Publications by authors named "Miha Modic"

15 Publications

  • Page 1 of 1

Recent evolution of a TET-controlled and DPPA3/STELLA-driven pathway of passive DNA demethylation in mammals.

Nat Commun 2020 11 24;11(1):5972. Epub 2020 Nov 24.

Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Human Biology and BioImaging, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany.

Genome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. Here, we describe a recently evolved pathway in which global hypomethylation is achieved by the coupling of active and passive demethylation. TET activity is required, albeit indirectly, for global demethylation, which mostly occurs at sites devoid of TET binding. Instead, TET-mediated active demethylation is locus-specific and necessary for activating a subset of genes, including the naïve pluripotency and germline marker Dppa3 (Stella, Pgc7). DPPA3 in turn drives large-scale passive demethylation by directly binding and displacing UHRF1 from chromatin, thereby inhibiting maintenance DNA methylation. Although unique to mammals, we show that DPPA3 alone is capable of inducing global DNA demethylation in non-mammalian species (Xenopus and medaka) despite their evolutionary divergence from mammals more than 300 million years ago. Our findings suggest that the evolution of Dppa3 facilitated the emergence of global DNA demethylation in mammals.
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http://dx.doi.org/10.1038/s41467-020-19603-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686362PMC
November 2020

Distinct and stage-specific contributions of TET1 and TET2 to stepwise cytosine oxidation in the transition from naive to primed pluripotency.

Sci Rep 2020 07 21;10(1):12066. Epub 2020 Jul 21.

Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany.

Cytosine DNA bases can be methylated by DNA methyltransferases and subsequently oxidized by TET proteins. The resulting 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are considered demethylation intermediates as well as stable epigenetic marks. To dissect the contributions of these cytosine modifying enzymes, we generated combinations of Tet knockout (KO) embryonic stem cells (ESCs) and systematically measured protein and DNA modification levels at the transition from naive to primed pluripotency. Whereas the increase of genomic 5-methylcytosine (5mC) levels during exit from pluripotency correlated with an upregulation of the de novo DNA methyltransferases DNMT3A and DNMT3B, the subsequent oxidation steps turned out to be far more complex. The strong increase of oxidized cytosine bases (5hmC, 5fC, and 5caC) was accompanied by a drop in TET2 levels, yet the analysis of KO cells suggested that TET2 is responsible for most 5fC formation. The comparison of modified cytosine and enzyme levels in Tet KO cells revealed distinct and differentiation-dependent contributions of TET1 and TET2 to 5hmC and 5fC formation arguing against a processive mechanism of 5mC oxidation. The apparent independent steps of 5hmC and 5fC formation suggest yet to be identified mechanisms regulating TET activity that may constitute another layer of epigenetic regulation.
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http://dx.doi.org/10.1038/s41598-020-68600-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7374584PMC
July 2020

In vitro capture and characterization of embryonic rosette-stage pluripotency between naive and primed states.

Nat Cell Biol 2020 05 4;22(5):534-545. Epub 2020 May 4.

Department of Cell Biology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands.

Following implantation, the naive pluripotent epiblast of the mouse blastocyst generates a rosette, undergoes lumenogenesis and forms the primed pluripotent egg cylinder, which is able to generate the embryonic tissues. How pluripotency progression and morphogenesis are linked and whether intermediate pluripotent states exist remain controversial. We identify here a rosette pluripotent state defined by the co-expression of naive factors with the transcription factor OTX2. Downregulation of blastocyst WNT signals drives the transition into rosette pluripotency by inducing OTX2. The rosette then activates MEK signals that induce lumenogenesis and drive progression to primed pluripotency. Consequently, combined WNT and MEK inhibition supports rosette-like stem cells, a self-renewing naive-primed intermediate. Rosette-like stem cells erase constitutive heterochromatin marks and display a primed chromatin landscape, with bivalently marked primed pluripotency genes. Nonetheless, WNT induces reversion to naive pluripotency. The rosette is therefore a reversible pluripotent intermediate whereby control over both pluripotency progression and morphogenesis pivots from WNT to MEK signals.
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http://dx.doi.org/10.1038/s41556-020-0508-xDOI Listing
May 2020

Integrative biology studies in pluripotent stem cells.

Stem Cell Res 2020 01 12;42:101686. Epub 2019 Dec 12.

Department of Cell Biology, Erasmus MC, University Medical Center Rotterdam, Postbus 2040, 3000 CA Rotterdam, The Netherlands. Electronic address:

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http://dx.doi.org/10.1016/j.scr.2019.101686DOI Listing
January 2020

Widespread FUS mislocalization is a molecular hallmark of amyotrophic lateral sclerosis.

Brain 2019 09;142(9):2572-2580

The Francis Crick Institute, 1 Midland Road, London, UK.

Mutations causing amyotrophic lateral sclerosis (ALS) clearly implicate ubiquitously expressed and predominantly nuclear RNA binding proteins, which form pathological cytoplasmic inclusions in this context. However, the possibility that wild-type RNA binding proteins mislocalize without necessarily becoming constituents of cytoplasmic inclusions themselves remains relatively unexplored. We hypothesized that nuclear-to-cytoplasmic mislocalization of the RNA binding protein fused in sarcoma (FUS), in an unaggregated state, may occur more widely in ALS than previously recognized. To address this hypothesis, we analysed motor neurons from a human ALS induced-pluripotent stem cell model caused by the VCP mutation. Additionally, we examined mouse transgenic models and post-mortem tissue from human sporadic ALS cases. We report nuclear-to-cytoplasmic mislocalization of FUS in both VCP-mutation related ALS and, crucially, in sporadic ALS spinal cord tissue from multiple cases. Furthermore, we provide evidence that FUS protein binds to an aberrantly retained intron within the SFPQ transcript, which is exported from the nucleus into the cytoplasm. Collectively, these data support a model for ALS pathogenesis whereby aberrant intron retention in SFPQ transcripts contributes to FUS mislocalization through their direct interaction and nuclear export. In summary, we report widespread mislocalization of the FUS protein in ALS and propose a putative underlying mechanism for this process.
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http://dx.doi.org/10.1093/brain/awz217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6735815PMC
September 2019

Choroid plexus-derived miR-204 regulates the number of quiescent neural stem cells in the adult brain.

EMBO J 2019 09 15;38(17):e100481. Epub 2019 Jul 15.

Institute of Stem Cell Research, Helmholtz Center Munich, Neuherberg, Germany.

Regulation of adult neural stem cell (NSC) number is critical for lifelong neurogenesis. Here, we identified a post-transcriptional control mechanism, centered around the microRNA 204 (miR-204), to control the maintenance of quiescent (q)NSCs. miR-204 regulates a spectrum of transcripts involved in cell cycle regulation, neuronal migration, and differentiation in qNSCs. Importantly, inhibition of miR-204 function reduced the number of qNSCs in the subependymal zone (SEZ) by inducing pre-mature activation and differentiation of NSCs without changing their neurogenic potential. Strikingly, we identified the choroid plexus of the mouse lateral ventricle as the major source of miR-204 that is released into the cerebrospinal fluid to control number of NSCs within the SEZ. Taken together, our results describe a novel mechanism to maintain adult somatic stem cells by a niche-specific miRNA repressing activation and differentiation of stem cells.
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http://dx.doi.org/10.15252/embj.2018100481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6717894PMC
September 2019

Cross-Regulation between TDP-43 and Paraspeckles Promotes Pluripotency-Differentiation Transition.

Mol Cell 2019 06 29;74(5):951-965.e13. Epub 2019 Apr 29.

Institute of Stem Cell Research, Helmholtz Zentrum München, 85764 Neuherberg, Germany; Comprehensive Pneumology Center (CPC-M), Ludwig-Maximilians-Universität München, Asklepios Fachkliniken München-Gauting und Helmholtz Zentrum München, Max-Lebsche-Platz 31, 81377 Munich, Germany. Electronic address:

RNA-binding proteins (RBPs) and long non-coding RNAs (lncRNAs) are key regulators of gene expression, but their joint functions in coordinating cell fate decisions are poorly understood. Here we show that the expression and activity of the RBP TDP-43 and the long isoform of the lncRNA Neat1, the scaffold of the nuclear compartment "paraspeckles," are reciprocal in pluripotent and differentiated cells because of their cross-regulation. In pluripotent cells, TDP-43 represses the formation of paraspeckles by enhancing the polyadenylated short isoform of Neat1. TDP-43 also promotes pluripotency by regulating alternative polyadenylation of transcripts encoding pluripotency factors, including Sox2, which partially protects its 3' UTR from miR-21-mediated degradation. Conversely, paraspeckles sequester TDP-43 and other RBPs from mRNAs and promote exit from pluripotency and embryonic patterning in the mouse. We demonstrate that cross-regulation between TDP-43 and Neat1 is essential for their efficient regulation of a broad network of genes and, therefore, of pluripotency and differentiation.
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http://dx.doi.org/10.1016/j.molcel.2019.03.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561722PMC
June 2019

Pathological ASXL1 Mutations and Protein Variants Impair Neural Crest Development.

Stem Cell Reports 2019 05 18;12(5):861-868. Epub 2019 Apr 18.

Institute for Stem Cell Research, Helmholtz Zentrum München GmbH, 85764 Neuherberg, Germany. Electronic address:

The neural crest (NC) gives rise to a multitude of fetal tissues, and its misregulation is implicated in congenital malformations. Here, we investigated molecular mechanisms pertaining to NC-related symptoms in Bohring-Opitz syndrome (BOS), a developmental disorder linked to mutations in the Polycomb group factor Additional sex combs-like 1 (ASXL1). Genetically edited human pluripotent stem cell lines that were differentiated to NC progenitors and then xenotransplanted into chicken embryos demonstrated an impairment of NC delamination and emigration. Molecular analysis showed that ASXL1 mutations correlated with reduced activation of the transcription factor ZIC1 and the NC gene regulatory network. These findings were supported by differentiation experiments using BOS patient-derived induced pluripotent stem cell lines. Expression of truncated ASXL1 isoforms (amino acids 1-900) recapitulated the NC phenotypes in vitro and in ovo, raising the possibility that truncated ASXL1 variants contribute to BOS pathology. Collectively, we expand the understanding of truncated ASXL1 in BOS and in the human NC.
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http://dx.doi.org/10.1016/j.stemcr.2019.03.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6524927PMC
May 2019

Nuclear RNA foci from expansion mutation form paraspeckle-like bodies.

J Cell Sci 2019 03 7;132(5). Epub 2019 Mar 7.

Department of Biotechnology, Jozef Stefan Institute, Ljubljana 1000, Slovenia

The GGGGCC (GC) repeat expansion mutation in the gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Transcription of the repeat and formation of nuclear RNA foci, which sequester specific RNA-binding proteins, is one of the possible pathological mechanisms. Here, we show that (GC) repeat RNA predominantly associates with essential paraspeckle proteins SFPQ, NONO, RBM14, FUS and hnRNPH and colocalizes with known paraspeckle-associated RNA As formation of paraspeckles in motor neurons has been associated with early phases of ALS, we investigated the extent of similarity between paraspeckles and (GC) RNA foci. Overexpression of (GC) RNA results in their increased number and colocalization with SFPQ-stained nuclear bodies. These paraspeckle-like (GC) RNA foci form independently of the known paraspeckle scaffold, the long non-coding RNA Moreover, the knockdown of SFPQ protein in expansion mutation-positive fibroblasts significantly reduces the number of (GC) RNA foci. In conclusion, (GC) RNA foci have characteristics of paraspeckles, which suggests that both RNA foci and paraspeckles play roles in FTD and ALS, and implies approaches for regulation of their formation.
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http://dx.doi.org/10.1242/jcs.224303DOI Listing
March 2019

Neuronal differentiation induces SNORD115 expression and is accompanied by post-transcriptional changes of serotonin receptor 2c mRNA.

Sci Rep 2018 03 23;8(1):5101. Epub 2018 Mar 23.

Jozef Stefan Institute, Department of Biotechnology, Jamova 39, 1000, Ljubljana, Slovenia.

The serotonin neurotransmitter system is widespread in the brain and implicated in modulation of neuronal responses to other neurotransmitters. Among 14 serotonin receptor subtypes, 5-HT2cR plays a pivotal role in controlling neuronal network excitability. Serotonergic activity conveyed through receptor 5-HT2cR is regulated post-transcriptionally via two mechanisms, alternative splicing and A-to-I RNA editing. Brain-specific small nucleolar RNA SNORD115 harbours a phylogenetically conserved 18-nucleotide antisense element with perfect complementarity to the region of 5ht2c primary transcript that undergoes post-transcriptional changes. Previous 5ht2c minigene studies have implicated SNORD115 in fine-tuning of both post-transcriptional events. We monitored post-transcriptional changes of endogenous 5ht2c transcripts during neuronal differentiation. Both SNORD115 and 5ht2c were upregulated upon neuronal commitment. We detected increased 5ht2c alternative exon Vb inclusion already at the stage of neuronal progenitors, and more extensive A-to-I editing of non-targeted sites A and B compared to adjacent adenosines at sites E, C and D throughout differentiation. As the extent of editing is known to positively correlate with exon Vb usage while it reduces receptor functionality, our data support the model where SNORD115 directly promotes alternative exon inclusion without the requirement for conversion of key adenosines to inosines, thereby favouring production of full-length receptor isoforms with higher potency.
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http://dx.doi.org/10.1038/s41598-018-23293-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5865145PMC
March 2018

High-Resolution RNA Maps Suggest Common Principles of Splicing and Polyadenylation Regulation by TDP-43.

Cell Rep 2017 05;19(5):1056-1067

MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK; UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK; The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. Electronic address:

Many RNA-binding proteins (RBPs) regulate both alternative exons and poly(A) site selection. To understand their regulatory principles, we developed expressRNA, a web platform encompassing computational tools for integration of iCLIP and RNA motif analyses with RNA-seq and 3' mRNA sequencing. This reveals at nucleotide resolution the "RNA maps" describing how the RNA binding positions of RBPs relate to their regulatory functions. We use this approach to examine how TDP-43, an RBP involved in several neurodegenerative diseases, binds around its regulated poly(A) sites. Binding close to the poly(A) site generally represses, whereas binding further downstream enhances use of the site, which is similar to TDP-43 binding around regulated exons. Our RNAmotifs2 software also identifies sequence motifs that cluster together with the binding motifs of TDP-43. We conclude that TDP-43 directly regulates diverse types of pre-mRNA processing according to common position-dependent principles.
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http://dx.doi.org/10.1016/j.celrep.2017.04.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5437728PMC
May 2017

Recursive splicing in long vertebrate genes.

Nature 2015 May 13;521(7552):371-375. Epub 2015 May 13.

Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, UK.

It is generally believed that splicing removes introns as single units from precursor messenger RNA transcripts. However, some long Drosophila melanogaster introns contain a cryptic site, known as a recursive splice site (RS-site), that enables a multi-step process of intron removal termed recursive splicing. The extent to which recursive splicing occurs in other species and its mechanistic basis have not been examined. Here we identify highly conserved RS-sites in genes expressed in the mammalian brain that encode proteins functioning in neuronal development. Moreover, the RS-sites are found in some of the longest introns across vertebrates. We find that vertebrate recursive splicing requires initial definition of an 'RS-exon' that follows the RS-site. The RS-exon is then excluded from the dominant mRNA isoform owing to competition with a reconstituted 5' splice site formed at the RS-site after the first splicing step. Conversely, the RS-exon is included when preceded by cryptic promoters or exons that fail to reconstitute an efficient 5' splice site. Most RS-exons contain a premature stop codon such that their inclusion can decrease mRNA stability. Thus, by establishing a binary splicing switch, RS-sites demarcate different mRNA isoforms emerging from long genes by coupling cryptic elements with inclusion of RS-exons.
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http://dx.doi.org/10.1038/nature14466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4471124PMC
May 2015

CLIPing the brain: studies of protein-RNA interactions important for neurodegenerative disorders.

Mol Cell Neurosci 2013 Sep 10;56:429-35. Epub 2013 Apr 10.

MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK; Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.

The fate of an mRNA is largely determined by its interactions with RNA binding proteins (RBPs). Post-transcriptional processing, RNA stability, localisation and translation are some of the events regulated by the plethora of RBPs present within cells. Mutations in various RBPs cause several diseases of the central nervous system, including frontotemporal lobar degeneration, amyotrophic lateral sclerosis and fragile X syndrome. Here we review the studies that integrated UV-induced cross-linked immunoprecipitation (CLIP) with other genome-wide methods to comprehensively characterise the function of diverse RBPs in the brain. We discuss the technical challenges of these studies and review the strategies that can be used to reliably identify the RNAs bound and regulated by an RBP. We conclude by highlighting how CLIP and related techniques have been instrumental in addressing the role of RBPs in neurologic diseases. This article is part of a Special Issue entitled: RNA and splicing regulation in neurodegeneration.
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http://dx.doi.org/10.1016/j.mcn.2013.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3793874PMC
September 2013

Widespread binding of FUS along nascent RNA regulates alternative splicing in the brain.

Sci Rep 2012 28;2:603. Epub 2012 Aug 28.

Centre for Neurodegeneration Research, King's College London, Institute of Psychiatry, De Crespigny Park, London, UK.

Fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP-43) are RNA-binding proteins pathogenetically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), but it is not known if they regulate the same transcripts. We addressed this question using crosslinking and immunoprecipitation (iCLIP) in mouse brain, which showed that FUS binds along the whole length of the nascent RNA with limited sequence specificity to GGU and related motifs. A saw-tooth binding pattern in long genes demonstrated that FUS remains bound to pre-mRNAs until splicing is completed. Analysis of FUS(-/-) brain demonstrated a role for FUS in alternative splicing, with increased crosslinking of FUS in introns around the repressed exons. We did not observe a significant overlap in the RNA binding sites or the exons regulated by FUS and TDP-43. Nevertheless, we found that both proteins regulate genes that function in neuronal development.
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http://dx.doi.org/10.1038/srep00603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3429604PMC
March 2013