH, Dirac-Svejstrup AB, Roma S, Stewart A, Aguilera A, Svejstrup JQ. Elongation Factor TFIIS Prevents Transcription Stress and R-Loop Accumulation to Maintain Genome Stability. Mol Cell. 2019 March 3, 76(1), 57-69
- Gregersen LH, Mitter R, Ugalde AP, Nojima T, Proudfoot NJ, Agami R, Stewart A, Svejstrup JQ. SCAF4 and SCAF8, mRNA Anti-Terminator Proteins. Cell. 2019 Jun 13;177(7):1797-1813.e18.
- Gregersen LH, Svejstrup JQ. The Cellular Response to Transcription-Blocking DNA Damage. Trends Biochem Sci. 2018 May;43(5):327-341.
- Gregersen LH, Schueler M, Munschauer M, Mastrobuoni G, Chen W, Kempa S, Dieterich C, Landthaler M. MOV10 Is a 5' to 3' RNA helicase contributing to UPF1 mRNA target degradation by translocation along 3' UTRs. Mol Cell. 2014 May 22;54(4):573-85.
We always welcome applications from motivated postdoctoral and predoctoral researchers as well as master students. Please contact us at firstname.lastname@example.org
Lea H. Gregersen