Sequence-Dependent nucleosome positioning and chromatin remolleing of hormone-responsive genes

Evidències recents han remarcat la importància del paper de l'estructura de la cromatina i el posicionament de nucleosomes en processos cel·lulars bàsics com és la regulació de la transcripció gènica. L'avenç de noves tecnologies en el camp ha permès l'estudi detallat de la disposició de nucleosomes en genomes sencers. Hi ha hagut també molts intents per definir les característiques de la seqüència de l'ADN que podrien arribar a guiar el posicionament dels nucleosomes; sent el conjunt d'aquestes característiques l'anomenat "codi nucleosòmic". En la present tesis doctoral, s'aporten evidències experimentals sobre l'existència d'un grup de nucleosomes molt ben posicionats en el genoma humà. Això ha estat possible mitjançant tècniques com els microarray i la seqüenciació massiva en paral·lel. Aquest treball mostra com aquests nucleosomes, que anomenem "nucleosomes clau", tenen tendència a ocupar regions del genoma amb funcions específiques, la qual cosa indica el seu paper especial. L'ADN els "nucleosomes clau" resulta tenir una alta simetria de curvatura. Aquesta característica inherent a la seqüència fa que sigui possible la predicció in silico de les posicions de nucleosomes d'aquest tipus. En la segona part de la present tesi doctoral, aporto noves evidències experimentals que fan avançar el camp de la organització de la cromatina i la seva dinàmica en el context de la regulació gènica. Les hormones esteroidees indueixen la transcripció dels seus gens diana a través de la unió dels receptors hormonals amb els seu corresponent motiu de reconeixement a l'ADN (HREs), així com el reclutament d'una gran varietat de co- eguladors. El promotor del Virus de Tumor Mamari de Ratolí (MMTV) ha estat un model molt usat per l'estudi dels efectes en l'activació gènica dels receptors hormonals. És conegut que el receptor de progesterona (PR) s'uniex a l'HRE1, accessible, i recluta maquinàries de remodelament de la cromatina que utilitzen l'ATP com a font energètica per expulsar els dimers d'histones H2A/H2B del nucleosoma B del promotor de l'MMTV. Aquí demostro que la màquinaria de remodelament reclutada és específicament BAF. Treballs anteriors van demostrar que la kinasa Msk1 és la responsable de la fosforilació de la serina 10 de la histona H3. En aquesta tesi es demostra que l'acetilació de la Lysina 14 de la histona H3 és essencial per l'activació del promotor, així com per l'anclatge de BAF. Després de l'expulsió dels dimers d'H2A/H2B, la unió d'NF1 al promotor és indispensable per estabilitzar la forma remodelada del nucleosoma. Per l'estudi en més detall de l'activació del promotor de l'MMTV he utilitzat el sistema de minicromosomes, mononucleosomes i tetràmers d'histones H3/H4 reconstituïts en seqüències salvatges i mutants del promotor de l'MMTV. He demostrat que, només quan un fragment de la seqüencia de l'MMTV està reconstituïda en tetràmers, el PR i l'NF1 poden estar units simultàniament a la seva seqüència de reconeixement en el promotor. També aporto evidències on es demostra que la unió d'NF1 al promotor facilita la posterior unió de més mol·lècules de PR als HREs interns (llocs 2 i 3) caracterizant en més detall el sinergisme funcional que existeix entre el PR i l'NF1 en aquestes condicions.Evidence has been accumulating over the last few years pointing to the importance of chromatin structure and nucleosome positioning in cellular processes such as transcriptional regulation. Recent technological advances in the field have allowed the construction of detailed genome-scale maps of nucleosome positions, and there have been several attempts to define the sequence characteristics that guide the positioning of nucleosomes, the so-called "nucleosome code". In this thesis I give experimental evidence for the existence of a subset of very well-positioned nucleosomes in the human genome based on nucleosome-resolution tilling arrays and on deep sequencing of MNase-digested chromatin using the Solexa-Illumina platform. I show that these nucleosomes, which we have named "key nucleosomes", tend to occupy genomic locations of specific function, indicative of their special role. The DNA of these "key nucleosomes" exhibits a high symmetry of curvature, allowing their precise position on the human genome to be predicted in silico based on the structural attributes of the primary DNA sequence. The second part of this thesis provides new insights into the importance of chromatin organization and dynamics in the context of gene regulation. Steroid hormones induce transcription of their target genes by a complex mechanism requiring binding of the hormone receptors to hormone responsive elements (HREs) and the recruitment of a variety of coregulators. The Mouse Mammary Tumor Virus (MMTV) promoter has long been used as a model for the study of hormone receptormediated gene activation. It is known that progesterone receptor (PR) binds the exposed HRE1 of the MMTV promoter chromatin and recruits chromatin remodellers that catalyse ATP-dependent histone H2A/H2B displacement. I show that the ATP- ependent chromatin remodelling complex BAF, but not PBAF, is recruited after hormone treatment and is necessary for MMTV promoter activation. Along with the previously reported osphorylation of H3S10 by Msk, I show that an early PCAF- ediated acetylation of H3K14 is essential for the activation of the promoter by anchoring the BAF complex. Following transient displacement of H2A/H2B dimers, binding of NF1 is required for stabilizing the remodelled conformation of the MMTVnucleosome. To further study the activation process I have used MMTVminichromosomes, mononucleosomes and H3/H4 tetramer particles reconstituted on wild type MMTV and MMTV promoter fragments with point mutations disrupting binding of PR and NF1. I show that only when MMTV sequences are assembled on H3/H4 tetramer particles can PR bind to all five HREs while allowing NF1 access to its cognate site. Furthermore, I found that binding of NF1 facilitates access of PR to the central HREs 2 and 3, thus contributing to the reciprocal synergism between PR and NF1

scTAM-seq enables targeted high-confidence analysis of DNA methylation in single cells

Single-cell DNA methylation profiling currently suffers from excessive noise and/or limited cellular throughput. We developed scTAM-seq, a targeted bisulfite-free method for profiling up to 650 CpGs in up to 10,000 cells per experiment, with a dropout rate as low as 7%. We demonstrate that scTAM-seq can resolve DNA methylation dynamics across B-cell differentiation in blood and bone marrow, identifying intermediate differentiation states that were previously masked. scTAM-seq additionally queries surface-protein expression, thus enabling integration of single-cell DNA methylation information with cell atlas data. In summary, scTAM-seq is a high-throughput, high-confidence method for analyzing DNA methylation at single-CpG resolution across thousands of single cells.We acknowledge support of the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Programme/Generalitat de Catalunya. We acknowledge support from the CRG/CNAG/UPF core facilities (cytometry and genomics unit). A.B. was supported by an FPI fellowship from the Spanish Ministry of Science and Innovation (PRE2019-087574), R.B. was supported by a Junior Leader Fellowship from the la Caixa foundation. M.S. was supported through the Walter Benjamin Fellowship funded by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - 493935791. This work was supported by grants from the Spanish Ministry of Science and Innovation (RTI2018-096359-A-I00) and the European Hematology Association (EHA, Advanced Research Grants to L.V. and R.B.)

Hormone-induced repression of genes requires BRG1-mediated H1.2 deposition at target promoters

Eukaryotic gene regulation is associated with changes in chromatin compaction that modulate access to DNA regulatory sequences relevant for transcriptional activation or repression. Although much is known about the mechanism of chromatin remodeling in hormonal gene activation, how repression is accomplished is much less understood. Here we report that in breast cancer cells, ligand-activated progesterone receptor (PR) is directly recruited to transcriptionally repressed genes involved in cell proliferation along with the kinases ERK1/2 and MSK1. PR recruits BRG1 associated with the HP1γ-LSD1 complex repressor complex, which is further anchored via binding of HP1γ to the H3K9me3 signal deposited by SUV39H2. In contrast to what is observed during gene activation, only BRG1 and not the BAF complex is recruited to repressed promoters, likely due to local enrichment of the pioneer factor FOXA1. BRG1 participates in gene repression by interacting with H1.2, facilitating its deposition and stabilizing nucleosome positioning around the transcription start site. Our results uncover a mechanism of hormone-dependent transcriptional repression and a novel role for BRG1 in progestin regulation of breast cancer cell growth.The experimental work was supported by grants from the Departament d’Innovació Universitat i Empresa (DIUiE). We acknowledge support of the Spanish Ministry of Economy and Competitiveness (SAF2013-42497-P), “Centro de Excelencia Severo Ochoa 2013–2017”, SEV-2012-0208 and ERC Synergy Grant “4DGenome” nr 60998

Nucleosome-driven transcription factor binding and gene regulation

In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)-dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint. When fission yeast cells are treated with DNA-damaging agents, Chk1 is activated and phosphorylates Cdc10 at its carboxy-terminal domain. This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin. This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents. Thus Yox1 and Cdc10 couple normal cell cycle regulation in unperturbed conditions and the DNA replication and DNA damage checkpoints into a single transcriptional complex.The experimental work was supported by grants from the Spanish government (BMC 2003-02902 and 2010-15313; CSD2006-00049), the European Union (IP HEROIC), and the Catalan government (AGAUR). L.G. was a recipient of a fellowship from the International PhD program of LaCaixa; G.P.V. was a recipient of a fellowship from the Ramón y Cajal program

Nucleosome-driven transcription factor binding and gene regulation

In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)-dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint. When fission yeast cells are treated with DNA-damaging agents, Chk1 is activated and phosphorylates Cdc10 at its carboxy-terminal domain. This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin. This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents. Thus Yox1 and Cdc10 couple normal cell cycle regulation in unperturbed conditions and the DNA replication and DNA damage checkpoints into a single transcriptional complex.The experimental work was supported by grants from the Spanish government (BMC 2003-02902 and 2010-15313; CSD2006-00049), the European Union (IP HEROIC), and the Catalan government (AGAUR). L.G. was a recipient of a fellowship from the International PhD program of LaCaixa; G.P.V. was a recipient of a fellowship from the Ramón y Cajal program

A set of accessible enhancers enables the initial response of breast cancer cells to physiological progestin concentrations

Here, we report that in T47D breast cancer cells 50 pM progestin is sufficient to activate cell cycle entry and the progesterone gene expression program. At this concentration, equivalent to the progesterone blood levels found around the menopause, progesterone receptor (PR) binds only to 2800 genomic sites, which are accessible to ATAC cleavage prior to hormone exposure. These highly accessible sites (HAs) are surrounded by well-organized nucleosomes and exhibit breast enhancer features, including estrogen receptor alpha (ERα), higher FOXA1 and BRD4 (bromodomain containing 4) occupancy. Although HAs are enriched in RAD21 and CTCF, PR binding is the driving force for the most robust interactions with hormone-regulated genes. HAs show higher frequency of 3D contacts among themselves than with other PR binding sites, indicating colocalization in similar compartments. Gene regulation via HAs is independent of classical coregulators and ATP-activated remodelers, relying mainly on MAP kinase activation that enables PR nuclear engagement. HAs are also preferentially occupied by PR and ERα in breast cancer xenografts derived from MCF-7 cells as well as from patients, indicating their potential usefulness as targets for therapeutic intervention.Funding: Ministerio de Ciencia e Innovación [PID2019-105173RB-I00 and PID2019-110384GB-C21]; Spanish Ministry of Economy and Competitiveness [SAF2016-75006-P and G62426937]; Consejo Superior de Investigaciones Científicas [201820I131]; Centro de Excelencia Severo Ochoa [SEV-2012-2018]; European Research Council [609989]. Funding for open access charge: Ministerio de Ciencia e Innovació