Telomeres are transcribed generating long non-coding RNAs known as TERRA. Deciphering the role of TERRA has been one of the unsolved issues of telomere biology in the past decade. This has been, in part, due to lack of knowledge on the TERRA loci, thus preventing functional genetic studies. Here, we describe that long non-coding RNAs with TERRA features are transcribed from the human 20q and Xp subtelomeres. Deletion of the 20q locus by using the CRISPR-Cas9 technology causes a dramatic decrease in TERRA levels, while deletion of the Xp locus does not result in decreased TERRA levels. Strikingly, 20q-TERRA ablation leads to dramatic loss of telomere sequences and the induction of a massive DNA damage response. These findings identify chromosome 20q as a main TERRA locus in human cells and represent the first demonstration in any organism of the essential role of TERRA in the maintenance of telomeres. ; We are indebted to R. Torres and S. Rodriguez for advice in the CRISPR-Cas9 technology and helpful discussions. We thank D. Megías for the quantification of confocal imagesignals and to O. Dominguez for troubleshooting with cloning and sequencing. We thank S. Llanos for the p21 promoter reporter (described in ref. 33). Research in the Blasco lab is funded by the Spanish Ministry of Economy and Competitiveness Project (SAF2013-45111-R), the Madrid Regional Government Project S2010/BMD-2303 (ReCaRe),Fundacio´n Botı´n (Spain) and AXA Research Fund (AXA 2011, Spain). ; Sí
Telomeric RNAs (TERRAs) are UUAGGG repeat-containing RNAs that are transcribed from the subtelomere towards the telomere. The precise genomic origin of TERRA has remained elusive. Using a whole-genome RNA-sequencing approach, we identify novel mouse transcripts arising mainly from the subtelomere of chromosome 18, and to a lesser extend chromosome 9, that resemble TERRA in several key aspects. Those transcripts contain UUAGGG-repeats and are heterogeneous in size, fluctuate in abundance in a TERRA-like manner during the cell cycle, are bound by TERRA RNA-binding proteins and are regulated in a manner similar to TERRA in response to stress and the induction of pluripotency. These transcripts are also found to associate with nearly all chromosome ends and downregulation of the transcripts that originate from chromosome 18 causes a reduction in TERRA abundance. Interestingly, downregulation of either chromosome 18 transcripts or TERRA results in increased number of telomere dysfunction-induced foci, suggesting a protective role at telomeres. ; We are indebted to Stefan Schoeftner and Susana Llanos for reagents and to Manuel Serrano, Maria Elisa Varela and Antonio Maraver for very helpful suggestions and discussion on the manuscript. We thank Diego Megias for confocal image acquisition and to Miguel Angel Grillo, Maria del Carmen Carralero and Juan Cruz Cigudosa for the Spectral Karyotyping (SKY). We thank Luis E. Donate for manuscript preparation. External RNAseq data were generated and analysed by the UW ENCODE group and by the transcriptome group at Cold Spring Harbor Laboratories and the Center for Genomic Regulation (CRG in Barcelona), who are participants in the ENCODE Transcriptome Group. ChIP data of transcription factors binding site were generated and analysed by the laboratories of Michael Snyder at Stanford University and Sherman Weissman at Yale University within the ENCODE Project. Histone marks data belong to the Caltech/ENCODE project in which cell growth, ChIP and Illumina library construction were done in the laboratory of Barbara Wold (California Institute of Technology). Sequencing was done at the Millard and Muriel Jacobs Genetics and Genomics Laboratory at the California Institute of Technology, initial HiSeq data were generated at Illumina Inc., Hawyard, CA. Cell growth and ChIP of histone marks were carried out by Georgi Marinov, Katherine Fisher, Gordon Kwan, Antony Kirilusha, Ali Mortazavi, Gilberto DeSalvo and Brian Williams. Library Construction, Sequencing and Primary Data Handling by Lorianne Schaeffer, Diane Trout, Igor Antoschechkin (California Institute of Technology), Lu Zhang and Gary Schroth (Illumina Inc.). Data processing and submission by Georgi Marinov and Diane Trout. Research in the Blasco laboratory is funded by the Spanish Ministry of Economy and Competitiveness Projects SAF2008-05384 and CSD2007-00017, the Madrid Regional Government Project S2010/BMD-2303 (ReCaRe), the European Union FP7 Project FHEALTH-2010-259749 (EuroBATS), the European Research Council (ERC) Project GA#232854 (TEL STEM CELL), the Preclinical Research Award from Fundacion Lilly (Spain), Fundacion Botin (Spain) and AXA Research Fund. ; Sí
The RNA polymerase II-associated protein 1 (RPAP1) is conserved across metazoa and required for stem cell differentiation in plants; however, very little is known about its mechanism of action or its role in mammalian cells. Here, we report that RPAP1 is essential for the expression of cell identity genes and for cell viability. Depletion of RPAP1 triggers cell de-differentiation, facilitates reprogramming toward pluripotency, and impairs differentiation. Mechanistically, we show that RPAP1 is essential for the interaction between RNA polymerase II (RNA Pol II) and Mediator, as well as for the recruitment of important regulators, such as the Mediator-specific RNA Pol II factor Gdown1 and the C-terminal domain (CTD) phosphatase RPAP2. In agreement, depletion of RPAP1 diminishes the loading of total and Ser5-phosphorylated RNA Pol II on many genes, with super-enhancer-driven genes among the most significantly downregulated. We conclude that Mediator/RPAP1/RNA Pol II is an ancient module, conserved from plants to mammals, critical for establishing and maintaining cell identity. Lynch et al. report a regulator of RNA Pol II called RPAP1, displaying functional conservation from plants to mammals. RPAP1 is required to establish and maintain cell identity. Mechanistically, RPAP1 is critical for the Mediator-RNA Pol II interaction, thereby preserving normal transcription at enhancer-driven genes ; Work in the laboratory of M.S. is funded by the CNIO and the IRB and by grants from the Spanish Ministry of Economy co-funded by the European Regional Development Fund (ERDF) (SAF2013-48256-R), the European Research Council (ERC-2014-AdG/669622), the Regional Government of Madrid co-funded by the European Social Fund (ReCaRe project), the European Union (RISK-IR project), the Botin Foundation and Banco Santander (Santander Universities Global Division), the Ramon Areces Foundation, and the AXA Foundation. S.R. was funded by a contract from the Ramon y Cajal Program(RYC-2011-09242) and by the Spanish Ministry of Economy cofunded by the ERDF (SAF2013-49147-P and SAF2016-80874-P).
The RNA polymerase II-associated protein 1 (RPAP1) is conserved across metazoa and required for stem cell differentiation in plants; however, very little is known about its mechanism of action or its role in mammalian cells. Here, we report that RPAP1 is essential for the expression of cell identity genes and for cell viability. Depletion of RPAP1 triggers cell de-differentiation, facilitates reprogramming toward pluripotency, and impairs differentiation. Mechanistically, we show that RPAP1 is essential for the interaction between RNA polymerase II (RNA Pol II) and Mediator, as well as for the recruitment of important regulators, such as the Mediator-specific RNA Pol II factor Gdown1 and the C-terminal domain (CTD) phosphatase RPAP2. In agreement, depletion of RPAP1 diminishes the loading of total and Ser5-phosphorylated RNA Pol II on many genes, with super-enhancer-driven genes among the most significantly downregulated. We conclude that Mediator/RPAP1/RNA Pol II is an ancient module, conserved from plants to mammals, critical for establishing and maintaining cell identity. ; We are grateful to Elisa Varela for assistance with morula and blastocyst fixa- tion. Work in the laboratory of M.S. is funded by the CNIO and the IRB and by grants from the Spanish Ministry of Economy co-funded by the European Regional Development Fund (ERDF) (SAF2013-48256-R), the European Research Co uncil (ERC-2014-AdG/66 9622), the Region al Government of Ma- drid co-funded by the Euro pean Social Fund (ReCaRe project), the Euro pean Union (RISK-IR project), the Botin Foundation and Banco Santander (Santander Universities Glo bal Division), the Ramon Areces Found ation, and the AXA Foundation. S.R. was funded by a contract from the Ramon y Cajal Program(RYC-2011-09242) and by the Spanish Ministry of Economy co- funded by the ERDF (SAF2013-49147- P and SAF2016-80874-P ; Sí
Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here we analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines were enriched in nerve growth factor (NGF) receptor (NGFR, p75NTR), spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK kinase, nuclear factor (NF)-κB activation and intracellular adhesion molecule (ICAM)-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to that in matched primary tumors, and the frequency of NGFR+ metastatic melanoma cells in lymph nodes correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs, reinforcing lymph node pre-metastatic niche formation and metastasis. ; we apologize to those authors whose work could not be cited due to size restrictions. We thank M. S. Soengas and the members of her laboratory for melanoma cells, primary melanocyte preparations and helpful discussions. We thank M. Detmar and S. Proulx for the mouse B16-F1R2 cell line. We are grateful to M. Yañez-Mo and M. Valés for antibodies against sEV markers. We thank D. Grela and A. Escobar from IESMAT for their support with the Zetasizer analysis. We thank G. Roncador, L. Maestre and J. L. Martinez Torrecuadrada for their help with the development and characterization of anti-NGFR antibodies and C. Villarroya Beltri for her help in flow cytometry analysis. This work was funded by the Starr Cancer Consortium (B.J.M., D.L. and H.P.), the US NIH (R01-CA169416), the Nancy C. and Daniel P. Paduano Foundation, the Children's Cancer and Blood Foundation (H.P. and D.L.), the Melanoma Research Alliance, the Feldstein Foundation, RETOS SAF2017-82924-R (AEI/10.13039/501100011033/FEDER-UE), the Fundación Ramón Areces, the Fundación Bancaria 'la Caixa' (HR18-00256), ATRES-MEDIA AXA Foundation (CONSTANTES Y VITALES, una iniciativa de laSexta y Fundación AXA) and the Fundación Científica AECC (LABAE19027PEIN, GCB15152978SOEN-HP) (H.P.), the Malcolm Hewitt Wiener Foundation, the AHEPA Fifth District Cancer Research Foundation, the Hartwell Foundation and the Manning Foundation (D.L.). We are also grateful for the support of the Translational Network for the Clinical Application of Extracellular Vesicles (TeNTaCLES), RED2018-102411-T (AEI/10.13039/501100011033), the Ramón y Cajal Programme, the FERO Foundation, Comunidad of Madrid 2017-T2/BMD6026 (L.N.) and La Caixa Foundation (ID100010434, fellowship LCF/BQ/ES17/11600007) (A.H.-B.). The CNIO, certified as a Severo Ochoa Excellence Centre, is supported by the Spanish government through the ISCIII. ; No
Histone H4 acetylation at Lysine 16 (H4K16ac) is a key epigenetic mark involved in gene regulation, DNA repair and chromatin remodeling, and though it is known to be essential for embryonic development, its role during adult life is still poorly understood. Here we show that this lysine is massively hyperacetylated in peripheral neutrophils. Genome-wide mapping of H4K16ac in terminally differentiated blood cells, along with functional experiments, supported a role for this histone post-translational modification in the regulation of cell differentiation and apoptosis in the hematopoietic system. Furthermore, in neutrophils, H4K16ac was enriched at specific DNA repeats. These DNA regions presented an accessible chromatin conformation and were associated with the cleavage sites that generate the 50 kb DNA fragments during the first stages of programmed cell death. Our results thus suggest that H4K16ac plays a dual role in myeloid cells as it not only regulates differentiation and apoptosis, but it also exhibits a non-canonical structural role in poising chromatin for cleavage at an early stage of neutrophil cell death. ; Plan Nacional de I+D+I co-funding FEDER [PI15/00892 and PI18/01527 to M.F.F. and A.F.F.; PI16/01318 and PI14/01244 to C.L.]; ISCIII-Subdireccion General de Evaluacion y Fomento de la Investigacion, and Plan Nacional de I+D+I 2008–2011/FEDER [CP11/00131 to A.F.F.]; IUOPA (to G.F.B. and M.I.S.); Fundacion Cientifica de la AECC (to R.G.U.); Ministry of Economy and Competitiveness Juan de la Cierva postdoctoral fellowships [FJCI-2015-26965 to J.R.T., IJCI-2015- 23316 to V.L.]; Fundacion Ramon Areces (to M.F.F); FICYT (to E.G.T., M.G.G., A.C.); Asturias Regional Government [GRUPIN14-052 to M.F.F.]; Gobierno del Principado de Asturias, PCTI-Plan de Ciencia, Tecnologia e Innovacion co-funding Fondos FEDER (grant number IDI/2018/146 to M.F.F. and IDI/2018/144 to C.L.); Deutsche Forschungsgemeinschaft (DFG) [SFB960 to A.V.G., R.D.]; European Research Council [CoG-2014-646903]; Spanish Ministry of Economy and Competitiveness [SAF-SAF2013-43065 to P.M.]; Asociacion Española Contra el Cancer [AECC-CI-2015]; FERO Foundation, and the ISCIII [PI14-01191 to C.B.]; P.M. acknowledges financial support from The Obra Social La Caixa Fundacio Josep Carreras and The Generalitat de Catalunya (SGR330). P.M. an investigator from the Spanish Cell Therapy cooperative network (TERCEL). The IUOPA is supported by the Obra Social Liberbank-Cajastur, Spain. Funding for open access charge: Plan Nacional de I+D+I co-funding FEDER [PI18/01527].
