Chromatin and Gene Expression

The group explores how eukaryotic cells respond to external cues; more specifically, how signals are transduced to the nucleus and modulate chromatin structure and gene expression. Previously, we found that gene regulation by progestins involves the communication of membrane-attached and nuclear progesterone receptor (PR) associated with ERK and MSK1 kinases leading to two consecutive cycles of chromatin remodelling: a very rapid displacement of histone H1, followed by a slower displacement of histones H2A/H2B. We found that the Mb topological associating domains (TADs) in chromatin behave as units of hormone response in that all their genes respond in a similar manner to hormone exposure. Within regulated TADs large clusters of hormone receptor binding sites –so-called Hormone Control Regions – organize the folding of the chromatin fibre even before hormone exposure and interact with each other at long distances (Le Dily et al., 2017). Similarly, transcription factors act as genome structural organizers in the transition from B cells to iPSC (Stadhouders et al., 2017). We found that the ATP required for chromatin remodelling is generated in the nucleus from ADP-Ribose and PPi by the enzyme NUDIX5. We are now studying the role of nuclear ATP in DNA repair, early embryogenesis and cell fate decisions. Our final aim is to integrate the signalling network with the changes in the topological organization of chromatin and the transcriptional response to generate a multidimensional network that will reveal the logic of the hormonal control of cancer cell proliferation.

• Role of transcription factors and hormone receptors in shaping genome architecture
  Ferrari, Le Dily, Lioutas, Nacht, Quilez, Sharma, Vicent, Vidal, Wright
  Progestin (Pg) addition to T47D breast cancer cells activates the Progesterone Receptor (PR) that binds to nucleosomaly organized promoter/enhancer regions of target genes along with kinases, histone modifying enzymes and chromatin remodeller, leading to coordinate activation or repression of genes clustered in topological associating domains (TADs) (Le Dily et al. Genes Dev 2014). In the context of the ERC Synergy Grant “4D Genome” and in collaboration with Thomas Graf, Marc Marti-Renom and Guillaume Filion and the 4D Genome Unit (Francois Le Dily, Enrique Vidal) we continue exploring the relevance of genome architecture for cell identity, the response to perturbations – such as inhibition of transcription, energy supply or signalling – and the epigenetic memory of various normal and cancer cells. Differential changes in loop organization within TADs in response to various hormones is one of the most advanced lines of research. We have generated a catalogue of lncRNAs expressed in the breast cancer cell line T47D and have identified a few that change expression in response to hormone. We want to explore their potential contribution to genome architecture using CRISPR-cas9 approaches.
• ADP-Ribose derived nuclear of ATP generation in DNA in damage repair and cell reprogramming
  Lioutas, Vicent, Wright
  PARP1 mediated protein parylation is essential for hormonal gene regulation, but degradation of PAR by PARG is also important. We found that in cells exposed to hormone the levels of ATP increase transiently in the cell nuclei, but not in mitochondria or in the cytoplasm. The nuclear ATP increase depends on PARP1 and PARG activity and cannot be blocked by blocking mitochondrial energy generation 10 min after hormone exposure. We identified NUDT5/NUDIX5 as a PAR-interacting enzyme that co-IP with PR and PARG and is needed for chromatin remodelling, gene regulation, and hormone-dependent cell proliferation. NUDIX5 forms a stable homodimer that hydrolyses ADPR to AMP and R-5-P. In response to hormone NUDIX5 is rapidly dephosphorylated at T45 leading to destabilization of the homodimer that facilities the reaction of ADPR with pyrophosphate to generate ATP and R-5-P. We are exploring the structure of the enzyme complex involved in nuclear ATP synthesis, including NMNAT1, PARP1, PARG and NUDIX5, and their role in the DNA damage response and transcriptional activation. NUDIX5 is overexpressed in breast cancer samples and is a biomarker for poor prognosis. Therefore, we want to develop small molecular weight inhibitors of ATP synthesis by NUDIX5 that could be of use in cancer therapy, alone or in combination with PARP inhibitors.
• High affinity PR binding sites
  Nacht, Quilez, Carbonell, Vicent, Wright, Zaurin
  The hormone concentration used for most studies on progestin action (R5020) on breast cancer cells is 10nM. By lowering the hormone concentration, we identified a small subset (around 1000) of high-affinity PR binding sites that are almost saturated at concentrations in the order of 0.05 to 0.1 nM. Cells exposed to these levels of hormones respond with increase DNA synthesis and regulate hundreds of genes. We are studying how this few sites may influence 3D genome structure to trigger the hormonal response
• Role of TFIIIC binding to Alu-elements in genome organization upon serum starvation
  Ferrari, Llobet
  TFIIIC is a large transcription factor of RNA-Pol3 involved in transcription of tRNA genes, that has been reported to act as an insulator that participates in 3D genome organization. We found an intimate relationship between TFIIIC and PR in breast cancer cells. Upon serum starvation TFIIIC relocates from tRNA genes to repetitive ALU/SINE elements near RNA-Pol2 transcribed genes, and upon hormone exposure PR binds nearby and activates transcription. We hypothesized that TFIIIC might be a sensor of growth signals that contributes to genomic rearrangements needed for rapid response to nutrient changes and we will explore whether its contribution to the breast cancer phenotype.
• Role of PADI2-mediated citrullination of the Pol-II CTD in transcription elongation
  Sharma, Vicent
  Among the family of ­peptidyl arginine de-iminases (PADIs), T47D cells only express PADI2 and PADI3. PADI2 interacts with RNA-Pol2 and citrullinates R1810 in RNA-Pol2 CTD repeat 31. A RNA-Pol2 R1810K mutant is defective in promoter release in a subset of genes control cell proliferation. In response to hormone PADI2 interacts with PR and is needed for hormonal gene regulation. As PADI2 is overexpressed in several cancer types, including breast cancer, and depletion of PADI2 block proliferation of breast cancer cells we are planning to develop specific inhibitors of PADI2 action on the RNA-Pol2 CTD.