Key nuclear processes in eukaryotes, including DNA replication, repair, and gene regulation, require extensive chromatin remodeling catalyzed by energy-consuming enzymes. It remains unclear how the ATP demands of such processes are met in response to rapid stimuli. We analyzed this question in the context of the massive gene regulation changes induced by progestins in breast cancer cells and found that ATP is generated in the cell nucleus via the hydrolysis of poly(ADP-ribose) to ADP-ribose. In the presence of pyrophosphate, ADP-ribose is used by the pyrophosphatase NUDIX5 to generate nuclear ATP. The nuclear source of ATP is essential for hormone-induced chromatin remodeling, transcriptional regulation, and cell proliferation.

That genome function may respond to its three-dimensional (3D) organization highlights the need for methods that can image genomes with superior coverage as well as greater genomic and optical resolution. Here, we push toward this goal by introducing OligoFISSEQ, a suite of three methods that leverage fluorescent in situ sequencing of barcoded Oligopaint probes to enable the rapid visualization of many targeted genomic regions. Applying OligoFISSEQ to human diploid fibroblast cells, we show how only four rounds of sequencing are sufficient to produce 3D maps of 66 genomic targets across 6 chromosomes in hundreds to thousands of cells. We then use OligoFISSEQ to trace chromosomes at finer resolution, following the path of the X chromosome through 46 regions, with separate studies showing compatibility of OligoFISSEQ with immunochemistry. Finally, we combined OligoFISSEQ with OligoSTORM, laying the foundation for accelerated single-molecule super-resolution imaging of large swaths of, if not entire, human genomes.

Cell division entails a marked reorganization of the microtubule network to form the spindle, a molecular machine that ensures accurate chromosome segregation to the daughter cells. Spindle organization is highly dynamic throughout mitosis and requires the activity of several kinases and complex regulatory mechanisms. Aurora A (AurA) kinase is essential for the assembly of the metaphase bipolar spindle and, thus, it has been difficult to address its function during the last phases of mitosis. Here, we examine the consequences of inhibiting AurA in cells undergoing anaphase, and show that AurA kinase activity is necessary for the assembly of a robust central spindle during anaphase. We also identify TACC3 as an AurA substrate essential in central spindle formation.

The ordered arrangement of cortical microtubules in growing plant cells is essential for anisotropic cell expansion and, hence, for plant morphogenesis. These arrays are dismantled when the microtubule cytoskeleton is rearranged during mitosis and reassembled following completion of cytokinesis. The reassembly of the cortical array has often been considered as initiating from a state of randomness, from which order arises at least partly through self-organizing mechanisms. However, some studies have shown evidence for ordering at early stages of array assembly. To investigate how cortical arrays are initiated in higher plant cells, we performed live-cell imaging studies of cortical array assembly in tobacco (Nicotiana tabacum) Bright Yellow-2 cells after cytokinesis and drug-induced disassembly. We found that cortical arrays in both cases did not initiate randomly but with a significant overrepresentation of microtubules at diagonal angles with respect to the cell axis, which coincides with the predominant orientation of the microtubules before their disappearance from the cell cortex in preprophase. In Arabidopsis (Arabidopsis thaliana) root cells, recovery from drug-induced disassembly was also nonrandom and correlated with the organization of the previous array, although no diagonal bias was observed in these cells. Surprisingly, during initiation, only about one-half of the new microtubules were nucleated from locations marked by green fluorescent protein-g-tubulin complex protein2-tagged g-nucleation complexes (g-tubulin ring complex), therefore indicating that a large proportion of early polymers was initiated by a noncanonical mechanism not involving g-tubulin ring complex. Simulation studies indicate that the high rate of noncanonical initiation of new microtubules has the potential to accelerate the rate of array repopulation.

Highlights d Serum starvation recruits TFIIIC at ADNP-bound Alu Elements (AEs) near Pol II genes d TFIIIC-associated histone acetylase activity acetylates H3K18 over the bound AEs d TFIIIC-bound acetylated AEs loop to contact CTCF at distal cell-cycle genes' promoters d CTCF-TFIIIC interaction ensures rapid cell-cycle genes' reactivation on serum exposure

Graphical AbstractHighlights d PADI2 citrullinates arginine1810 (cit1810) at CTD of RNA polymerase II (RNAP2) d PADI2 and R1810 regulate transcription and proliferation of breast cancer cells d Absence of cit1810 leads to RNAP2 accumulation at proximal promoter regions d Cit1810 facilitates interaction of RNAP2 with P-TEFb complex SUMMARYThe post-translational modification of key residues at the C-terminal domain of RNA polymerase II (RNAP2-CTD) coordinates transcription, splicing, and RNA processing by modulating its capacity to act as a landing platform for a variety of protein complexes.Here, we identify a new modification at the CTD, the deimination of arginine and its conversion to citrulline by peptidyl arginine deiminase 2 (PADI2), an enzyme that has been associated with several diseases, including cancer. We show that, among PADI family members, only PADI2 citrullinates R1810 (Cit1810) at repeat 31 of the CTD. Depletion of PADI2 or loss of R1810 results in accumulation of RNAP2 at transcription start sites, reduced gene expression, and inhibition of cell proliferation. Cit1810 is needed for interaction with the P-TEFb (positive transcription elongation factor b) kinase complex and for its recruitment to chromatin. In this way, CTD-Cit1810 favors RNAP2 pause release and efficient transcription in breast cancer cells.

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-...

The growth of cancer cells as oncospheres in three-dimensional (3D) culture provides a robust cell model for understanding cancer progression, as well as for early drug discovery and validation. We have previously described a novel pathway in breast cancer cells, whereby ADP (Adenosine diphosphate)-ribose derived from hydrolysis of poly (ADP-Ribose) and pyrophosphate (PPi) are converted to ATP, catalysed by the enzyme NUDT5 (nucleotide diphosphate hydrolase type 5). Overexpression of the NUDT5 gene in breast and other cancer types is associated with poor prognosis, increased risk of recurrence and metastasis. In order to understand the role of NUDT5 in cancer cell growth, we performed phenotypic and global expression analysis in breast cancer cells grown as oncospheres. Comparison of two-dimensional (2D) versus 3D cancer cell cultures from different tissues of origin suggest that NUDT5 increases the aggressiveness of the disease via the modulation of several key driver genes, including ubiquitin specific peptidase 22 (USP22), RAB35B, focadhesin (FOCAD) and prostagladin E synthase (PTGES). NUDT5 functions as a master regulator of key oncogenic pathways and of genes involved in cell adhesion, cancer stem cell (CSC) maintenance and epithelial to mesenchyme transition (EMT). Inhibiting the enzymatic activities of NUDT5 prevents oncosphere formation and precludes the activation of cancer driver genes. These findings highlight NUDT5 as an upstream regulator of tumour drivers and may provide a biomarker for cancer stratification, as well as a novel target for drug discovery for combinatorial drug regimens for the treatment of aggressive cancer types and metastasis.