Highlights-Peptidyl arginine deiminase 2 (PADI2) deiminates arginine (R1810) present at Cterminal domain of RNA polymerase II (RNAP2-CTD).-PADI2 and R1810 of RNAP2-CTD regulate transcription of breast cancer cells.-PADI2 depletion and absence of R1810 deimination at RNAP2-CTD inhibits proliferation of breast cancer cells.-PADI2 mediated deimination of R1810 at RNAP2-CTD favors pause release.not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/216143 doi: bioRxiv preprint first posted online Nov. 8, 2017; 2 SummaryThe C-terminal domain of the large subunit of RNA polymerase II (RNAP2-CTD) coordinates transcription and associated processes by acting as a landing platform for a variety of protein complexes. In mammals, the RNAP2-CTD comprises 52 heptapeptide repeats, the first half of which (1-27) exhibit the consensus repeat sequence Y 1 -S 2 -P 3 -T 4 -S 5 -P 6 -S 7 , whereas the second half (28-52) contains deviations from this consensus [1][2][3] . The residues present on CTD undergo posttranslational modifications to determine which factors will be recruited to process nascent transcripts and modify chromatin. Dynamic phosphorylation, mainly on serines 2 and 5 mediates selective recruitment of protein complexes 4-7 , but recently, modifications of lysines and arginines in non-consensus repeats have expanded the functional complexity of the CTD code [8][9][10][11] . Here we show that R1810 can be deiminated by PADI2 favoring RNAP2 pause release at highly expressed genes relevant for proliferation of breast cancer cells. Depletion of PADI2 reduced expression of these genes accompanied by accumulation of RNAP2 at transcriptional start sites and resulted in inhibition of cell proliferation. As PADI2 is overexpressed in several cancers and is related to poor prognosis, selective inhibitors may help to prevent cancer progression. Deimination of R1810 at RNAP2-CTDArginine deimination, also known as citrullination, is a post-translational modification of arginine residues that generates the non-coded amino acid citrulline. This reaction is catalyzed by enzymes called peptidyl arginine deiminases (PADIs) [12][13][14] . Arginine 1810 in repeat 31 can be asymmetrically 10 or symmetrically 11 dimethylated leading to reduced expression of small nuclear and nucleolar RNAs 10 , or facilitating transcription termination 11 , respectively. We explored the possibility that it could be deiminated and functionally affect the transcription. Immunoprecipitation from nuclear extracts of the breast cancer cell line T47D-MTVL 15 using an α-citrulline detects two bands migrating as the non-phosphorylated (IIA) and week phosphorylated (IIO) forms of the large subunit of RNAP2 (Fig.1a). The IIO form reacted with α-citrulline to much higher proportion than IIA form of RNAP2. Next, we raised a specific antibody against a 13 residues peptide centered on R1810, which was replaced by citrulline (Extended Data not peer-...