bioRxiv preprint doi: https://doi.org/10.1101/717769; this version posted July 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Role for Merlin/NF2 in transcription elongation through interaction with the PAF complex Anne E. Roehrig1¶, Kristina Klupsch1¶, Juan A. Oses-Prieto2, Selim Chaib1, Stephen Henderson1, Warren Emmett3 , Lucy C.Young1, Silvia Surinova 1, Andreas Blees1, Anett Pfeiffer1, Maha Tijani1, Fabian Brunk1, Nicole Hartig1, Marta Muñoz-Alegre1, Alexander Hergovich1, Barbara H. Jennings1, Alma L. Burlingame2 and Pablo Rodriguez-Viciana1* 1 UCL Cancer Institute, University College London, London WC1E 6BT, United Kingdom 2 Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, USA 3 University College London Genetics Institute, Gower Street, London WC1E 6BT, UK ¶ These authors contributed equally to this work *Lead contact and corresponding author E-mail: [email protected] Phone: +44-(0)207-7679-6931 Running Title: Role for Merlin/NF2 in elongation through interaction with the PAF complex Key words: Merlin, NF2, PAF complex, CDC73, FAT, CHD1, transcription 1 bioRxiv preprint doi: https://doi.org/10.1101/717769; this version posted July 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. SUMMARY The PAF complex (PAFC) coordinates transcription elongation and mRNA processing events and its CDC73/parafibromin subunit functions as a tumour suppressor. The NF2/Merlin tumour suppressor functions at the cell cortex and nucleus and is a key mediator of contact inhibition. Here we provide a direct link between nuclear Merlin and transcription elongation controlled by cell-cell adhesion. Merlin interacts with the PAFC in a cell density-dependent manner and tumour- derived inactivating mutations in both Merlin and CDC73 mutually disrupt their interaction. Growth suppression by Merlin requires CDC73 and Merlin regulates PAFC association with chromatin in a subset of genes. We also identify by CDC73 affinity-proteomics a role for FAT cadherins in regulating the Merlin-PAFC interaction. Our results suggest that in addition to its function within the Hippo pathway, nuclear Merlin is part of a tumour suppressor network which coordinates post- initiation steps of the transcription cycle of genes mediating in contact inhibition. 2 bioRxiv preprint doi: https://doi.org/10.1101/717769; this version posted July 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. INTRODUCTION Normal cells cease to proliferate when they come into contact with each other and assemble intercellular junctions. Contact-dependent inhibition of proliferation is essential for normal tissue homeostasis and its loss is one of the hallmarks of cancer (Hanahan and Weinberg, 2011; McClatchey and Yap, 2012). The molecular mechanisms underlying contact inhibition remain unclear but Merlin, the protein encoded by the NF2 tumour suppressor gene is known to be an important mediator (Cooper and Giancotti, 2014). Germline mutations in NF2 cause Neurofibromatosis type 2, a cancer syndrome characterized by the development of schwannomas, meningiomas and ependymomas. Somatic NF2 mutations are frequently found in these tumour types as well as at lower frequency in several others, suggesting that NF2 has a broad tumour suppression function (Cooper and Giancotti, 2014; Dalgliesh et al., 2010; Forbes et al., 2015). Merlin functions as an upstream regulator of the Hippo tumour suppressor pathway to inactivate the YAP/TAZ transcriptional co-activators (Li et al., 2014; Yin et al., 2013; Zhang et al., 2010). In the absence of Hippo signalling, YAP/TAZ translocate to the nucleus to promote transcription of genes that regulate tissue homeostasis, organ size and tumorigenesis (Ma et al., 2019). Merlin has also been implicated in controlling the surface availability of membrane receptors and in the regulation of multiple signalling pathways (Li et al., 2012). Merlin can localize to different cellular compartments. It is found at the cell cortex in lamellipodia and membrane ruffles and at cell–cell contacts in dense cultures where it can interact with a-catenin and Par3 at nascent adherens junctions and with AMOT/Angiomotin at tight junctions (Gladden et al., 2010; Yi et al., 2011). Merlin can also exhibit a punctate distribution that has been attributed to intracellular vesicles or particles that can move along microtubules in a kinesin and dynein-dependent manner (Bensenor et al., 2010; McCartney and Fehon, 1996; McClatchey and Giovannini, 2005). In addition, Merlin can also localize to the nucleus in a density and cell cycle dependent manner (Furukawa et al., 2017; Gronholm et al., 2006; Kressel and Schmucker, 2002; Li et al., 2010; Muranen et al., 2005). Transcription of mRNAs is a tightly regulated process consisting of discrete stages of RNA polymerase II (Pol II) recruitment, initiation, elongation and termination. Transcription elongation is now recognized to play a key role in regulated gene expression. In particular, promoter-proximal pausing of Pol II and its release to initiate productive elongation are key regulatory steps in transcription with its precise intensity and the mechanisms regulating pause release likely varying from gene to gene (Adelman and Lis, 2012; Jonkers and Lis, 2015; Nechaev and Adelman, 2011). After promoter-proximal pause release, elongation rates also vary between genes and are closely linked to co-transcriptional RNA processing steps such as splicing, cleavage and polyadenylation (Jonkers and Lis, 2015). Chromatin represents a barrier to transcription by Pol II that has to be overcome as well as restored following polymerase passage. Accordingly, elongation is also tightly coupled with modulation of chromatin structure through the concerted actions of chromatin remodelers, histone-modifying enzymes and histone chaperones (Petesch and Lis, 2012). The Pol II-associated factor 1 complex (PAFC) has been linked to multiple transcription related processes including communicating with transcriptional activators, recruiting histone modification factors, release from promoter-proximal pausing, facilitating elongation across chromatin templates, recruitment of 3’ end processing factors and mRNA stability (Chen et al., 2015; Jaehning, 2010; Jo et al., 2014; Yang et al., 2016; Yu et al., 2015). In humans it is composed of 5 subunits, PAF1, CTR9, LEO1, CDC73 and SKI8. CDC73, also called Parafibromin, is inactivated in Hyperparathyroidism-jaw tumour (HPT-JT) syndrome, an autosomal dominant disorder characterized by the development of parathyroid tumours as well as uterine and renal cancers. (Newey et al., 2010). The PAF1 subunit localizes to 19q13, a locus amplified in several tumours and has transforming properties highlighting a role for the PAFC in cancer (Chaudhary et al., 2007). 3 bioRxiv preprint doi: https://doi.org/10.1101/717769; this version posted July 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Here we show that Merlin interacts with multiple proteins involved in transcription elongation and mRNA processing including the PAFC, the CHD1 chromatin remodeller and the TAT-SF1 elongation/splicing factor suggesting a role of Merlin in coordinating transcription elongation and mRNA processing. We use genomics to identify Merlin-regulated genes and proteomics to show Merlin can regulate the PAFC interactome and identify an interaction between CDC73 and FAT cadherins that is required for Merlin interaction with the PAFC. Our results suggest that Merlin is part of a tumour suppressor network which helps regulate expression of genes mediating contact inhibition by coordinating initiation and post-initiation steps of their transcription cycle in response to cell adhesion. 4 bioRxiv preprint doi: https://doi.org/10.1101/717769; this version posted July 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. RESULTS Merlin interacts with proteins that function in transcription elongation and splicing To identify Merlin interacting proteins that may shed light on its function, overexpressed Merlin was affinity purified from HEK293T cells and co-purifying proteins identified by mass spectrometry (Figure 1A and S1A). We identified several known Merlin-binding partners such as the VPRBP/DCAF1 and DDB1, members of a Cullin 4 containing E3 ubiquitin ligase complex (CRL4VPRBP) (Huang and Chen, 2008; Li et al., 2010; Yi et al., 2011)and AMOTL1, a tight-junction associated protein (Huang and Chen, 2008; Li et al., 2010; Yi et al., 2011). We also identified here new proteins that co-purified specifically with Merlin (but not other control baits) such as: CDC73,