Publications by authors named "David Landeira"

15 Publications

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The molecular clock protein Bmal1 regulates cell differentiation in mouse embryonic stem cells.

Life Sci Alliance 2020 05 13;3(5). Epub 2020 Apr 13.

Centre for Genomics and Oncological Research (GENYO), Granada, Spain

Mammals optimize their physiology to the light-dark cycle by synchronization of the master circadian clock in the brain with peripheral clocks in the rest of the tissues of the body. Circadian oscillations rely on a negative feedback loop exerted by the molecular clock that is composed by transcriptional activators Bmal1 and Clock, and their negative regulators Period and Cryptochrome. Components of the molecular clock are expressed during early development, but onset of robust circadian oscillations is only detected later during embryogenesis. Here, we have used naïve pluripotent mouse embryonic stem cells (mESCs) to study the role of Bmal1 during early development. We found that, compared to wild-type cells, -/- mESCs express higher levels of Nanog protein and altered expression of pluripotency-associated signalling pathways. Importantly, mESCs display deficient multi-lineage cell differentiation capacity during the formation of teratomas and gastrula-like organoids. Overall, we reveal that Bmal1 regulates pluripotent cell differentiation and propose that the molecular clock is an hitherto unrecognized regulator of mammalian development.
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http://dx.doi.org/10.26508/lsa.201900535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156284PMC
May 2020

Polycomb regulation is coupled to cell cycle transition in pluripotent stem cells.

Sci Adv 2020 03 4;6(10):eaay4768. Epub 2020 Mar 4.

Centre for Genomics and Oncological Research (GENYO), Avenue de la Ilustración 114, 18016 Granada, Spain.

When self-renewing pluripotent cells receive a differentiation signal, ongoing cell duplication needs to be coordinated with entry into a differentiation program. Accordingly, transcriptional activation of lineage specifier genes and cell differentiation is confined to the G phase of the cell cycle by unknown mechanisms. We found that Polycomb repressive complex 2 (PRC2) subunits are differentially recruited to lineage specifier gene promoters across cell cycle in mouse embryonic stem cells (mESCs). Jarid2 and the catalytic subunit Ezh2 are markedly accumulated at target promoters during S and G phases, while the transcriptionally activating subunits EPOP and EloB are enriched during G phase. Fluctuations in the recruitment of PRC2 subunits promote changes in RNA synthesis and RNA polymerase II binding that are compromised in Jarid2 -/- mESCs. Overall, we show that differential recruitment of PRC2 subunits across cell cycle enables the establishment of a chromatin state that facilitates the induction of cell differentiation in G phase.
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http://dx.doi.org/10.1126/sciadv.aay4768DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056320PMC
March 2020

NOMePlot: analysis of DNA methylation and nucleosome occupancy at the single molecule.

Sci Rep 2019 05 31;9(1):8140. Epub 2019 May 31.

Centre for Genomics and Oncological Research (GENYO), Avenue de la Ilustración 114, 18016, Granada, Spain.

Recent technical advances highlight that to understand mammalian development and human disease we need to consider transcriptional and epigenetic cell-to-cell differences within cell populations. This is particularly important in key areas of biomedicine like stem cell differentiation and intratumor heterogeneity. The recently developed nucleosome occupancy and methylome (NOMe) assay facilitates the simultaneous study of DNA methylation and nucleosome positioning on the same DNA strand. NOMe-treated DNA can be sequenced by sanger (NOMe-PCR) or high throughput approaches (NOMe-seq). NOMe-PCR provides information for a single locus at the single molecule while NOMe-seq delivers genome-wide data that is usually interrogated to obtain population-averaged measures. Here, we have developed a bioinformatic tool that allow us to easily obtain locus-specific information at the single molecule using genome-wide NOMe-seq datasets obtained from bulk populations. We have used NOMePlot to study mouse embryonic stem cells and found that polycomb-repressed bivalent gene promoters coexist in two different epigenetic states, as defined by the nucleosome binding pattern detected around their transcriptional start site.
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http://dx.doi.org/10.1038/s41598-019-44597-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544651PMC
May 2019

Activating Transcription Factor 4 Modulates TGFβ-Induced Aggressiveness in Triple-Negative Breast Cancer via SMAD2/3/4 and mTORC2 Signaling.

Clin Cancer Res 2018 11 16;24(22):5697-5709. Epub 2018 Jul 16.

UGC de Oncología Médica, Complejo Hospitalario de Jaén, Jaén, Spain.

On the basis of the identified stress-independent cellular functions of activating transcription factor 4 (ATF4), we reported enhanced ATF4 levels in MCF10A cells treated with TGFβ1. is overexpressed in patients with triple-negative breast cancer (TNBC), but its impact on patient survival and the underlying mechanisms remain unknown. We aimed to determine effects on patients with breast cancer survival and TNBC aggressiveness, and the relationships between TGFβ and ATF4. Defining the signaling pathways may help us identify a cell signaling-tailored gene signature. Patient survival data were determined by Kaplan-Meier analysis. Relationship between TGFβ and ATF4, their effects on aggressiveness (tumor proliferation, metastasis, and stemness), and the underlying pathways were analyzed in three TNBC cell lines and using patient-derived xenografts (PDX). overexpression correlated with TNBC patient survival decrease and a SMAD-dependent crosstalk between ATF4 and TGFβ was identified. expression inhibition reduced migration, invasiveness, mammosphere-forming efficiency, proliferation, epithelial-mesenchymal transition, and antiapoptotic and stemness marker levels. In PDX models, silencing decreased metastases, tumor growth, and relapse after chemotherapy. ATF4 was shown to be active downstream of SMAD2/3/4 and mTORC2, regulating TGFβ/SMAD and mTOR/RAC1-RHOA pathways independently of stress. We defined an eight-gene signature with prognostic potential, altered in 45% of 2,509 patients with breast cancer. ATF4 may represent a valuable prognostic biomarker and therapeutic target in patients with TNBC, and we identified a cell signaling pathway-based gene signature that may contribute to the development of combinatorial targeted therapies for breast cancer. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-3125DOI Listing
November 2018

Jarid2 Coordinates Nanog Expression and PCP/Wnt Signaling Required for Efficient ESC Differentiation and Early Embryo Development.

Cell Rep 2015 Jul 16;12(4):573-86. Epub 2015 Jul 16.

Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK. Electronic address:

Jarid2 is part of the Polycomb Repressor complex 2 (PRC2) responsible for genome-wide H3K27me3 deposition. Unlike other PRC2-deficient embryonic stem cells (ESCs), however, Jarid2-deficient ESCs show a severe differentiation block, altered colony morphology, and distinctive patterns of deregulated gene expression. Here, we show that Jarid2(-/-) ESCs express constitutively high levels of Nanog but reduced PCP signaling components Wnt9a, Prickle1, and Fzd2 and lowered β-catenin activity. Depletion of Wnt9a/Prickle1/Fzd2 from wild-type ESCs or overexpression of Nanog largely phenocopies these cellular defects. Co-culture of Jarid2(-/-) with wild-type ESCs restores variable Nanog expression and β-catenin activity and can partially rescue the differentiation block of mutant cells. In addition, we show that ESCs lacking Jarid2 or Wnt9a/Prickle1/Fzd2 or overexpressing Nanog induce multiple ICM formation when injected into normal E3.5 blastocysts. These data describe a previously unrecognized role for Jarid2 in regulating a core pluripotency and Wnt/PCP signaling circuit that is important for ESC differentiation and for pre-implantation development.
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http://dx.doi.org/10.1016/j.celrep.2015.06.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4534826PMC
July 2015

Different roles for Tet1 and Tet2 proteins in reprogramming-mediated erasure of imprints induced by EGC fusion.

Mol Cell 2013 Mar 28;49(6):1023-33. Epub 2013 Feb 28.

Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK.

Genomic imprinting directs the allele-specific marking and expression of loci according to their parental origin. Differential DNA methylation at imprinted control regions (ICRs) is established in gametes and, although largely preserved through development, can be experimentally reset by fusing somatic cells with embryonic germ cell (EGC) lines. Here, we show that the Ten-Eleven Translocation proteins Tet1 and Tet2 participate in the efficient erasure of imprints in this model system. The fusion of B cells with EGCs initiates pluripotent reprogramming, in which rapid re-expression of Oct4 is accompanied by an accumulation of 5-hydroxymethylcytosine (5hmC) at several ICRs. Tet2 was required for the efficient reprogramming capacity of EGCs, whereas Tet1 was necessary to induce 5-methylcytosine oxidation specifically at ICRs. These data show that the Tet1 and Tet2 proteins have discrete roles in cell-fusion-mediated pluripotent reprogramming and imprint erasure in somatic cells.
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http://dx.doi.org/10.1016/j.molcel.2013.01.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3613797PMC
March 2013

DNA synthesis is required for reprogramming mediated by stem cell fusion.

Cell 2013 Feb;152(4):873-83

Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College London, Du Cane Road, London W12 0NN, UK.

Embryonic stem cells (ESCs) can instruct the conversion of differentiated cells toward pluripotency following cell-to-cell fusion by a mechanism that is rapid but poorly understood. Here, we used centrifugal elutriation to enrich for mouse ESCs at sequential stages of the cell cycle and showed that ESCs in S/G2 phases have an enhanced capacity to dominantly reprogram lymphocytes and fibroblasts in heterokaryon and hybrid assays. Reprogramming success was associated with an ability to induce precocious nucleotide incorporation within the somatic partner nuclei in heterokaryons. BrdU pulse-labeling experiments revealed that virtually all successfully reprogrammed somatic nuclei, identified on the basis of Oct4 re-expression, had undergone DNA synthesis within 24 hr of fusion with ESCs. This was essential for successful reprogramming because drugs that inhibited DNA polymerase activity effectively blocked pluripotent conversion. These data indicate that nucleotide incorporation is an early and critical event in the epigenetic reprogramming of somatic cells in experimental ESC-heterokaryons.
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http://dx.doi.org/10.1016/j.cell.2013.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3605571PMC
February 2013

Inactive yet indispensable: the tale of Jarid2.

Trends Cell Biol 2011 Feb 10;21(2):74-80. Epub 2010 Nov 10.

Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK.

Methylation of histone tails is believed to be important for the establishment and inheritance of gene expression programs during development. Jarid2/Jumonji is the founding member of a family of chromatin modifiers with histone demethylase activity. Although Jarid2 contains amino acid substitutions that are thought to abolish its catalytic activity, it is essential for the development of multiple organs in mice. Recent studies have shown that Jarid2 is a component of the polycomb repressive complex 2 and is required for embryonic stem (ES) cell differentiation. Here, we discuss current literature on the function of Jarid2 and hypothesize that defects resulting from Jarid2 deficiency arise from a failure to correctly prime genes in ES cells that are required for later stages in development.
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http://dx.doi.org/10.1016/j.tcb.2010.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3034028PMC
February 2011

ESCs require PRC2 to direct the successful reprogramming of differentiated cells toward pluripotency.

Cell Stem Cell 2010 Jun;6(6):547-56

MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, London, UK.

Embryonic stem cells (ESCs) are pluripotent, self-renewing, and have the ability to reprogram differentiated cell types to pluripotency upon cellular fusion. Polycomb-group (PcG) proteins are important for restraining the inappropriate expression of lineage-specifying factors in ESCs. To investigate whether PcG proteins are required for establishing, rather than maintaining, the pluripotent state, we compared the ability of wild-type, PRC1-, and PRC2-depleted ESCs to reprogram human lymphocytes. We show that ESCs lacking either PRC1 or PRC2 are unable to successfully reprogram B cells toward pluripotency. This defect is a direct consequence of the lack of PcG activity because it could be efficiently rescued by reconstituting PRC2 activity in PRC2-deficient ESCs. Surprisingly, the failure of PRC2-deficient ESCs to reprogram somatic cells is functionally dominant, demonstrating a critical requirement for PcG proteins in the chromatin-remodeling events required for the direct conversion of differentiated cells toward pluripotency.
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http://dx.doi.org/10.1016/j.stem.2010.04.013DOI Listing
June 2010

Jarid2 is a PRC2 component in embryonic stem cells required for multi-lineage differentiation and recruitment of PRC1 and RNA Polymerase II to developmental regulators.

Nat Cell Biol 2010 Jun 16;12(6):618-24. Epub 2010 May 16.

Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN UK.

Polycomb Repressor Complexes (PRCs) are important regulators of embryogenesis. In embryonic stem (ES) cells many genes that regulate subsequent stages in development are enriched at their promoters for PRC1, PRC2 and Ser 5-phosphorylated RNA Polymerase II (RNAP), and contain domains of 'bivalent' chromatin (enriched for H3K4me3; histone H3 di- or trimethylated at Lys 4 and H3K27me3; histone H3 trimethylated at Lys 27). Loss of individual PRC components in ES cells can lead to gene de-repression and to unscheduled differentiation. Here we show that Jarid2 is a novel subunit of PRC2 that is required for the co-recruitment of PRC1 and RNAP to genes that regulate development in ES cells. Jarid2-deficient ES cells showed reduced H3K4me2/me3 and H3K27me3 marking and PRC1/PRC2 recruitment, and did not efficiently establish Ser 5-phosporylated RNAP at target genes. ES cells lacking Jarid2, in contrast to previously characterized PRC1 and PRC2 mutants, did not inappropriately express PRC2 target genes. Instead, they show a severely compromised capacity for successful differentiation towards neural or mesodermal fates and failed to correctly initiate lineage-specific gene expression in vitro. Collectively, these data indicate that transcriptional priming of bivalent genes in pluripotent ES cells is Jarid2-dependent, and suggests that priming is critical for subsequent multi-lineage differentiation.
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http://dx.doi.org/10.1038/ncb2065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3572404PMC
June 2010

Cohesin regulates VSG monoallelic expression in trypanosomes.

J Cell Biol 2009 Jul;186(2):243-54

Instituto de Parasitología y Biomedicina López-Neyra, Consejo Superior de Investigaciones Cientificas, 18100 Granada, Spain.

Antigenic variation allows Trypanosoma brucei to evade the host immune response by switching the expression of 1 out of approximately 15 telomeric variant surface glycoprotein (VSG) expression sites (ESs). VSG ES transcription is mediated by RNA polymerase I in a discrete nuclear site named the ES body (ESB). However, nothing is known about how the monoallelic VSG ES transcriptional state is maintained over generations. In this study, we show that during S and G2 phases and early mitosis, the active VSG ES locus remains associated with the single ESB and exhibits a delay in the separation of sister chromatids relative to control loci. This delay is dependent on the cohesin complex, as partial knockdown of cohesin subunits resulted in premature separation of sister chromatids of the active VSG ES. Cohesin depletion also prompted transcriptional switching from the active to previously inactive VSG ESs. Thus, in addition to maintaining sister chromatid cohesion during mitosis, the cohesin complex plays an essential role in the correct epigenetic inheritance of the active transcriptional VSG ES state.
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http://dx.doi.org/10.1083/jcb.200902119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2717648PMC
July 2009

RNA pol II subunit RPB7 is required for RNA pol I-mediated transcription in Trypanosoma brucei.

EMBO Rep 2009 Mar 23;10(3):252-7. Epub 2009 Jan 23.

Instituto de Parasitología y Biomedicina López-Neyra, Consejo Superior de Investigaciones Científicas, CSIC (Spanish National Research Council), Avda. del Conocimiento s/n, 18100 Granada, Spain.

In the protozoan parasite Trypanosoma brucei, the two main surface glycoprotein genes are transcribed by RNA polymerase I (pol I) instead of RNA pol II, the polymerase committed to the production of mRNA in eukaryotes. This unusual feature might be accomplished by the recruitment of specific subunits or cofactors that allow pol I to transcribe protein-coding RNAs. Here, we report that transcription mediated by pol I requires TbRPB7, a dissociable subunit of the pol II complex. TbRPB7 was found to interact with two pol I-specific subunits, TbRPA1 and TbRPB6z. Pol I-specific transcription was affected on depletion of TbRPB7 in run-on assays, whereas recombinant TbRPB7 increased transcription driven by a pol I promoter. These results represent a unique example of a functional RNA polymerase chimaera consisting of a core pol I complex that recruits a specific pol II subunit.
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http://dx.doi.org/10.1038/embor.2008.244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2658562PMC
March 2009

Nuclear architecture underlying gene expression in Trypanosoma brucei.

Trends Microbiol 2007 Jun 4;15(6):263-70. Epub 2007 May 4.

Instituto de Parasitología y Biomedicina López-Neyra, Consejo Superior de Investigaciones Científicas (Spanish National Research Council), Avda. del Conocimiento s/n, 18100 Granada, Spain.

The influence of nuclear architecture on the regulation of developmental gene expression has recently become evident in many organisms ranging from yeast to humans. During interphase, chromosomes and nuclear structures are in constant motion; therefore, correct temporal association is needed to meet the requirements of gene expression. Trypanosoma brucei is an excellent model system in which to analyze nuclear spatial implications in the regulation of gene expression because the two main surface-protein genes (procyclin and VSG) are transcribed by the highly compartmentalized RNA polymerase I and undergo distinct transcriptional activation or downregulation during developmental differentiation. Furthermore, the infective bloodstream form of the parasite undergoes antigenic variation, displaying sequentially different types of VSG by allelic exclusion. Here, we discuss recent advances in understanding the role of chromosomal nuclear positioning in the regulation of gene expression in T. brucei.
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http://dx.doi.org/10.1016/j.tim.2007.04.004DOI Listing
June 2007

Nuclear repositioning of the VSG promoter during developmental silencing in Trypanosoma brucei.

J Cell Biol 2007 Jan 8;176(2):133-9. Epub 2007 Jan 8.

Instituto de Parasitologia y Biomedicina Lopez-Neyra, Consejo Superior de Investigaciones Científicas, Spanish National Research Council, 18100 Granada, Spain.

Interphase nuclear repositioning of chromosomes has been implicated in the epigenetic regulation of RNA polymerase (pol) II transcription. However, little is known about the nuclear position-dependent regulation of RNA pol I-transcribed loci. Trypanosoma brucei is an excellent model system to address this question because its two main surface protein genes, procyclin and variant surface glycoprotein (VSG), are transcribed by pol I and undergo distinct transcriptional activation or downregulation events during developmental differentiation. Although the monoallelically expressed VSG locus is exclusively localized to an extranucleolar body in the bloodstream form, in this study, we report that nonmutually exclusive procyclin genes are located at the nucleolar periphery. Interestingly, ribosomal DNA loci and pol I transcription activity are restricted to similar perinucleolar positions. Upon developmental transcriptional downregulation, however, the active VSG promoter selectively undergoes a rapid and dramatic repositioning to the nuclear envelope. Subsequently, the VSG promoter region was subjected to chromatin condensation. We propose a model whereby the VSG expression site pol I promoter is selectively targeted by temporal nuclear repositioning during developmental silencing.
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http://dx.doi.org/10.1083/jcb.200607174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2063932PMC
January 2007