Women’s contribution in understanding how topoisomerases, supercoiling, and transcription control genome organization

One of the biggest paradoxes in biology is that human genome is roughly 2 m long, while the nucleus containing it is almost one million times smaller. To fit into the nucleus, DNA twists, bends and folds into several hierarchical levels of compaction. Still, DNA has to maintain a high degree of accessibility to be readily replicated and transcribed by proteins. How compaction and accessibility co-exist functionally in human cells is still a matter of debate. Here, we discuss how the torsional stress of the DNA helix acts as a buffer, regulating both chromatin compaction and accessibility. We will focus on chromatin supercoiling and on the emerging role of topoisomerases as pivotal regulators of genome organization. We will mainly highlight the major breakthrough studies led by women, with the intention of celebrating the work of this group that remains a minority within the scientific community

Cohesin-independent STAG proteins interact with RNA and localise to R-loops to promote complex loading

Most studies of cohesin function consider the Stromalin Antigen (STAG/SA) proteins as core complex members given their ubiquitous interaction with the cohesin ring. Here, we provide functional data to support the notion that the SA subunit is not a mere passenger in this structure, but instead plays a key role in the localization of cohesin to diverse biological processes and promotes loading of the complex at these sites. We show that in cells acutely depleted for RAD21, SA proteins remain bound to chromatin, cluster in 3D and interact with CTCF, as well as with a wide range of RNA binding proteins involved in multiple RNA processing mechanisms. Accordingly, SA proteins interact with RNA and are localised to R-loops where they contribute to R-loop regulation. Our results place SA1 within R-loop domains upstream of the cohesin complex and reveal a role for SA1 in cohesin loading which is independent of NIPBL, the canonical cohesin loader. We propose that SA1 takes advantage of structural R-loop platforms to link cohesin loading and chromatin structure with diverse functions. Since SA proteins are pan-cancer targets, and R-loops play an increasingly prevalent role in cancer biology, our results have important implications for the mechanistic understanding of SA proteins in cancer and disease

Cohesin-independent STAG proteins interact with RNA and R-loops and promote complex loading

Most studies of cohesin function consider the Stromalin Antigen (STAG/SA) proteins as core complex members given their ubiquitous interaction with the cohesin ring. Here, we provide functional data to support the notion that the SA subunit is not a mere passenger in this structure, but instead plays a key role in the localization of cohesin to diverse biological processes and promotes loading of the complex at these sites. We show that in cells acutely depleted for RAD21, SA proteins remain bound to chromatin, cluster in 3D and interact with CTCF, as well as with a wide range of RNA binding proteins involved in multiple RNA processing mechanisms. Accordingly, SA proteins interact with RNA, RNA binding proteins and R-loops, even in the absence of cohesin. Our results place SA1 on chromatin upstream of the cohesin ring and reveal a role for SA1 in cohesin loading which is independent of NIPBL, the canonical cohesin loader. We propose that SA1 takes advantage of structural R-loop platforms to link cohesin loading and chromatin structure with diverse functions. Since SA proteins are pan-cancer targets, and R-loops play an increasingly prevalent role in cancer biology, our results have important implications for the mechanistic understanding of SA proteins in cancer and disease

Cohesin controls X chromosome structure remodeling and X-reactivation during mouse iPSC-reprogramming

Reactivation of the inactive X chromosome is a hallmark epigenetic event during reprogramming of mouse female somatic cells to induced pluripotent stem cells (iPSCs). This involves global structural remodeling from a condensed, heterochromatic into an open, euchromatic state, thereby changing a transcriptionally inactive into an active chromosome. Despite recent advances, very little is currently known about the molecular players mediating this process and how this relates to iPSC-reprogramming in general. To gain more insight, here we perform a RNAi-based knockdown screen during iPSC-reprogramming of mouse fibroblasts. We discover factors important for X chromosome reactivation (XCR) and iPSC-reprogramming. Among those, we identify the cohesin complex member SMC1a as a key molecule with a specific function in XCR, as its knockdown greatly affects XCR without interfering with iPSC-reprogramming. Using super-resolution microscopy, we find SMC1a to be preferentially enriched on the active compared with the inactive X chromosome and that SMC1a is critical for the decompacted state of the active X. Specifically, depletion of SMC1a leads to contraction of the active X both in differentiated and in pluripotent cells, where it normally is in its most open state. In summary, we reveal cohesin as a key factor for remodeling of the X chromosome from an inactive to an active structure and that this is a critical step for XCR during iPSC-reprogramming

The magic of unraveling genome architecture and function

Data de publicació electrònica: 13-04-2023Over the last decades, technological breakthroughs in super-resolution microscopy have allowed us to reach molecular resolution and design experiments of unprecedented complexity. Investigating how chromatin is folded in 3D, from the nucleosome level up to the entire genome, is becoming possible by "magic" (imaging genomic), i.e., the combination of imaging and genomic approaches. This offers endless opportunities to delve into the relationship between genome structure and function. Here, we review recently achieved objectives and the conceptual and technical challenges the field of genome architecture is currently undertaking. We discuss what we have learned so far and where we are heading. We elucidate how the different super-resolution microscopy approaches and, more specifically, live-cell imaging have contributed to the understanding of genome folding. Moreover, we discuss how future technical developments could address remaining open questions.We acknowledge the support from the European Union’s Horizon 2020 Research and Innovation Program (no. 964342 to M.P.C.); the Ministerio de Ciencia e Innovación (grant no. 008506-PID2020- 114080GB-I00 to M.P.C.); an AGAUR grant from Secretaria d’Universitats i Recerca del Departament d’Empresa iConeixement de la Generalitat de Catalunya (grant no. 006712 BFU2017-86760-P [AEI/FEDER, UE] to M.P.C.); the CERCA Program/Generalitat de Catalunya (to CRG); ICREA (Institucio Catalana de Recerca i Estudis Avançats) (to M.P.C.); the National Natural Science Foundation of China (31971177 and 32270577 to M.P.C.); Guangzhou Key Projects of Brain Science and Brain-Like Intelligence Technology (20200730009 to M.P.C.); Barcelona Institute of Science and Technology (BIST) Ignite Grants (Seeding Stage 2017 and Second Phase 2018, to M.V.N.); the People Program (Marie Curie Actions) FP7/2007–2013 under REA (grant no. 608959 to M.V.N.); and Juan de la Cierva-Incorporación 2017 (to M.V.N.) and the Spanish Ministry of Science and Innovation through the Centro de Excelencia Severo Ochoa (CEX2020-001049-S, MCIN/AEI/10.13039/501100011033 to CRG)

Women's contribution in understanding how topoisomerases, supercoiling, and transcription control genome organization

One of the biggest paradoxes in biology is that human genome is roughly 2 m long, while the nucleus containing it is almost one million times smaller. To fit into the nucleus, DNA twists, bends and folds into several hierarchical levels of compaction. Still, DNA has to maintain a high degree of accessibility to be readily replicated and transcribed by proteins. How compaction and accessibility co-exist functionally in human cells is still a matter of debate. Here, we discuss how the torsional stress of the DNA helix acts as a buffer, regulating both chromatin compaction and accessibility. We will focus on chromatin supercoiling and on the emerging role of topoisomerases as pivotal regulators of genome organization. We will mainly highlight the major breakthrough studies led by women, with the intention of celebrating the work of this group that remains a minority within the scientific community.We acknowledge the support from the European Union’s Horizon 2020 Research and Innovation Programme [CellViewer no. 686637 and Ecabox no. 964342 to MPC]; Ministerio de Ciencia e Innovación [grant no. 008506-PID2020- 114080 GB-I00 to MPC], and an AGAUR grant from Secretaria d’Universitats i Recerca del Departament d’Empresa iConeixement de la Generalitat de Catalunya (grant no. 006712 BFU 2017-86760-P (AEI/FEDER, UE) to MPC); ICREA (Institució Catalana de Recerca i Estudis Avançats) [to MPC]; The National Natural Science Foundation of China [grants no. 31971177 and 32270577 to MPC]; Innovative Team Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory [2018GZR110103001 to MPC]; Guangzhou Key Projects of Brain Science and Brain-Like Intelligence Technology [grant no. 20200730009 to MPC]; Centro de Excelencia Severo Ochoa [grants no. CEX 2020-001049-S, MCIN/AEI/10.13039/501100011033 to CRG]; CERCA Programme/Generalitat de Catalunya [to CRG]; The Spanish Ministry of Science and Innovation to the EMBL partnership [to MPC]; Fundació La Marató de TV3 [grant no. 202027-10 to MPC]; The Barcelona Institute of Science and Technology (BIST) Ignite Grants [Seeding Stage 2017 and Second Phase 2018, to MVN]; The People Program (Marie Curie Actions) FP7/2007–2013 under REA [grant no. 608959 to MVN]; Juan de la Cierva-Incorporación 2017 [to MVN]; Grant for the recruitment of early-stage research staff FI-2020 [Operational Program of Catalonia 2014-2020 CCI grant no. 2014ES05SFOP007 of the European Social Fund to LM] and La Caixa’ Foundation fellowship [grant no. LCF/BQ/DR20/11790016 to LM]

A protocol to quantify chromatin compaction with confocal and super-resolution microscopy in cultured cells

Here, we describe three complementary microscopy-based approaches to quantify morphological changes of chromatin organization in cultured adherent cells: the analysis of the coefficient of variation of DNA, the measurement of DNA-free nuclear areas, and the quantification of chromatin-associated proteins at the nuclear edge. These approaches rely on confocal imaging and stochastic optical reconstruction microscopy and allow a fast and robust quantification of chromatin compaction. These approaches circumvent inter-variability between imaging conditions and apply to every type of adherent cells. For complete details on the use and execution of this protocol, please refer to Neguembor et al. (2021).The authors acknowledge the support from European Union's Horizon 2020 Research and Innovation Programme (CellViewer no. 686637 to M.P.C. and M.L.); Ministerio de Ciencia e Innovación, grant (BFU2017-86760-P [AEI/FEDER, UE] to M.P.C.), and an AGAUR grant from Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya ([2017 SGR 689] to M.P.C.); Centro de Excelencia Severo Ochoa (2013–2017 to M.P.C.); CERCA Programme/Generalitat de Catalunya (to M.P.C.); the Spanish Ministry of Science and Innovation to the EMBL partnership (to M.P.C.); National Natural Science Foundation of China (31971177 to M.P.C.); Innovative Team Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory (2018GZR110103001 to M.P.C.); People Program (Marie Curie Actions) FP7/2007–2013 under REA grant 608959 (to M.V.N.); Juan de la Cierva-Incorporación 2017 (to M.V.N.); grant for the recruitment of early-stage research staff FI-2020 (Operational Program of Catalonia 2014-2020 CCI 2014ES05SFOP007 of the European Social Fund to L.M.); "La Caixa" Foundation Fellowship (ID 100010434, #LCF/BQ/DR20/11790016) (to L.M.); and "La Caixa-Severo Ochoa" pre-doctoral fellowship (to A.C.-G.

(Po)STAC (Polycistronic SunTAg modified CRISPR) enables live-cell and fixed-cell super-resolution imaging of multiple genes

CRISPR/dCas9-based labeling has allowed direct visualization of genomic regions in living cells. However, poor labeling efficiency and signal-to-background ratio have limited its application to visualize genome organization using super-resolution microscopy. We developed (Po)STAC (Polycistronic SunTAg modified CRISPR) by combining CRISPR/dCas9 with SunTag labeling and polycistronic vectors. (Po)STAC enhances both labeling efficiency and fluorescence signal detected from labeled loci enabling live cell imaging as well as super-resolution fixed-cell imaging of multiple genes with high spatiotemporal resolution.European Union’s Horizon 2020 Research and Innovation Programme [CellViewer No 686637 to M.L., M.P.C.]; Ministerio de Economia y Competitividad [BFU2013–49867-EXP to M.L., M.P.C.]; Fundació Cellex Barcelona (to M.L); European Union Seventh Framework Programme under the European Research Council Grants [337191-MOTORS to M.L.]; ‘Severo Ochoa’ Programme for Centres of Excellence in R&D [SEV-2015- 0522 to M.L.]; Ministerio de Economia y Competitividad and FEDER Funds [BFU2014–54717-P, BFU2015–71984-ERC to M.P.C.]; AGAUR Grant [2014 SGR1137 to M.P.C.]; Spanish Ministry of Economy and Competitiveness (to M.P.C.); Centro de Excelencia Severo Ochoa [2013–2017 to M.P.C.]; CERCA Programme/Generalitat de Catalunya (to M.P.C); Ministerio de Ciencia e Innovacion FPI (to F.A.); People Program (Marie Curie Actions) FP7/2007–2013 under REA grant [608959 to M.V.N.]. Funding for open access charge: European Union’s Horizon 2020 Research and Innovation Programme [CellViewer No 686637]

Super resolution microscopy reveals how elongating RNA polymerase II and nascent RNA interact with nucleosome clutches

Transcription and genome architecture are interdependent, but it is still unclear how nucleosomes in the chromatin fiber interact with nascent RNA, and which is the relative nuclear distribution of these RNAs and elongating RNA polymerase II (RNAP II). Using super-resolution (SR) microscopy, we visualized the nascent transcriptome, in both nucleoplasm and nucleolus, with nanoscale resolution. We found that nascent RNAs organize in structures we termed RNA nanodomains, whose characteristics are independent of the number of transcripts produced over time. Dual-color SR imaging of nascent RNAs, together with elongating RNAP II and H2B, shows the physical relation between nucleosome clutches, RNAP II, and RNA nanodomains. The distance between nucleosome clutches and RNA nanodomains is larger than the distance measured between elongating RNAP II and RNA nanodomains. Elongating RNAP II stands between nascent RNAs and the small, transcriptionally active, nucleosome clutches. Moreover, RNA factories are small and largely formed by few RNAP II. Finally, we describe a novel approach to quantify the transcriptional activity at an individual gene locus. By measuring local nascent RNA accumulation upon transcriptional activation at single alleles, we confirm the measurements made at the global nuclear level.Innovative Team Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory [2018GZR110103001 to M.P.C.]; Guangzhou Key Projects of Brain Science and Brain-Like Intelligence Technology [20200730009 to M.P.C.]; National Natural Science Foundation of China [31971177 to M.P.C.]; Science and Technology Program of Guangzhou, China [202002030146 to M.P.C.]; European Union's Horizon 2020 Research and Innovation Programme [CellViewer No. 686637 to M.P.C. and M.L.]; Ministerio de Ciencia e Innovación [BFU2017-86760-P (AEI/FEDER, UE) to M.P.C.]; AGAUR grant from Secretaria d’Universitats i Recerca del Departament d’Empresa I Coneixement de la Generalitat de Catalunya [2017 SGR 689 to M.P.C.]; Centro de Excelencia Severo Ochoa [2013–2017 to M.P.C.]; CERCA Programme/Generalitat de Catalunya [to M.P.C.]; People Program (Marie Curie Actions) FP7/2007–2013 under REA grant [608959 to M.V.N.]; Spanish Ministry of Science and Innovation to the European Molecular Biology Laboratory (EMBL) partnership [to M.P.C.]; Juan de la Cierva-Incorporación 2017 [to M.V.N.]. Funding for open access charge: National Natural Science Foundation of China (NSFC) grant [31971177 to M.P.C]

Active transcription and epigenetic reactions synergistically regulate meso-scale genomic organization

Abstract In interphase nuclei, chromatin forms dense domains of characteristic sizes, but the influence of transcription and histone modifications on domain size is not understood. We present a theoretical model exploring this relationship, considering chromatin-chromatin interactions, histone modifications, and chromatin extrusion. We predict that the size of heterochromatic domains is governed by a balance among the diffusive flux of methylated histones sustaining them and the acetylation reactions in the domains and the process of loop extrusion via supercoiling by RNAPII at their periphery, which contributes to size reduction. Super-resolution and nano-imaging of five distinct cell lines confirm the predictions indicating that the absence of transcription leads to larger heterochromatin domains. Furthermore, the model accurately reproduces the findings regarding how transcription-mediated supercoiling loss can mitigate the impacts of excessive cohesin loading. Our findings shed light on the role of transcription in genome organization, offering insights into chromatin dynamics and potential therapeutic targets