Dynamics of alternative splicing during somatic cell reprogramming reveals functions for RNA-binding proteins CPSF3, hnRNP UL1, and TIA1

C.V. was recipient of an FPI-Severo Ochoa Fellowship from the Spanish Ministry of Economy and Competitiveness. Work in J.V. laboratory is supported by the European Research Council (ERC AdvG 670146), AGAUR, Spanish Ministry of Economy and Competitiveness (BFU 2017 89308-P) and the Centre of Excellence Severo Ochoa. Work in T.G.'s laboratory was supported by the European Research Council FP7/2007-2013 (ERC Synergy Grant 4D-Genome) the Ministerio de Educación y Ciencia (SAF.2012-37167) and AGAUR. We acknowledge support of the Spanish Ministry of Science and Innovation to the EMBL partnership and the CERCA Programme / Generalitat de Catalunya.UDTRIASBackground: Somatic cell reprogramming is the process that allows differentiated cells to revert to a pluripotent state. In contrast to the extensively studied rewiring of epigenetic and transcriptional programs required for reprogramming, the dynamics of post-transcriptional changes and their associated regulatory mechanisms remain poorly understood. Here we study the dynamics of alternative splicing changes occurring during efficient reprogramming of mouse B cells into induced pluripotent stem (iPS) cells and compare them to those occurring during reprogramming of mouse embryonic fibroblasts. Results: We observe a significant overlap between alternative splicing changes detected in the two reprogramming systems, which are generally uncoupled from changes in transcriptional levels. Correlation between gene expression of potential regulators and specific clusters of alternative splicing changes enables the identification and subsequent validation of CPSF3 and hnRNP UL1 as facilitators, and TIA1 as repressor of mouse embryonic fibroblasts reprogramming. We further find that these RNA-binding proteins control partially overlapping programs of splicing regulation, involving genes relevant for developmental and morphogenetic processes. Conclusions: Our results reveal common programs of splicing regulation during reprogramming of different cell types and identify three novel regulators of this process and their targets

A Screen for novel factors involved in pluripotency and X-chromosome reactivation

X-Chromosome Reactivation (XCR) occurs in the epiblast cells of the blastocyst and in germ cells, thereby coupling XCR with pluripotency. We performed a screen in iPS cells by knocking down the expression of candidate genes picked from a single cell microarray expression profile in blastocysts. We thereby identified candidates which had an effect on both pluripotency and X-Reactivation. However, we also identified factors with a specific role in XCR. This suggests that XCR is not an absolute requirement for iPSC reprogramming and that the two processes can be uncoupled. Among these factors, there was the cohesin complex member Smc1a. In experiments based on Super resolution microscopy (STORM), we observed a preferential enrichment of Smc1a on the active compared to inactive X, suggesting a role in shaping the Xa structure. Therefore, we conclude that cohesin-mediated changes in X-chromosome structure are a key step during the XCR process.La reactivación del cromosoma X (XCR) ocurre en las células epiblásticas del blastocisto y en las células germinales, acoplando XCR con la pluripotencia. Se realizó un cribaje durante la reprogramación de iPSC reduciendo la expresión de genes candidatos, seleccionados a partir de un microarray de expresión en blastocitos. Se identificaron factores con un efecto tanto en la pluripotencia como en la XCR y factores con un rol específico en la XCR. Esto sugiere que la XCR no es un requisito absoluto para la reprogramación de las iPSC, y que los dos procesos se pueden desacoplar. Se identificó el miembro Smc1a del complejo de cohesina. Mediante microscopía de súper resolución (STORM) se observó un enriquecimiento preferencial de Smc1a en el cromosoma X activo en comparación con el X inactivo, lo que sugiere un papel en la configuración de la estructura del X activo. Por lo tanto, concluimos que los cambios mediados por cohesina en la estructura del cromosoma X son un paso clave durante el proceso de XCR

A Screen for novel factors involved in pluripotency and X-chromosome reactivation

X-Chromosome Reactivation (XCR) occurs in the epiblast cells of the blastocyst and in germ cells, thereby coupling XCR with pluripotency. We performed a screen in iPS cells by knocking down the expression of candidate genes picked from a single cell microarray expression profile in blastocysts. We thereby identified candidates which had an effect on both pluripotency and X-Reactivation. However, we also identified factors with a specific role in XCR. This suggests that XCR is not an absolute requirement for iPSC reprogramming and that the two processes can be uncoupled. Among these factors, there was the cohesin complex member Smc1a. In experiments based on Super resolution microscopy (STORM), we observed a preferential enrichment of Smc1a on the active compared to inactive X, suggesting a role in shaping the Xa structure. Therefore, we conclude that cohesin-mediated changes in X-chromosome structure are a key step during the XCR process.La reactivación del cromosoma X (XCR) ocurre en las células epiblásticas del blastocisto y en las células germinales, acoplando XCR con la pluripotencia. Se realizó un cribaje durante la reprogramación de iPSC reduciendo la expresión de genes candidatos, seleccionados a partir de un microarray de expresión en blastocitos. Se identificaron factores con un efecto tanto en la pluripotencia como en la XCR y factores con un rol específico en la XCR. Esto sugiere que la XCR no es un requisito absoluto para la reprogramación de las iPSC, y que los dos procesos se pueden desacoplar. Se identificó el miembro Smc1a del complejo de cohesina. Mediante microscopía de súper resolución (STORM) se observó un enriquecimiento preferencial de Smc1a en el cromosoma X activo en comparación con el X inactivo, lo que sugiere un papel en la configuración de la estructura del X activo. Por lo tanto, concluimos que los cambios mediados por cohesina en la estructura del cromosoma X son un paso clave durante el proceso de XCR

A conserved expression signature predicts growth rate and reveals cell & lineage-specific differences

Isogenic cells cultured together show heterogeneity in their proliferation rate. To determine the differences between fast and slow-proliferating cells, we developed a method to sort cells by proliferation rate, and performed RNA-seq on slow and fast proliferating subpopulations of pluripotent mouse embryonic stem cells (mESCs) and mouse fibroblasts. We found that slowly proliferating mESCs have a more naïve pluripotent character. We identified an evolutionarily conserved proliferation-correlated transcriptomic signature that is common to all eukaryotes: fast cells have higher expression of genes for protein synthesis and protein degradation. This signature accurately predicted growth rate in yeast and cancer cells, and identified lineage-specific proliferation dynamics during development, using C. elegans scRNA-seq data. In contrast, sorting by mitochondria membrane potential revealed a highly cell-type specific mitochondria-state related transcriptome. mESCs with hyperpolarized mitochondria are fast proliferating, while the opposite is true for fibroblasts. The mitochondrial electron transport chain inhibitor antimycin affected slow and fast subpopulations differently. While a major transcriptional-signature associated with cell-to-cell heterogeneity in proliferation is conserved, the metabolic and energetic dependency of cell proliferation is cell-type specific.This work has been funded by the Spanish Ministry of Science, Innovation and Universities (BFU2014-55275-P and BFU2017-88407-P to B.P. and BFU2015-68351-P to L.B.C.), the AXA Research Fund and the Agencia de Gestio d’Ajuts Universitaris i de Recerca (AGAUR, 2017 SGR 346 to B.P. and 2014 SGR 0974 & 2017 SGR 1054 to L.B.C.), the National Natural Science Foundation of China (31950410537 to L.B.C.). We would like to thank the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, to the ‘Centro de Excelencia Severo Ochoa’, and the Unidad de Excelencia María de Maeztu, funded by the MINECO (MDM-2014-0370). We also acknowledge the support of the CERCA Programme of the Generalitat de Catalunya. L.B.C. was supported by funding from Peking University and from the Peking-Tsinghua Center for Life Sciences, and from the Research Fund for International Young Scientists (National Natural Science Foundation of China

Molecular basis and clinical management of Pompe disease

Pompe disease (glycogenosis type II) is a rare autosomal recessive lysosomal storage disorder due to mutations of the GAA gene, leading to the deficiency of acid α-glucosidase and consequent glycogen storage in various tissues, mainly in the skeletal muscle, heart and liver. The consequent clinical picture is mainly due to the muscle and heart involvement, although clinical manifestations may be multi-systemic. The phenotype of patients is heterogeneous and the severity is inversely related to the residual enzymatic activity of acid α-glucosidase. More than 200 different mutations of GAA gene have been described and genotype/phenotype correlations have been established for some of them. Traditionally three forms have been described, i.e. early onset classical and non-classical forms and late onset attenuated forms. A severe hypertrophic cardiomyopathy in combination with conduction disorder in newborns represents a typical feature in the classic infantile presentation, while clinical picture in late onset forms is dominated by skeletal muscle dysfunction, resulting in mobility and respiratory problems. Enzyme replacement therapy with recombinant human GAA is the approved therapeutic approach in Pompe disease patients. Clinical trials on enzyme replacement therapy (ERT) support the efficacy in improving survival and hypertrophic cardiomyopathy, while efficacy seems to be variable on manifestations due to skeletal muscle involvement, mainly in lateonset patients. Considering the limitations of ERT and its high costs, innovative therapeutic approaches are now under development

Dynamics of alternative splicing during somatic cell reprogramming reveals functions for RNA-binding proteins CPSF3, hnRNP UL1, and TIA1

Background: Somatic cell reprogramming is the process that allows differentiated cells to revert to a pluripotent state. In contrast to the extensively studied rewiring of epigenetic and transcriptional programs required for reprogramming, the dynamics of post-transcriptional changes and their associated regulatory mechanisms remain poorly understood. Here we study the dynamics of alternative splicing changes occurring during efficient reprogramming of mouse B cells into induced pluripotent stem (iPS) cells and compare them to those occurring during reprogramming of mouse embryonic fibroblasts. Results: We observe a significant overlap between alternative splicing changes detected in the two reprogramming systems, which are generally uncoupled from changes in transcriptional levels. Correlation between gene expression of potential regulators and specific clusters of alternative splicing changes enables the identification and subsequent validation of CPSF3 and hnRNP UL1 as facilitators, and TIA1 as repressor of mouse embryonic fibroblasts reprogramming. We further find that these RNA-binding proteins control partially overlapping programs of splicing regulation, involving genes relevant for developmental and morphogenetic processes. Conclusions: Our results reveal common programs of splicing regulation during reprogramming of different cell types and identify three novel regulators of this process and their targets.C.V. was recipient of an FPI-Severo Ochoa Fellowship from the Spanish Ministry of Economy and Competitiveness. Work in J.V. laboratory is supported by the European Research Council (ERC AdvG 670146), AGAUR, Spanish Ministry of Economy and Competitiveness (BFU 2017 89308-P) and the Centre of Excellence Severo Ochoa. Work in T.G.’s laboratory was supported by the European Research Council FP7/2007-2013 (ERC Synergy Grant 4D-Genome) the Ministerio de Educación y Ciencia (SAF.2012-37167) and AGAUR. We acknowledge support of the Spanish Ministry of Science and Innovation to the EMBL partnership and the CERCA Programme / Generalitat de Catalunya

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.This work was supported by the Spanish Agencia Estatal de Investigación (AEI) of the Ministry of Science and Innovation (PID2020-114080GB-I00 and BFU2017-86760-P (AEI/FEDER, UE) to M.P.C.; and BFU2014-55275-P, BFU2017-88407-P, EUR2019-103817, PID2021-123383NB-I00 to B.P.), the AXA Research Fund (to B.P.) and the Agencia de Gestio d’Ajuts Universitaris i de Recerca (AGAUR, 2017 SGR 689 to M.P.C. and 2017 SGR 346 to B.P.). M.P.C. is supported by ICREA (Institucio Catalana de Recerca i Estudis Avançats). We would like to thank the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership and to the “Centro de Excelencia Severo Ochoa”. We also acknowledge support of the CERCA Programme of the Generalitat de Catalunya. M.V.N. has been supported by the People Program (Marie Curie Actions) FP7/2007–2013 under REA grant [608959] from the European Union and a Juan de la Cierva-Incorporación 2017 from the Spanish Ministry of Science and Innovation. P.A. has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 747488. M.B. has been supported by an EMBO postdoctoral fellowship (ALTF682–2021). J.T.L. is supported by the U.S. NIH grant, R01-MH118351. Portions of this work have appeared as part of the PhD dissertation “Screen for novel factors involved in pluripotency and X chromosome reactivation.“ by S.F.G. (Universitat Pompeu Fabra, 2019)