MYC-dependent co-ordination of RNA polymerase activities during ribosome biogenesis and cell growth
Final Report Abstract
Cellular growth is a fundamental process of life, which is determined by the synthesis of ribosomes. Ribosomal biogenesis requires the coordinated activity of all three RNA polymerases. Myc – one of the most potent cellular proto-oncoproteins – is a key regulator of growth. As a promiscuous transcription factor, binding to the majority of all genes, Myc represents an attractive candidate to understand how induction of cellular growth is coordinated. We used advanced genomic and proteomic methods to investigate how Myc regulates the transcription of growth genes. As originally proposed, the work was divided into four sub-projects and the following results have been achieved during the project: A) The dominating physiological function of Myc is the induction of cellular growth. In contrast, oncogenic Myc does not induce an increase in cellular mass. The aim of this project was to understand why Myc induces cellular growth in untransformed but not in cancer cells. We hypothesized that Myc regulates distinct sets of genes at low physiological levels as compared to the oncogenic levels found in cancer cells. To test this model, we manipulated Myc levels in subtle steps over two orders of magnitude by both overexpression and depletion. We then analyzed the genome-wide binding of Myc to chromatin by ChIP–sequencing. Strikingly, occupancy of Myc did not uniformly change at all promoters. Instead, Myc binding remained constant at promoters that were already strongly occupied at physiological Myc levels upon overexpression, while Myc was strongly recruited to genes that were weakly bound at low levels of Myc. This observation prompted us to estimate affinities for all Myc-bound promoters. Genes encoding proteins involved in growth showed the highest affinity for Myc, while genes involved in signal transduction and migration had low affinity for Myc. By combining Myc-binding with gene expression data, we concluded that induction of growth genes takes place already at low physiological Myc concentrations, while the oncogenic effects of Myc result from regulation of distinct genes with low-affinity promoters. B) Growth genes with high affinity promoters consist of two classes of genes, ribosomal biogenesis genes (RiBi) and ribosomal protein genes (Ribo). Unexpectedly, the Myc binding sites differ between these two types: While the majority of RiBi genes contain E-boxes, most Ribo genes do not. The aim of the second project was to characterize the different Myc binding complexes at these two classes of genes. The central goal was to set up a genetic screen to identify novel factors involved in Myc binding. We have now successfully performed the screen and identified several new regulators of ribosome biogenesis. One interesting example is the glycolytic enzyme AldoA. In agreement with published data, we found a fraction of AldoA localized in the nucleus and demonstrated that it regulates the transcription of endogenous genes important for ribosome biogenesis and cell growth (Schwarz et al, in preparation). C) Induction of growth gene transcription is a core function of Myc. The precise nature and the molecular mechanisms underlying Myc-mediated transcription activation remained elusive despite extensive research. In this project we analyzed the mechanistic details of how Myc activates RNA polymerase II (RNAPII). Using mass spectrometry, we showed that Myc controls the assembly of RNAPII with a set of transcription elongation factors, including SPT5 and SPT6. The Myc-mediated transfer of both proteins is required for fast and processive transcription elongation. Altogether, these results argue that Myc controls the productive assembly of processive RNAPII elongation complexes and provide insight into how Myc permits uncontrolled cellular growth. D) Based on the observation that Myc also increases the translation of its target genes, and in agreement with our novel observations that Myc is a key regulator of transcription elongation, we hypothesize that Myc regulates the fate of transcripts in addition to changing its abundance. We have established all required methods to study splicing kinetics and will continue exploring the role of Myc in this context in the future. So far, the results of project A and D have been published in 7 publications with me as a corresponding author, amongst others in Nature Chemical Biology, eLife, and Molecular Cell (2x). Emmy-Noether-funded members of my lab contributed as co-authors to ten more publications, amongst others in Nature.
Publications
- Different promoter affinities account for specificity in MYC-dependent gene regulation, eLife, 2016, 5
Lorenzin F, Benary U, Baluapuri A, Walz S, Jung LA, von Eyß B, Kisker C, Wolf J, Eilers M, Wolf E
(See online at https://doi.org/10.7554/elife.15161) - The ubiquitin ligase Huwe1 regulates the maintenance and lymphoid commitment of hematopoietic stem cells, Nature Immunology, 2016, 17, 1312-1321
King B, Boccalatte F, Moran-Crusio K, Wolf E, Wang J, Kayembe C, Lazaris C, Yu X, Aranda-Orgilles B, Lasorella A, Aifantis I
(See online at https://doi.org/10.1038/ni.3559) - PAF1 complex component Leo1 helps recruit Drosophila MYC to promoters, PNAS, 2017, 114, 9224-9232
Gerlach JM, Furrer M, Gallant M, Birkel D, Baluapuri A, Wolf E, Gallant P
(See online at https://doi.org/10.1073/pnas.1705816114) - A MYC–GCN2–eIF2α negative feedback loop limits protein synthesis to prevent MYC-dependent apoptosis in colorectal cancer, Nature Cell Biology, 2019, 21, 1413-1424
Schmidt S, Gay D, Uthe FW, Denk S, Paauwe M, Matthes N, Diefenbacher ME, Bryson S, Warrander FC, Erhard F, Ade CP, Baluapuri A, Walz S, Jackstadt R, Ford C, Vlachogiannis G, Valeri N, Otto C, Schülein-Völk C, Maurus K, Schmitz W, Knight JRP, Wolf E, Strathdee D, Schulze A, Germer CT, Rosenwald A, Sansom OJ, Eilers M, Wiegering A
(See online at https://doi.org/10.1038/s41556-019-0408-0) - MYC recruits SPT5 to RNA polymerase II to promote processive transcription elongation, Molecular Cell, 2019, 74, 674-687
Baluapuri A, Hofstetter J, Dudvarski N, Endres T, Bhandare P, Vos SM, Adhikari B Schwarz JD, Narain A, Vogt M, Wang SJ, Düster R, Jung LA, Vanselow JT, Wiegering A, Geyer M, Maric HM, Gallant P, Walz S, Schlosser A, Cramer P, Eilers M, Wolf E
(See online at https://doi.org/10.1016/j.molcel.2019.02.031) - Recruitment of BRCA1 limits MYCN-driven accumulation of stalled RNA polymerase, Nature, 2019, 567,545-549
Herold S, Kalb J, Büchel G, Ade CP, Baluapuri A, Xu J, Koster J, Solvie D, Carstensen A, Klotz C, Rodewald S, Schülein-Völk C, Dobbelstein M, Wolf E, Molenaar J, Versteeg R, Walz S, Eilers M
(See online at https://doi.org/10.1038/s41586-019-1030-9) - PROTAC-mediated degradation reveals a non-catalytic function of AURORA-A kinase, Nature Chemical Biology, 2020, 5, 1390-1402
Adhikari B, Bozilovic J, Diebold M, Schwarz JD, Hofstetter J, Schröder M, Wanior M, Narain A, Vogt M, Dudvarski Stankovic N, Baluapuri A, Schönemann L, Eing L, Bhandare P, Kuster B, Schlosser A, Heinzlmeir S, Sotriffer C, Knapp S Wolf E
(See online at https://doi.org/10.1038/s41589-020-00652-y) - Design, Synthesis, and Evaluation of WD-Repeat-Containing Protein 5 (WDR5) Degraders, Journal of Medicinal Chemistry, 2021, 64, 10682-10710
Dölle A, Adhikari B, Krämer A, Weckesser J, Berner N, Berger L-M, Diebold M, Szewczyk MM, Barsyte-Lovejoy D, Arrowsmith CH, Gebel J, Löhr F, Dötsch V, Eilers M, Heinzlmeir S, Kuster B, Sotriffer C, Wolf E, Knapp S
(See online at https://doi.org/10.1021/acs.jmedchem.1c00146) - Generation of AID knock-in cell lines for targeted protein degradation in mammalian cells, Star Protocols, 2021, 2
Adhikari B, Narain A, Wolf E
(See online at https://doi.org/10.1016/j.xpro.2021.100949) - Targeted protein degradation reveals a direct role of SPT6 in POL2 elongation and termination, Molecular Cell, 2021, 81
Narain A, Bhandare P, Adhikari B, Backes S, Eilers M, Dölken L, Schlosser A, Erhard F, Baluapuri A, Wolf E
(See online at https://doi.org/10.1016/j.molcel.2021.06.016)
