Chromatin and Gene Expression

The group explores how eukaryotic cells respond to external cues; in particular, how signals are transduced to the nucleus and modulate chromatin structure and gene expression. Previously, we found that progestins involves via the cell membrane attached nuclear progesterone receptor (PR) activate ERK and MSK1 kinases leading to two consecutive cycles of chromatin remodelling involving H1 displacement followed displacement of histones H2A/H2B. We found that 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. We now found that within regulated TADs large clusters of hormone receptor binding sites –so-called Hormone Control Regions- organise the folding of the chromatin fibre even before hormone exposure and interact with each other at long distances (Le Dily et al., 2018). Similarly, transcription factors act as genome structural organisers in the transition from B cells to iPSC (Stadhouders et al., 2018). 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 organisation 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 (TF) in shaping genome architecture (Le Dily)
Carbonell, Ferrari, Lioutas, Nacht, Quilez, Sharma, Vicent, Vidal, Wright (collaboration with, CNAG/CRG)

• In collaboration with Thomas Graf, Marc Marti-Renom & Guillaume Filion (CRG) and the 4D Genome Unit we studied the relevance of genome architecture for gene expression changes in B cells induced to form iPSCs by a pulse of C/EBP followed by expression of the four Yamanaka factors. In many cases changes in the genome topology precede the changes in transcription and the initial TFs induce genome structural changes required for action of the following TF (1. Stadhouders et al., Nature Genetics 2018).
• Similarly, in breast cancer cells several hormonally regulated TADs contain so-called Hormone Control Regions (HCR) exhibiting clusters of occupied hormone receptors sites, which interact with all the promoters of their TAD. TADs containing HCRs interact preferentially with each other at distances longer that 10Mb, even prior to hormone exposure. These long-distance interactions are dependent on the expression of hormone receptors, which organise the 3D genome topology (2. Le Dily et al., Genome Res. 2018.

Inhibition of ADP-Ribose derived nuclear of ATP generation (Wright)
Lioutas, Vicent
In cells exposed to hormone the levels of nuclear ATP increase transiently in a way that depends on PARP1, PARG and NUDIX5 but not on concomitant mitochondrial energy generation (Wright et al., Science 2016). 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 a conformational change of the homodimer that forms an hexamer and facilities the reaction of ADPR with pyrophosphate to generate ATP and R-5-P (Wright et al., Nucleus 2016). NUDIX5 is over-expressed in breast cancer samples and is a biomarker for poor prognosis. In collaboration with the Karolinska Institute we have developed a NUDIX5 specific inhibitor that block AMP and ATP synthesis (3. Page et al., Nature Commun. 2018) and are trying to create an inhibitor that blocks only ATP synthesis. To this end we have obtained a Proof of Concept grant from the ERC.

PADI2-mediated citrullination of the Pol-II CTD favours transcription elongation (Sharma)
Lioutas (collaboration with Dirk Eick, Munich and Baldomero Oliva & Narcis Fernandez, UPF)
In breast cancer cells PADI2 citrullinates R1810 in RNA-Pol2 CTD repeat 31 promoting pTEFB recruitment and transcription of genes involved in cell proliferation. As PADI2 is over-expressed in several cancer types, including breast cancer, and depletion of PADI2 blocks proliferation of breast cancer cells we are planning to develop specific inhibitors of PADI2 action on the RNA-Pol2 CTD (4. Sharma et al., Molecular Cell 2018).

Progesterone receptor maintains basal levels of oestrogen receptor expression in the absence of hormone (Verde)
(De Llobet, Wright, Quilez, Le Dily)
In collaboration with Sandra Peiró ((Vall d’Hebron Institute of Oncology, Barcelona) we found that PR binds to the promoter of the ESR1 gene and maintains its basal expression in the absence of hormone by preventing its methylation, suggesting that the analysis of the methylation state of the ESR1 gene may provide useful information for breast cancer management (5. Verde et al., Cancers 2018).

Stadhouders R, Vidal E, Serra F, Di Stefano B, Le Dily F, Quilez J, Gomez A, Collombet S, Berenguer C, Cuartero Y, Hecht J, Filion G, Beato M, Marti-Renom MA, Graf T.
“Genome topology can direct gene regulatory path choice during cell reprogramming”
Nature Genetics, 50:238-49, 2018. doi:10.1038/s41588-017-0030-7.

LeDily F, Vidal E, Cuartero Y, Quilez J, Nacht S, Vicent GP, Sharma P, Verde G, Marti-Renom M, Beato M.
“Hormone control regions mediate opposing steroid receptor-dependent genome organization.”
Genome Research, Dec 14, 2018. doi:10.1101/gr.243824.118

Page BDG, Valerie NCK, Wright RHG, Wallner O, Isaksson R, Carter M, Rudd SG, Loseva O, Jemth AS, Almlöf I, Font-Mateu J, Llona-Minguez S, Baranczewski P, Jeppsson F, Homan E, Almqvist H, Axelsson H, Regmi S, Lundbäck T, Scobie M, Strömberg K, Stenmark K, Beato M, Helleday T.
^“Potent inhibitors of NUDT5 block hormonal gene regulation in breast cancer cells.”
Nature Communications, 9:250, 2018. doi:10.1038/s41467-017-02293-7.

Sharma P, Lioutas A, Fernandez-Fuentes N, Quilez J, Carbonell J, Wright RHG, Di Vona C, Le Dily F, Schüller R, Eick D, Oliva B, Beato M.
“Arginine citrullination of the C-terminal domain controls RNA polymerase II transcription.”
Molecular Cell, Nov 21, 2018. doi:10.1101/gr.243824.118

Verde G, De Llobet L, Wright RHG, Quilez J, Peiró S, Le Dily F, Beato M.
“Unliganded progesterone receptor governs estrogen receptor gene expression by regulating DNA methylation in breast cancer cells.”
Cancers, 10:371, 2018.