Nascent RNA Bru-seq to explore regulation of gene expression
We have developed four new techniques based on bromouridine labeling of nascent RNA that allows us to estimate both the rate of transcription (Bru-seq) and the rate of degradation (BruChase-seq) of any mRNA or ncRNA. In addition, the use of UV light to introduce random transcription-blocking lesions and to suppress RNA exosome activity allow us to map transcription start sites and active enhancer elements genome-wide (BruUV-seq). Finally, BruDRB-seq allows for the assessment of transcription elongation rates genome-wide. We participate in the ENCODE IV as a mapping center to provide nascent RNA Bru-seq data across many human cell lines. We are currently collaborating with more than 50 labs in 10 countries to run and analyze various Bru-seq experiments and as part of the ENCODE Consortium we are engaged in many collaborations to elucidate the fundamental relationships between form (chromatin organization and epigenetics) and function (nascent RNA synthesis).
Therapeutic targeting of the RNA exosome in cancer
The RNA exosome plays a critical role in RNA quality control in the nucleus. Transcripts not completely or incorrectly spliced are degraded by the nuclear RNA exosome. This complex also plays roles in transcription elongation and the resolution of R-loops formed during transcription. In many cancers the splicing machinery is compromised by mutations or aberrant expression resulting in the generation of increased levels of aberrant transcripts. We hypothezise that cancer cells may be highly dependent on RNA exosome activity to remove this excess of aberrant transcripts and would therefore be uniquely sensitive to therapeutic intervention to inhibit its activity. We have together with the Neamati lab identified candidate first-in-class small molecule inhibitors of the RNA exosome and have for the first one shown that it is well tolerated and has tumor-specific targeting activity. We are designing high throughput screening assays to identify additional small molecule inhibitors to be used in pre-clinical studies of pancreatic cancer and in basic fundamental studies to explore the role of the RNA exosome in transcription elongation, splicing, termination and RNA turnover.
Mechanisms of DNA damage-induced gene expression
Exposure of cells to DNA-damaging agents is known to alter gene expression as part of a DNA damage response pathway. However, whether gene expression alterations occur at the level of transcription, splicing and/or RNA stability is not well understood. Using our new Bru-seq techniques we have found that cells modify their gene expression both at the level of synthesis and stability and we have been able to map a number of enhancer elements responding to the DNA damage insult. We are currently exploring the role of p53 in mRNA stabilization and enhancer element regulation and these studies may lead to the identification of new potential therapeutic targets for cancer treatment.
Mechanisms of gene expression alterations in cancer
A major component of the carcinogenesis process is the reprogramming of gene expression. Extensive studies have documented gene expression reprogramming in cancer cells but it cannot be determined from these studies whether such changes are due to alterations in the synthesis or degradation of mRNAs. We are currently exploring whether cancer cells have altered their gene expression of mRNAs and ncRNAs at the level of transcription and/or RNA stability. We are exploring splicing efficiencies and the regulation of alternative splicing. Furthermore, we are mapping the cancer-specific utilization of promoter and enhancer elements genome-wide. We are exploring the dynamic transcriptional and post-transcriptional regulation of gene expression following exposure of cells to TGFb to induce epithelial to mesenchymal transition (EMT) and following other cellular transitions such as reprogramming of fibroblasts into iPSCs.
Exploration of regulation of gene expression during the cell cycle
It is thought that the progression of cells through the cell cycle depends on timely regulation of gene expression. We are currently using our BrU-labeling techniques to explore gene expression alterations at the level of transcription and RNA stability as well as mapping the cell cycle-specific activity of enhancer elements and transcription elongation rates on a genome-wide scale. We are using a "baby machine" to specifically collect "newborn" cells in early G1 and we then age these cells into different phases of the cell cycle without any chemical perturbations.
Single cell nascent RNA Bru-seq
Most of our accumulated knowledge regarding mechanisms of transcription regulation has been obtained from cell population studies where average assessments of gene expression have been performed. We are developing techniques to assess nascent RNA synthesis in single cells for the first assessment of transcription rules and restrictions in a single cell.
- Frank, S.B., Berger, P.L., Ljungman, M. and Miranti, C.K. (2017) Human prostate luminal cell differentiation requires NOTCH3 induction by p38-MAPK and MYC. J Cell Sci, 130, 1952-1964.
- Galban, S., Apfelbaum, A.A., Espinoza, C., Heist, K., Haley, H., Bedi, K., Ljungman, M., Galban, C.J., Luker, G.D., Dort, M.V. et al. (2017) A Bifunctional MAPK/PI3K Antagonist for Inhibition of Tumor Growth and Metastasis. Mol Cancer Ther, 16, 2340-2350.
- Iannelli, F., Galbiati, A., Capozzo, I., Nguyen, Q., Magnuson, B., Michelini, F., D'Alessandro, G., Cabrini, M., Roncador, M., Francia, S. et al. (2017) A damaged genome's transcriptional landscape through multilayered expression profiling around in situ-mapped DNA double-strand breaks. Nature Comm, 8, 15656.
- Kirkconnell, K.S., Magnuson, B., Paulsen, M.T., Lu, B., Bedi, K. and Ljungman, M. (2017) Gene length as a biological timer to establish temporal transcriptional regulation. Cell Cycle, 16, 259-270.
- Venkata Narayanan, I., Paulsen, M.T., Bedi, K., Berg, N., Ljungman, E.A., Francia, S., Veloso, A., Magnuson, B., di Fagagna, F.D., Wilson, T.E. et al. (2017) Transcriptional and post-transcriptional regulation of the ionizing radiation response by ATM and p53. Sci Reports, 7, 43598.
- Zhang, H., Gan, H., Wang, Z., Lee, J.H., Zhou, H., Ordog, T., Wold, M.S., Ljungman, M. and Zhang, Z. (2017) RPA Interacts with HIRA and Regulates H3.3 Deposition at Gene Regulatory Elements in Mammalian Cells. Mol Cell, 65, 272-284.
- Ljungman, M., Parks, L., Hulbatte, R. and Bedi, K. ((in press)) The role of H3K79 methylation in transcription and the DNA damage response. Mutat Res
Please contact Mats Ljungman, PhD (firstname.lastname@example.org) if you are interested in working with the Ljungman Lab.