Myc-mediated reprogramming of RNA polymerases.
Growth is a fundamental process of life and its de-regulation is implicated in multiple human diseases. Cellular growth is determined by the content and synthesis of ribosomes. Ribosomal biogenesis requires the coordinated activity of all three nuclear RNA polymerases (RNAPI, -II, -III). The oncogene MYC is indispensable for development and maintenance of most human tumors. MYC´s evolutionary conserved physiological function is to promote cell growth. MYC binds to thousands of genes transcribed by all three RNAPs. The mechanisms through which MYC induces cellular growth remains unclear and four aspects will be analyzed in my group:
(I) MYC is a central regulator of cell growth and global regulator (“amplifier”) of transcription in primary cells, yet MYC-driven tumors are characterized by specific gene expression profiles that suggest a much more restricted and gene-specific role in transcription during oncogenesis. In the first set of experiments, I will develop and test a quantitative model of MYC binding to chromatin that aims to resolve this apparent discrepancy.
(II) Two chromatin-binding complexes of MYC are known: MYC/MAX complexes bind to Eboxes and their predominant targets are genes involved in ribosome biogenesis and translation (RiBi genes). Genes transcribed by RNAPIII are bound by MYC/TFIIIB complexes that do not require Max. Neither of these complexes is responsible for binding of MYC to the majority of ribosomal protein (RP) gene promoters and I aim to identify and characterize the missing complex of MYC.
(III) The existence of different chromatin-binding complexes suggests that MYC not only induces a general increase in ribosomal biogenesis, but also co-ordinates the synthesis of different ribosomal components. MYC has been suggested to act at the elongation step of transcription, but this remains to be directly demonstrated and I will use direct measurements of elongation to measure MYC function in a global manner. Our preliminary work has led to a model in which MYC catalyzes the transfer of elongation factors onto RNA polymerase to promote transcriptional elongation. I will test this model by purifying RNA polymerase holo-enzymes and analyze the effect of MYC on holoenzyme composition. Performing these experiments in response to an experimentally induced imbalance in the production of ribosome components (e.g. by selective blocking RNAPI transcription) will test whether MYC indeed co-ordinates rather than simply stimulates ribosome biogenesis.
(IV) Preliminary data obtained in collaboration with Reuven Agami (NKI) show that MYC not only activates the expression of ribosomal protein genes on a transcriptional level, but also enhances their translation. I hypothesize that enhancing translation of selected groups of mRNPs is also a direct function of Myc and propose to unravel the underlying mechanisms and its impact on cellular growth.
Targeting the oncogenic function of Myc for tumour therapy.
The transcription factor Myc plays a central role in the development of human tumours but is also an essential protein. Proof-of-principle studies using a dominant negative allele of Myc in mice have demonstrated the dependency of established tumours on Myc function. One goal of our research is to develop strategies to target Myc function for cancer therapy. We have already discovered that promoters differ in their affinity for Myc and that these differences enable Myc to regulate functionally distinct genes at different nuclear concentrations (Lorenzin et al, eLife, 2016). The new notion that Myc regulates distinct sets of genes at physiological and oncogenic levels opens the compelling possibility to specifically target the oncogenic functions of Myc.
We are exploring this concept in vivo and will identify and target crucial Myc target genes.
Using functional genomics to unravel transcriptional networks in tumour cells.
Role of the oncoprotein Myc has been well established as a transcription factor (Blackwood and Eisenman 1991). However, the exact mechanism by which Myc brings about this role had been long sought for. The precise binding pattern of Myc on the chromatin and the consequential control of the genes it thereby regulates was recently catalogued in rigorous detail (Walz et al. 2014) using ChIP-Seq and RNA-Seq techniques. Of considerable importance is also the fact that Myc drives the expression of these mentioned genes via an affinity based mechanism which further clarifies how Myc bring about its diverse roles (Lorenzin et al. 2016).One important distinction that must be mentioned here is that these analyses were restricted purely to poly-adenylated mRNA transcribing genes. Even though earlier works (Rahl et al. 2010) have tried to dissect the exact stage of transcription that is regulated by Myc, the quantification of effect of Myc on each stage of transcription remains elusive due to the inherent pitfall of ChIP-Seq technique being a static readout of the protein binding on chromatin. Thus, the effect on Myc on RNA Polymerase II (Pol2), which is a traveling protein during transcription, cannot be studied using ChIP-Seq and a new technique is warranted to study this question.
One way to study the active transcription in a cell is to read out the nascent mRNA formation in a specific time window. This has been attempted using a variety of analytical techniques like NET-Seq, TT-Seq, GRO-Seq and 4sU Seq (Schwalb et al. 2016)(Churchman and Weissman 2011)(Nojima et al. 2015)(Jonkers, Kwak, and Lis 2014)(Fuchs et al. 2015). To study specifically, the elongation stage of transcription and to read the effect of Myc on elongation rate, we took advantage of 4sU-DRB Seq, which has inherent advantages of being precise as compared to GRO-Seq and offers good enough resolution to study rate of transcriptional elongation with considerable accuracy (Fuchs et al. 2015). In this method, all Pol2 in a cell are synchronized using a reversible elongation inhibitor called DRB. This is followed by washout of DRB concomitant with metabolic labelling of nascent RNA using 4-thiouridine (4sU), which is eventually used to pull down the nascent RNA via biotin label and streptavidin beads. This nascent RNA is then read out using deep sequencing and thus, the exact end of transcriptional wave front can be pin pointed using exhaustive machine learning based bioinformatics algorithms.
Since all forms of mRNA apart from poly-A mRNA are captured using 4sU-Seq technique, it makes it possible to study transiently expressed and unstable mRNA as well. It has also been postulated that transcriptional control can be fine-tuned with ncRNA and enhancer RNAs (Skalska et al. 2017)(Lai et al. 2015). Hence, an additional output of this work could be an insight into the complete transcriptional landscape of the cell when viewed through the lens of Myc as oncoprotein.
1. To gather the quantitative effect of Myc on rate of transcription elongation using DRB-4sU Seq
2. To elucidate the percentage of control Myc has on individual stages of transcription using spiked-ChIP Seq and 4sU-Seq
3. To capture the role of ncRNA, eRNA and circRNA in regulation of transcription by Myc.
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