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1.
Genome Res ; 2023 Dec 21.
Article in English | MEDLINE | ID: mdl-38129077

ABSTRACT

Paternal genomes are compacted during spermiogenesis and decompacted following fertilization. These processes are fundamental for inheritance but incompletely understood. We analyzed these processes in the frog Xenopus laevis, whose sperm can be assembled into functional pronuclei in egg extracts in vitro. In such extracts, cohesin extrudes DNA into loops, but in vivo cohesin only assembles topologically associating domains (TADs) at the mid-blastula transition (MBT). Why cohesin assembles TADs only at this stage is unknown. We first analyzed genome architecture in frog sperm and compared it to human and mouse. Our results indicate that sperm genome organization is conserved between frogs and humans and occurs without formation of TADs. TADs can be detected in mouse sperm samples, as reported, but these structures might originate from somatic chromatin contaminations. We therefore discuss the possibility that the absence of TADs might be a general feature of vertebrate sperm. To analyze sperm genome remodeling upon fertilization, we reconstituted male pronuclei in Xenopus egg extracts. In pronuclei, chromatin compartmentalization increases, but cohesin does not accumulate at CTCF sites and assemble TADs. However, if pronuclei are formed in the presence of exogenous CTCF, CTCF binds to its consensus sites, and cohesin accumulates at these and forms short-range chromatin loops, which are preferentially anchored at CTCF's N terminus. These results indicate that TADs are only assembled at MBT because before this stage CTCF sites are not occupied and cohesin only forms short-range chromatin loops.

2.
Mol Cell ; 83(17): 3049-3063.e6, 2023 09 07.
Article in English | MEDLINE | ID: mdl-37591243

ABSTRACT

Cohesin connects CTCF-binding sites and other genomic loci in cis to form chromatin loops and replicated DNA molecules in trans to mediate sister chromatid cohesion. Whether cohesin uses distinct or related mechanisms to perform these functions is unknown. Here, we describe a cohesin hinge mutant that can extrude DNA into loops but is unable to mediate cohesion in human cells. Our results suggest that the latter defect arises during cohesion establishment. The observation that cohesin's cohesion and loop extrusion activities can be partially separated indicates that cohesin uses distinct mechanisms to perform these two functions. Unexpectedly, the same hinge mutant can also not be stopped by CTCF boundaries as well as wild-type cohesin. This suggests that cohesion establishment and cohesin's interaction with CTCF boundaries depend on related mechanisms and raises the possibility that both require transient hinge opening to entrap DNA inside the cohesin ring.


Subject(s)
Cell Cycle Proteins , Chromatids , Humans , Chromatids/genetics , Binding Sites , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , Cohesins
3.
Genome Biol ; 24(1): 158, 2023 07 05.
Article in English | MEDLINE | ID: mdl-37408019

ABSTRACT

Genome browsers facilitate integrated analysis of multiple genomics datasets yet visualize only a few regions at a time and lack statistical functions for extracting meaningful information. We present HiCognition, a visual exploration and machine-learning tool based on a new genomic region set concept, enabling detection of patterns and associations between 3D chromosome conformation and collections of 1D genomics profiles of any type. By revealing how transcription and cohesion subunit isoforms contribute to chromosome conformation, we showcase how the flexible user interface and machine learning tools of HiCognition help to understand the relationship between the structure and function of the genome.


Subject(s)
Genome, Human , Genomics , Software , Humans , Genomics/methods , Chromosomes, Human , Machine Learning
4.
Proc Natl Acad Sci U S A ; 120(11): e2210480120, 2023 03 14.
Article in English | MEDLINE | ID: mdl-36897969

ABSTRACT

Cohesin folds mammalian interphase chromosomes by extruding the chromatin fiber into numerous loops. "Loop extrusion" can be impeded by chromatin-bound factors, such as CTCF, which generates characteristic and functional chromatin organization patterns. It has been proposed that transcription relocalizes or interferes with cohesin and that active promoters are cohesin loading sites. However, the effects of transcription on cohesin have not been reconciled with observations of active extrusion by cohesin. To determine how transcription modulates extrusion, we studied mouse cells in which we could alter cohesin abundance, dynamics, and localization by genetic "knockouts" of the cohesin regulators CTCF and Wapl. Through Hi-C experiments, we discovered intricate, cohesin-dependent contact patterns near active genes. Chromatin organization around active genes exhibited hallmarks of interactions between transcribing RNA polymerases (RNAPs) and extruding cohesins. These observations could be reproduced by polymer simulations in which RNAPs were moving barriers to extrusion that obstructed, slowed, and pushed cohesins. The simulations predicted that preferential loading of cohesin at promoters is inconsistent with our experimental data. Additional ChIP-seq experiments showed that the putative cohesin loader Nipbl is not predominantly enriched at promoters. Therefore, we propose that cohesin is not preferentially loaded at promoters and that the barrier function of RNAP accounts for cohesin accumulation at active promoters. Altogether, we find that RNAP is an extrusion barrier that is not stationary, but rather, translocates and relocalizes cohesin. Loop extrusion and transcription might interact to dynamically generate and maintain gene interactions with regulatory elements and shape functional genomic organization.


Subject(s)
Cell Cycle Proteins , Chromatin , Animals , Mice , CCCTC-Binding Factor/genetics , Cell Cycle Proteins/metabolism , Chromosomes, Mammalian/metabolism , DNA-Directed RNA Polymerases/genetics , Mammals/genetics
5.
EMBO J ; 41(13): e110600, 2022 07 04.
Article in English | MEDLINE | ID: mdl-35703121

ABSTRACT

Germ cells are unique in engendering totipotency, yet the mechanisms underlying this capacity remain elusive. Here, we perform comprehensive and in-depth nucleome analysis of mouse germ-cell development in vitro, encompassing pluripotent precursors, primordial germ cells (PGCs) before and after epigenetic reprogramming, and spermatogonia/spermatogonial stem cells (SSCs). Although epigenetic reprogramming, including genome-wide DNA de-methylation, creates broadly open chromatin with abundant enhancer-like signatures, the augmented chromatin insulation safeguards transcriptional fidelity. These insulatory constraints are then erased en masse for spermatogonial development. Notably, despite distinguishing epigenetic programming, including global DNA re-methylation, the PGCs-to-spermatogonia/SSCs development entails further euchromatization. This accompanies substantial erasure of lamina-associated domains, generating spermatogonia/SSCs with a minimal peripheral attachment of chromatin except for pericentromeres-an architecture conserved in primates. Accordingly, faulty nucleome maturation, including persistent insulation and improper euchromatization, leads to impaired spermatogenic potential. Given that PGCs after epigenetic reprogramming serve as oogenic progenitors as well, our findings elucidate a principle for the nucleome programming that creates gametogenic progenitors in both sexes, defining a basis for nuclear totipotency.


Subject(s)
Epigenesis, Genetic , Germ Cells , Animals , Chromatin/genetics , Chromatin/metabolism , DNA Methylation , Epigenomics , Female , Germ Cells/metabolism , Male , Mammals/genetics , Mice , Spermatogonia
6.
Nature ; 606(7912): 197-203, 2022 06.
Article in English | MEDLINE | ID: mdl-35585235

ABSTRACT

Eukaryotic genomes are compacted into loops and topologically associating domains (TADs)1-3, which contribute to transcription, recombination and genomic stability4,5. Cohesin extrudes DNA into loops that are thought to lengthen until CTCF boundaries are encountered6-12. Little is known about whether loop extrusion is impeded by DNA-bound machines. Here we show that the minichromosome maintenance (MCM) complex is a barrier that restricts loop extrusion in G1 phase. Single-nucleus Hi-C (high-resolution chromosome conformation capture) of mouse zygotes reveals that MCM loading reduces CTCF-anchored loops and decreases TAD boundary insulation, which suggests that loop extrusion is impeded before reaching CTCF. This effect extends to HCT116 cells, in which MCMs affect the number of CTCF-anchored loops and gene expression. Simulations suggest that MCMs are abundant, randomly positioned and partially permeable barriers. Single-molecule imaging shows that MCMs are physical barriers that frequently constrain cohesin translocation in vitro. Notably, chimeric yeast MCMs that contain a cohesin-interaction motif from human MCM3 induce cohesin pausing, indicating that MCMs are 'active' barriers with binding sites. These findings raise the possibility that cohesin can arrive by loop extrusion at MCMs, which determine the genomic sites at which sister chromatid cohesion is established. On the basis of in vivo, in silico and in vitro data, we conclude that distinct loop extrusion barriers shape the three-dimensional genome.


Subject(s)
Cell Cycle Proteins , Chromosomal Proteins, Non-Histone , DNA , Minichromosome Maintenance Proteins , Animals , CCCTC-Binding Factor/metabolism , Cell Cycle Proteins/metabolism , Chromatids/chemistry , Chromatids/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA/chemistry , DNA/metabolism , G1 Phase , HCT116 Cells , Humans , Mice , Minichromosome Maintenance Complex Component 3/chemistry , Minichromosome Maintenance Complex Component 3/metabolism , Minichromosome Maintenance Proteins/chemistry , Minichromosome Maintenance Proteins/metabolism , Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Nucleic Acid Conformation , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/metabolism , Cohesins
7.
Cell Rep ; 32(3): 107929, 2020 07 21.
Article in English | MEDLINE | ID: mdl-32698000

ABSTRACT

It is currently assumed that 3D chromosomal organization plays a central role in transcriptional control. However, depletion of cohesin and CTCF affects the steady-state levels of only a minority of transcripts. Here, we use high-resolution Capture Hi-C to interrogate the dynamics of chromosomal contacts of all annotated human gene promoters upon degradation of cohesin and CTCF. We show that a majority of promoter-anchored contacts are lost in these conditions, but many contacts with distinct properties are maintained, and some new ones are gained. The rewiring of contacts between promoters and active enhancers upon cohesin degradation associates with rapid changes in target gene transcription as detected by SLAM sequencing (SLAM-seq). These results provide a mechanistic explanation for the limited, but consistent, effects of cohesin and CTCF depletion on steady-state transcription and suggest the existence of both cohesin-dependent and -independent mechanisms of enhancer-promoter pairing.


Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes/metabolism , Enhancer Elements, Genetic/genetics , Promoter Regions, Genetic , Chromatin , DNA-Binding Proteins/metabolism , Gene Expression Regulation , HeLa Cells , Humans , Transcription, Genetic , Cohesins
8.
J Cell Biol ; 219(4)2020 04 06.
Article in English | MEDLINE | ID: mdl-32328639

ABSTRACT

Cohesin is essential for genome folding and inheritance. In somatic cells, these functions are both mediated by Scc1-cohesin, which in mitosis is released from chromosomes by Wapl and separase. In mammalian oocytes, cohesion is mediated by Rec8-cohesin. Scc1 is expressed but neither required nor sufficient for cohesion, and its function remains unknown. Likewise, it is unknown whether Wapl regulates one or both cohesin complexes and chromosome segregation in mature oocytes. Here, we show that Wapl is required for accurate meiosis I chromosome segregation, predominantly releases Scc1-cohesin from chromosomes, and promotes production of euploid eggs. Using single-nucleus Hi-C, we found that Scc1 is essential for chromosome organization in oocytes. Increasing Scc1 residence time on chromosomes by Wapl depletion leads to vermicelli formation and intra-loop structures but, unlike in somatic cells, does not increase loop size. We conclude that distinct cohesin complexes generate loops and cohesion in oocytes and propose that the same principle applies to all cell types and species.


Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Mammalian/metabolism , DNA-Binding Proteins/metabolism , Oocytes/metabolism , Proteins/metabolism , Animals , Cells, Cultured , Mice , Mice, Inbred C57BL , Mice, Transgenic , Cohesins
9.
Elife ; 92020 02 17.
Article in English | MEDLINE | ID: mdl-32065581

ABSTRACT

Eukaryotic genomes are folded into loops. It is thought that these are formed by cohesin complexes via extrusion, either until loop expansion is arrested by CTCF or until cohesin is removed from DNA by WAPL. Although WAPL limits cohesin's chromatin residence time to minutes, it has been reported that some loops exist for hours. How these loops can persist is unknown. We show that during G1-phase, mammalian cells contain acetylated cohesinSTAG1 which binds chromatin for hours, whereas cohesinSTAG2 binds chromatin for minutes. Our results indicate that CTCF and the acetyltransferase ESCO1 protect a subset of cohesinSTAG1 complexes from WAPL, thereby enable formation of long and presumably long-lived loops, and that ESCO1, like CTCF, contributes to boundary formation in chromatin looping. Our data are consistent with a model of nested loop extrusion, in which acetylated cohesinSTAG1 forms stable loops between CTCF sites, demarcating the boundaries of more transient cohesinSTAG2 extrusion activity.


Subject(s)
Acetyltransferases/physiology , CCCTC-Binding Factor/physiology , Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Nuclear Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Acetylation , Carrier Proteins/genetics , Computer Simulation , G1 Phase , Genome, Human , Humans , Nuclear Proteins/genetics , Protein Binding , Proto-Oncogene Proteins/genetics , Cohesins
10.
Elife ; 82019 06 17.
Article in English | MEDLINE | ID: mdl-31204999

ABSTRACT

The organisation of mammalian genomes into loops and topologically associating domains (TADs) contributes to chromatin structure, gene expression and recombination. TADs and many loops are formed by cohesin and positioned by CTCF. In proliferating cells, cohesin also mediates sister chromatid cohesion, which is essential for chromosome segregation. Current models of chromatin folding and cohesion are based on assumptions of how many cohesin and CTCF molecules organise the genome. Here we have measured absolute copy numbers and dynamics of cohesin, CTCF, NIPBL, WAPL and sororin by mass spectrometry, fluorescence-correlation spectroscopy and fluorescence recovery after photobleaching in HeLa cells. In G1-phase, there are ~250,000 nuclear cohesin complexes, of which ~ 160,000 are chromatin-bound. Comparison with chromatin immunoprecipitation-sequencing data implies that some genomic cohesin and CTCF enrichment sites are unoccupied in single cells at any one time. We discuss the implications of these findings for how cohesin can contribute to genome organisation and cohesion.


Subject(s)
CCCTC-Binding Factor/genetics , Carrier Proteins/genetics , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , Gene Dosage , Gene Expression , Nuclear Proteins/genetics , Proto-Oncogene Proteins/genetics , CCCTC-Binding Factor/metabolism , Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , Cell Line , Chromatids/genetics , Chromatin/genetics , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Segregation/genetics , Fluorescence Recovery After Photobleaching/methods , G1 Phase/genetics , Genome, Human/genetics , HeLa Cells , Humans , Mass Spectrometry/methods , Nuclear Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Cohesins
11.
J Cell Biol ; 217(6): 1985-2004, 2018 06 04.
Article in English | MEDLINE | ID: mdl-29695489

ABSTRACT

Faithful genome transmission in dividing cells requires that the two copies of each chromosome's DNA package into separate but physically linked sister chromatids. The linkage between sister chromatids is mediated by cohesin, yet where sister chromatids are linked and how they resolve during cell cycle progression has remained unclear. In this study, we investigated sister chromatid organization in live human cells using dCas9-mEGFP labeling of endogenous genomic loci. We detected substantial sister locus separation during G2 phase irrespective of the proximity to cohesin enrichment sites. Almost all sister loci separated within a few hours after their respective replication and then rapidly equilibrated their average distances within dynamic chromatin polymers. Our findings explain why the topology of sister chromatid resolution in G2 largely reflects the DNA replication program. Furthermore, these data suggest that cohesin enrichment sites are not persistent cohesive sites in human cells. Rather, cohesion might occur at variable genomic positions within the cell population.


Subject(s)
Cell Cycle , Chromatids/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Cell Cycle Proteins/metabolism , Cell Line , Cell Survival , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA Replication Timing , Fluorescent Dyes/metabolism , Genetic Loci , Genome, Human , Humans , Interphase , Models, Biological , Cohesins
12.
EMBO J ; 36(24): 3573-3599, 2017 12 15.
Article in English | MEDLINE | ID: mdl-29217591

ABSTRACT

Mammalian genomes are spatially organized into compartments, topologically associating domains (TADs), and loops to facilitate gene regulation and other chromosomal functions. How compartments, TADs, and loops are generated is unknown. It has been proposed that cohesin forms TADs and loops by extruding chromatin loops until it encounters CTCF, but direct evidence for this hypothesis is missing. Here, we show that cohesin suppresses compartments but is required for TADs and loops, that CTCF defines their boundaries, and that the cohesin unloading factor WAPL and its PDS5 binding partners control the length of loops. In the absence of WAPL and PDS5 proteins, cohesin forms extended loops, presumably by passing CTCF sites, accumulates in axial chromosomal positions (vermicelli), and condenses chromosomes. Unexpectedly, PDS5 proteins are also required for boundary function. These results show that cohesin has an essential genome-wide function in mediating long-range chromatin interactions and support the hypothesis that cohesin creates these by loop extrusion, until it is delayed by CTCF in a manner dependent on PDS5 proteins, or until it is released from DNA by WAPL.


Subject(s)
CCCTC-Binding Factor/metabolism , Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , Chromatin/genetics , Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Transcription Factors/metabolism , CCCTC-Binding Factor/genetics , Carrier Proteins/genetics , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , Chromosomes/genetics , DNA-Binding Proteins/genetics , Genome, Human/genetics , HeLa Cells , Humans , Nuclear Proteins/genetics , Proto-Oncogene Proteins/genetics , Transcription Factors/genetics , Cohesins
13.
Nature ; 544(7651): 503-507, 2017 04 27.
Article in English | MEDLINE | ID: mdl-28424523

ABSTRACT

Mammalian genomes are spatially organized by CCCTC-binding factor (CTCF) and cohesin into chromatin loops and topologically associated domains, which have important roles in gene regulation and recombination. By binding to specific sequences, CTCF defines contact points for cohesin-mediated long-range chromosomal cis-interactions. Cohesin is also present at these sites, but has been proposed to be loaded onto DNA elsewhere and to extrude chromatin loops until it encounters CTCF bound to DNA. How cohesin is recruited to CTCF sites, according to this or other models, is unknown. Here we show that the distribution of cohesin in the mouse genome depends on transcription, CTCF and the cohesin release factor Wings apart-like (Wapl). In CTCF-depleted fibroblasts, cohesin cannot be properly recruited to CTCF sites but instead accumulates at transcription start sites of active genes, where the cohesin-loading complex is located. In the absence of both CTCF and Wapl, cohesin accumulates in up to 70 kilobase-long regions at 3'-ends of active genes, in particular if these converge on each other. Changing gene expression modulates the position of these 'cohesin islands'. These findings indicate that transcription can relocate mammalian cohesin over long distances on DNA, as previously reported for yeast cohesin, that this translocation contributes to positioning cohesin at CTCF sites, and that active genes can be freed from cohesin either by transcription-mediated translocation or by Wapl-mediated release.


Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Mammalian/metabolism , Genome/genetics , Proteins/metabolism , Repressor Proteins/metabolism , Transcription, Genetic/genetics , Animals , Binding Sites , CCCTC-Binding Factor , Cell Cycle Proteins/deficiency , Cell Cycle Proteins/genetics , Cells, Cultured , Chondroitin Sulfate Proteoglycans/deficiency , Chondroitin Sulfate Proteoglycans/genetics , Chromatin/genetics , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/deficiency , Chromosomal Proteins, Non-Histone/genetics , Chromosomes, Mammalian/genetics , DNA/genetics , DNA/metabolism , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Male , Mice , Protein Transport , Proteins/genetics , Repressor Proteins/deficiency , Repressor Proteins/genetics , Transcription Initiation Site , Cohesins
14.
EMBO J ; 33(22): 2643-58, 2014 Nov 18.
Article in English | MEDLINE | ID: mdl-25257309

ABSTRACT

Although splicing is essential for the expression of most eukaryotic genes, inactivation of splicing factors causes specific defects in mitosis. The molecular cause of this defect is unknown. Here, we show that the spliceosome subunits SNW1 and PRPF8 are essential for sister chromatid cohesion in human cells. A transcriptome-wide analysis revealed that SNW1 or PRPF8 depletion affects the splicing of specific introns in a subset of pre-mRNAs, including pre-mRNAs encoding the cohesion protein sororin and the APC/C subunit APC2. SNW1 depletion causes cohesion defects predominantly by reducing sororin levels, which causes destabilisation of cohesin on DNA. SNW1 depletion also reduces APC/C activity and contributes to cohesion defects indirectly by delaying mitosis and causing "cohesion fatigue". Simultaneous expression of sororin and APC2 from intron-less cDNAs restores cohesion in SNW1-depleted cells. These results indicate that the spliceosome is required for mitosis because it enables expression of genes essential for cohesion. Our transcriptome-wide identification of retained introns in SNW1- and PRPF8-depleted cells may help to understand the aetiology of diseases associated with splicing defects, such as retinosa pigmentosum and cancer.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Cycle Proteins/metabolism , Chromatids/metabolism , Cytoskeletal Proteins/metabolism , Nuclear Receptor Coactivators/metabolism , RNA Precursors/metabolism , RNA Splicing/physiology , Adaptor Proteins, Signal Transducing/genetics , Cell Cycle Proteins/genetics , Chromatids/genetics , Cytoskeletal Proteins/genetics , Gene Deletion , HeLa Cells , Humans , Nuclear Receptor Coactivators/genetics , RNA Precursors/genetics , Transcriptome/physiology
15.
Genome Res ; 24(11): 1808-20, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25135956

ABSTRACT

The selectivity of transcriptional responses to extracellular cues is reflected by the deposition of stimulus-specific chromatin marks. Although histone H3 phosphorylation is a target of numerous signaling pathways, its role in transcriptional regulation remains poorly understood. Here, for the first time, we report a genome-wide analysis of H3S28 phosphorylation in a mammalian system in the context of stress signaling. We found that this mark targets as many as 50% of all stress-induced genes, underlining its importance in signal-induced transcription. By combining ChIP-seq, RNA-seq, and mass spectrometry we identified the factors involved in the biological interpretation of this histone modification. We found that MSK1/2-mediated phosphorylation of H3S28 at stress-responsive promoters contributes to the dissociation of HDAC corepressor complexes and thereby to enhanced local histone acetylation and subsequent transcriptional activation of stress-induced genes. Our data reveal a novel function of the H3S28ph mark in the activation of mammalian genes in response to MAP kinase pathway activation.


Subject(s)
Histones/metabolism , Serine/metabolism , Stress, Physiological/genetics , Transcriptional Activation , 3T3 Cells , Acetylation , Animals , Chromatin Immunoprecipitation , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression Profiling , Gene Ontology , Genome-Wide Association Study , HeLa Cells , High-Throughput Nucleotide Sequencing , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Humans , MAP Kinase Signaling System/genetics , Mice , Oligonucleotide Array Sequence Analysis , Phosphorylation , Promoter Regions, Genetic/genetics , Ribosomal Protein S6 Kinases, 90-kDa/genetics , Ribosomal Protein S6 Kinases, 90-kDa/metabolism
16.
Science ; 338(6113): 1469-72, 2012 Dec 14.
Article in English | MEDLINE | ID: mdl-23239737

ABSTRACT

Mammalian imprinted genes often cluster with long noncoding (lnc) RNAs. Three lncRNAs that induce parental-specific silencing show hallmarks indicating that their transcription is more important than their product. To test whether Airn transcription or product silences the Igf2r gene, we shortened the endogenous lncRNA to different lengths. The results excluded a role for spliced and unspliced Airn lncRNA products and for Airn nuclear size and location in silencing Igf2r. Instead, silencing only required Airn transcriptional overlap of the Igf2r promoter, which interferes with RNA polymerase II recruitment in the absence of repressive chromatin. Such a repressor function for lncRNA transcriptional overlap reveals a gene silencing mechanism that may be widespread in the mammalian genome, given the abundance of lncRNA transcripts.


Subject(s)
Gene Silencing , Genomic Imprinting , RNA, Long Noncoding/metabolism , Receptor, IGF Type 2/genetics , Transcription, Genetic , Alternative Splicing , Animals , Cells, Cultured , Mice , Multigene Family , Promoter Regions, Genetic , RNA Polymerase II/metabolism , RNA, Long Noncoding/genetics
17.
Mol Biol Evol ; 27(11): 2507-21, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20530152

ABSTRACT

The use of secondary structures has been advocated to improve both the alignment and the tree reconstruction processes of ribosomal RNA (rRNA) data sets. We used simulated and empirical rRNA data to test the impact of secondary structure consideration in both steps of molecular phylogenetic analyses. A simulation approach was used to generate realistic rRNA data sets based on real 16S, 18S, and 28S sequences and structures in combination with different branch length and topologies. Alignment and tree reconstruction performance of four recent structural alignment methods was compared with exclusively sequence-based approaches. As empirical data, we used a hexapod rRNA data set to study the influence of nucleotide interdependencies in sequence alignment and tree reconstruction. Structural alignment methods delivered significantly better sequence alignments compared with pure sequence-based methods. Also, structural alignment methods delivered better trees judged by topological congruence to simulation base trees. However, the advantage of structural alignments was less pronounced and even vanished in several instances. For simulated data, application of mixed RNA/DNA models to stems and loops, respectively, led to significantly shorter branches. The application of mixed RNA/DNA models in the hexapod analyses delivered partly implausible relationships. This can be interpreted as a stronger sensitivity of mixed model setups to nonphylogenetic signal. Secondary structure consideration clearly influenced sequence alignment and tree reconstruction of ribosomal genes. Although sequence alignment quality can considerably be improved by the use of secondary structure information, the application of mixed models in tree reconstructions needs further studies to understand the observed effects.


Subject(s)
Arthropods/genetics , Computer Simulation , Nucleic Acid Conformation , Phylogeny , RNA, Ribosomal/chemistry , Sequence Alignment/methods , Animals , Bayes Theorem , RNA, Ribosomal/genetics
18.
Nucleic Acids Res ; 37(18): 6184-93, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19723687

ABSTRACT

Ribosomal RNA (rRNA) genes are probably the most frequently used data source in phylogenetic reconstruction. Individual columns of rRNA alignments are not independent as a consequence of their highly conserved secondary structures. Unless explicitly taken into account, these correlation can distort the phylogenetic signal and/or lead to gross overestimates of tree stability. Maximum likelihood and Bayesian approaches are of course amenable to using RNA-specific substitution models that treat conserved base pairs appropriately, but require accurate secondary structure models as input. So far, however, no accurate and easy-to-use tool has been available for computing structure-aware alignments and consensus structures that can deal with the large rRNAs. The RNAsalsa approach is designed to fill this gap. Capitalizing on the improved accuracy of pairwise consensus structures and informed by a priori knowledge of group-specific structural constraints, the tool provides both alignments and consensus structures that are of sufficient accuracy for routine phylogenetic analysis based on RNA-specific substitution models. The power of the approach is demonstrated using two rRNA data sets: a mitochondrial rRNA set of 26 Mammalia, and a collection of 28S nuclear rRNAs representative of the five major echinoderm groups.


Subject(s)
Phylogeny , RNA, Ribosomal/classification , Animals , Base Sequence , Echinodermata/genetics , Nucleic Acid Conformation , Primates/genetics , RNA, Ribosomal/chemistry , Sequence Alignment , Software
19.
Mol Phylogenet Evol ; 53(3): 758-71, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19654047

ABSTRACT

Secondary structure models of mitochondrial and nuclear (r)RNA sequences are frequently applied to aid the alignment of these molecules in phylogenetic analyses. Additionally, it is often speculated that structure variation of (r)RNA sequences might profitably be used as phylogenetic markers. The benefit of these approaches depends on the reliability of structure models. We used a recently developed approach to show that reliable inference of large (r)RNA secondary structures as a prerequisite of simultaneous sequence and structure alignment is feasible. The approach iteratively establishes local structure constraints of each sequence and infers fully folded individual structures by constrained MFE optimization. A comparison of structure edit distances of individual constraints and fully folded structures showed pronounced phylogenetic signal in fully folded structures. As model sequences we characterized secondary structures of 28S rRNA sequences of selected insects and examined their phylogenetic signal according to established phylogenetic hypotheses.


Subject(s)
Nucleic Acid Conformation , Phylogeny , RNA, Ribosomal, 28S/genetics , Animals , Genes, rRNA , Insecta/genetics , Sequence Alignment , Sequence Analysis, RNA
20.
BMC Evol Biol ; 9: 119, 2009 May 27.
Article in English | MEDLINE | ID: mdl-19473484

ABSTRACT

BACKGROUND: Whenever different data sets arrive at conflicting phylogenetic hypotheses, only testable causal explanations of sources of errors in at least one of the data sets allow us to critically choose among the conflicting hypotheses of relationships. The large (28S) and small (18S) subunit rRNAs are among the most popular markers for studies of deep phylogenies. However, some nodes supported by this data are suspected of being artifacts caused by peculiarities of the evolution of these molecules. Arthropod phylogeny is an especially controversial subject dotted with conflicting hypotheses which are dependent on data set and method of reconstruction. We assume that phylogenetic analyses based on these genes can be improved further i) by enlarging the taxon sample and ii) employing more realistic models of sequence evolution incorporating non-stationary substitution processes and iii) considering covariation and pairing of sites in rRNA-genes. RESULTS: We analyzed a large set of arthropod sequences, applied new tools for quality control of data prior to tree reconstruction, and increased the biological realism of substitution models. Although the split-decomposition network indicated a high noise content in the data set, our measures were able to both improve the analyses and give causal explanations for some incongruities mentioned from analyses of rRNA sequences. However, misleading effects did not completely disappear. CONCLUSION: Analyses of data sets that result in ambiguous phylogenetic hypotheses demand for methods, which do not only filter stochastic noise, but likewise allow to differentiate phylogenetic signal from systematic biases. Such methods can only rely on our findings regarding the evolution of the analyzed data. Analyses on independent data sets then are crucial to test the plausibility of the results. Our approach can easily be extended to genomic data, as well, whereby layers of quality assessment are set up applicable to phylogenetic reconstructions in general.


Subject(s)
Arthropods/genetics , Evolution, Molecular , Models, Genetic , Phylogeny , Animals , Nucleic Acid Conformation , RNA, Ribosomal, 18S/genetics , RNA, Ribosomal, 28S/genetics , Sequence Alignment , Sequence Analysis, RNA/methods
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