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Final Report Summary - TRXTERMSIGN (Genetic and epigenetic signature of transcription termination)

Most eukaryotic genes are transcribed by RNA polymerase II (Pol II) in a cycle of initiation, elongation and termination. Transcription termination, defined as the cessation of RNA synthesis and release of Pol II from its DNA template, depends on a functional poly(A) sequence as well as downstream terminator sequences. Correct termination is important for the prevention of transcriptional interference, efficient protein production and Pol II recycling. Further more, aberrant poly(A) usage and alterations of 3' UTR length have been functionally linked to cancer, and transcription termination mechanisms perturbed during viral infections.

Despite its vital importance, transcription termination mechanisms in mammalian cells have only been studied on a handful of genes at the time the project proposal had been drafted, and the general principles ruling the process were unknown.

The objective of this project was to elucidate the genetic and epigenetic signature of transcription termination genome-wide using a combination of computational, genetic and biochemical approaches.

The project aims were pursued in frame of both collaborative and independent studies.

The collaborative studies were initiated as soon as the fellow joined the host laboratory already before the formal start date of the fellowship and resulted in four co-authorship publications.
The first study addressed the mechanism of CoTC-type transcription termination, and resulted in the description of an AT-rich DNA motive promoting this type of termination (White et al., NAR 2013). The second study described the nuclear function of human DICER protein in restricting the deleterious accumulation of endogenous double-stranded RNA (White et al., NSMB 2014). The third study described a novel mechanism of R-loops inducing antisense transcription over pause terminator elements, leading to the recruitment of DICER, AGO1, AGO2 and the G9a histone methyltransferase, the formation of H3K9 repressive mark and heterochromatin protein gamma (HP1 gamma) recruitment, which together reinforce Pol II pausing before efficient transcriptional termination (Skourti-Stathaki et al., Nature 2014). The fourth study demonstrated that the tumour suppressor protein BRCA1 is recruited to RNA Pol II pause sites in transcription termination regions where it is required for R-loop-driven DNA damage repair (Hatchi et al., Mol Cell 2015).

Further to these collaborative studies, the fellow has also investigated the proposed subject in independent research.

One of the major achievements has been the generation of the first genome-wide data on the chromatin binding of a cleavage and polyadenylation factor performed in the mammalian system. We were able to create a chromatin binding map for the protein PCF11, which has multiple functions in RNA 3’ processing as well as in transcription termination. Another major advancement has been assaying of the termination defects upon PCF11 knock-down measured by changes in RNA Pol II occupancy and nascent transcriptome. Based on the obtained data we propose a model in which PCF11 preferentially binds to chromatin at the 3’ end of genes that require particularly efficient termination, especially in gene dense loci prone to transcriptional interference.

Additionally, using mass spectrometry we have been able to map a number of post-translational modifications on important transcription termination factors potentially regulating their function. For example, we have identified a phosphorylation site on PCF11 that regulates its binding to RNA Pol II and the ability to terminate transcription. This will subject to further research.

In summary, the research objectives of the project have been fully met. The signature of transcription termination has been studied using an array of different yet complementary approaches. Novel insights into the mechanism of both CoTC-type and pause-type transcription termination have been obtained. The genome-wide chromatin binding of the first human cleavage and polyadenylation complex factor has been described along with transcription termination defects arising from its downregulation. Multiple additional factors have been added to the transcription termination map, including DICER, G9A, HP1 gamma, H3K9me2 and BRCA1. The obtained results address the genetic (DNA motives), epigenetic (chromatin modifications) and transcription factor binding aspects of transcription termination which were set out in the project proposal. Furthermore, the work performed lead to the realization of an additional layer of regulation of transcriptional termination in form of the post-translational modification of crucial termination factors.

The conducted research has the potential to have impact on medicine given the fact that perturbations in RNA 3’ processing and transcription termination have been linked to cancer and viral infections.

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United Kingdom
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