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1.
Cell ; 185(20): 3689-3704.e21, 2022 09 29.
Artículo en Inglés | MEDLINE | ID: mdl-36179666

RESUMEN

Regulatory landscapes drive complex developmental gene expression, but it remains unclear how their integrity is maintained when incorporating novel genes and functions during evolution. Here, we investigated how a placental mammal-specific gene, Zfp42, emerged in an ancient vertebrate topologically associated domain (TAD) without adopting or disrupting the conserved expression of its gene, Fat1. In ESCs, physical TAD partitioning separates Zfp42 and Fat1 with distinct local enhancers that drive their independent expression. This separation is driven by chromatin activity and not CTCF/cohesin. In contrast, in embryonic limbs, inactive Zfp42 shares Fat1's intact TAD without responding to active Fat1 enhancers. However, neither Fat1 enhancer-incompatibility nor nuclear envelope-attachment account for Zfp42's unresponsiveness. Rather, Zfp42's promoter is rendered inert to enhancers by context-dependent DNA methylation. Thus, diverse mechanisms enabled the integration of independent Zfp42 regulation in the Fat1 locus. Critically, such regulatory complexity appears common in evolution as, genome wide, most TADs contain multiple independently expressed genes.


Asunto(s)
Cromatina , Placenta , Animales , Factor de Unión a CCCTC/metabolismo , Ensamble y Desensamble de Cromatina , Elementos de Facilitación Genéticos , Evolución Molecular , Femenino , Genoma , Mamíferos/metabolismo , Placenta/metabolismo , Embarazo , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
2.
Mol Cell ; 82(1): 190-208.e17, 2022 01 06.
Artículo en Inglés | MEDLINE | ID: mdl-34932975

RESUMEN

Developmental genes such as Xist, which initiates X chromosome inactivation, are controlled by complex cis-regulatory landscapes, which decode multiple signals to establish specific spatiotemporal expression patterns. Xist integrates information on X chromosome dosage and developmental stage to trigger X inactivation in the epiblast specifically in female embryos. Through a pooled CRISPR screen in differentiating mouse embryonic stem cells, we identify functional enhancer elements of Xist at the onset of random X inactivation. Chromatin profiling reveals that X-dosage controls the promoter-proximal region, while differentiation cues activate several distal enhancers. The strongest distal element lies in an enhancer cluster associated with a previously unannotated Xist-enhancing regulatory transcript, which we named Xert. Developmental cues and X-dosage are thus decoded by distinct regulatory regions, which cooperate to ensure female-specific Xist upregulation at the correct developmental time. With this study, we start to disentangle how multiple, functionally distinct regulatory elements interact to generate complex expression patterns in mammals.


Asunto(s)
Elementos de Facilitación Genéticos , Sitios Genéticos , Células Madre Embrionarias de Ratones/metabolismo , Regiones Promotoras Genéticas , ARN Largo no Codificante/genética , Inactivación del Cromosoma X , Cromosoma X , Animales , Diferenciación Celular , Línea Celular , Femenino , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Regulación hacia Arriba
3.
Nature ; 592(7852): 93-98, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33568816

RESUMEN

Long non-coding RNAs (lncRNAs) can be important components in gene-regulatory networks1, but the exact nature and extent of their involvement in human Mendelian disease is largely unknown. Here we show that genetic ablation of a lncRNA locus on human chromosome 2 causes a severe congenital limb malformation. We identified homozygous 27-63-kilobase deletions located 300 kilobases upstream of the engrailed-1 gene (EN1) in patients with a complex limb malformation featuring mesomelic shortening, syndactyly and ventral nails (dorsal dimelia). Re-engineering of the human deletions in mice resulted in a complete loss of En1 expression in the limb and a double dorsal-limb phenotype that recapitulates the human disease phenotype. Genome-wide transcriptome analysis in the developing mouse limb revealed a four-exon-long non-coding transcript within the deleted region, which we named Maenli. Functional dissection of the Maenli locus showed that its transcriptional activity is required for limb-specific En1 activation in cis, thereby fine-tuning the gene-regulatory networks controlling dorso-ventral polarity in the developing limb bud. Its loss results in the En1-related dorsal ventral limb phenotype, a subset of the full En1-associated phenotype. Our findings demonstrate that mutations involving lncRNA loci can result in human Mendelian disease.


Asunto(s)
Extremidades , Proteínas de Homeodominio/genética , Deformidades Congénitas de las Extremidades/genética , ARN Largo no Codificante/genética , Eliminación de Secuencia/genética , Transcripción Genética , Activación Transcripcional/genética , Animales , Línea Celular , Cromatina/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Transgénicos
4.
Nature ; 600(7890): 731-736, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34819668

RESUMEN

Extrachromosomal DNA (ecDNA) is prevalent in human cancers and mediates high expression of oncogenes through gene amplification and altered gene regulation1. Gene induction typically involves cis-regulatory elements that contact and activate genes on the same chromosome2,3. Here we show that ecDNA hubs-clusters of around 10-100 ecDNAs within the nucleus-enable intermolecular enhancer-gene interactions to promote oncogene overexpression. ecDNAs that encode multiple distinct oncogenes form hubs in diverse cancer cell types and primary tumours. Each ecDNA is more likely to transcribe the oncogene when spatially clustered with additional ecDNAs. ecDNA hubs are tethered by the bromodomain and extraterminal domain (BET) protein BRD4 in a MYC-amplified colorectal cancer cell line. The BET inhibitor JQ1 disperses ecDNA hubs and preferentially inhibits ecDNA-derived-oncogene transcription. The BRD4-bound PVT1 promoter is ectopically fused to MYC and duplicated in ecDNA, receiving promiscuous enhancer input to drive potent expression of MYC. Furthermore, the PVT1 promoter on an exogenous episome suffices to mediate gene activation in trans by ecDNA hubs in a JQ1-sensitive manner. Systematic silencing of ecDNA enhancers by CRISPR interference reveals intermolecular enhancer-gene activation among multiple oncogene loci that are amplified on distinct ecDNAs. Thus, protein-tethered ecDNA hubs enable intermolecular transcriptional regulation and may serve as units of oncogene function and cooperative evolution and as potential targets for cancer therapy.


Asunto(s)
Neoplasias , Proteínas Nucleares , Azepinas/farmacología , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/genética , Proteínas Nucleares/genética , Oncogenes/genética , Factores de Transcripción/genética
5.
Am J Hum Genet ; 108(9): 1725-1734, 2021 09 02.
Artículo en Inglés | MEDLINE | ID: mdl-34433009

RESUMEN

Copy-number variations (CNVs) are a common cause of congenital limb malformations and are interpreted primarily on the basis of their effect on gene dosage. However, recent studies show that CNVs also influence the 3D genome chromatin organization. The functional interpretation of whether a phenotype is the result of gene dosage or a regulatory position effect remains challenging. Here, we report on two unrelated families with individuals affected by bilateral hypoplasia of the femoral bones, both harboring de novo duplications on chromosome 10q24.32. The ∼0.5 Mb duplications include FGF8, a key regulator of limb development and several limb enhancer elements. To functionally characterize these variants, we analyzed the local chromatin architecture in the affected individuals' cells and re-engineered the duplications in mice by using CRISPR-Cas9 genome editing. We found that the duplications were associated with ectopic chromatin contacts and increased FGF8 expression. Transgenic mice carrying the heterozygous tandem duplication including Fgf8 exhibited proximal shortening of the limbs, resembling the human phenotype. To evaluate whether the phenotype was a result of gene dosage, we generated another transgenic mice line, carrying the duplication on one allele and a concurrent Fgf8 deletion on the other allele, as a control. Surprisingly, the same malformations were observed. Capture Hi-C experiments revealed ectopic interaction with the duplicated region and Fgf8, indicating a position effect. In summary, we show that duplications at the FGF8 locus are associated with femoral hypoplasia and that the phenotype is most likely the result of position effects altering FGF8 expression rather than gene dosage effects.


Asunto(s)
Duplicación Cromosómica , Cromosomas Humanos Par 10/química , Variaciones en el Número de Copia de ADN , Factor 8 de Crecimiento de Fibroblastos/genética , Deformidades Congénitas de las Extremidades Inferiores/genética , Adolescente , Alelos , Animales , Sistemas CRISPR-Cas , Preescolar , Cromatina/química , Cromatina/metabolismo , Cromosomas Humanos Par 10/metabolismo , Elementos de Facilitación Genéticos , Familia , Femenino , Fémur/anomalías , Fémur/diagnóstico por imagen , Fémur/metabolismo , Factor 8 de Crecimiento de Fibroblastos/metabolismo , Edición Génica , Heterocigoto , Humanos , Lactante , Deformidades Congénitas de las Extremidades Inferiores/diagnóstico por imagen , Deformidades Congénitas de las Extremidades Inferiores/metabolismo , Deformidades Congénitas de las Extremidades Inferiores/patología , Masculino , Ratones , Ratones Transgénicos , Linaje , Fenotipo
6.
Am J Hum Genet ; 106(6): 872-884, 2020 06 04.
Artículo en Inglés | MEDLINE | ID: mdl-32470376

RESUMEN

Genome-wide analysis methods, such as array comparative genomic hybridization (CGH) and whole-genome sequencing (WGS), have greatly advanced the identification of structural variants (SVs) in the human genome. However, even with standard high-throughput sequencing techniques, complex rearrangements with multiple breakpoints are often difficult to resolve, and predicting their effects on gene expression and phenotype remains a challenge. Here, we address these problems by using high-throughput chromosome conformation capture (Hi-C) generated from cultured cells of nine individuals with developmental disorders (DDs). Three individuals had previously been identified as harboring duplications at the SOX9 locus and six had been identified with translocations. Hi-C resolved the positions of the duplications and was instructive in interpreting their distinct pathogenic effects, including the formation of new topologically associating domains (neo-TADs). Hi-C was very sensitive in detecting translocations, and it revealed previously unrecognized complex rearrangements at the breakpoints. In several cases, we observed the formation of fused-TADs promoting ectopic enhancer-promoter interactions that were likely to be involved in the disease pathology. In summary, we show that Hi-C is a sensible method for the detection of complex SVs in a clinical setting. The results help interpret the possible pathogenic effects of the SVs in individuals with DDs.


Asunto(s)
Cromosomas Humanos/genética , Discapacidades del Desarrollo/genética , Genoma Humano/genética , Conformación Molecular , Translocación Genética/genética , Ensamble y Desensamble de Cromatina/genética , Puntos de Rotura del Cromosoma , Estudios de Cohortes , Humanos , Factor de Transcripción SOX9/genética , Duplicaciones Segmentarias en el Genoma/genética
7.
Nature ; 538(7624): 265-269, 2016 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-27706140

RESUMEN

Chromosome conformation capture methods have identified subchromosomal structures of higher-order chromatin interactions called topologically associated domains (TADs) that are separated from each other by boundary regions. By subdividing the genome into discrete regulatory units, TADs restrict the contacts that enhancers establish with their target genes. However, the mechanisms that underlie partitioning of the genome into TADs remain poorly understood. Here we show by chromosome conformation capture (capture Hi-C and 4C-seq methods) that genomic duplications in patient cells and genetically modified mice can result in the formation of new chromatin domains (neo-TADs) and that this process determines their molecular pathology. Duplications of non-coding DNA within the mouse Sox9 TAD (intra-TAD) that cause female to male sex reversal in humans, showed increased contact of the duplicated regions within the TAD, but no change in the overall TAD structure. In contrast, overlapping duplications that extended over the next boundary into the neighbouring TAD (inter-TAD), resulted in the formation of a new chromatin domain (neo-TAD) that was isolated from the rest of the genome. As a consequence of this insulation, inter-TAD duplications had no phenotypic effect. However, incorporation of the next flanking gene, Kcnj2, in the neo-TAD resulted in ectopic contacts of Kcnj2 with the duplicated part of the Sox9 regulatory region, consecutive misexpression of Kcnj2, and a limb malformation phenotype. Our findings provide evidence that TADs are genomic regulatory units with a high degree of internal stability that can be sculptured by structural genomic variations. This process is important for the interpretation of copy number variations, as these variations are routinely detected in diagnostic tests for genetic disease and cancer. This finding also has relevance in an evolutionary setting because copy-number differences are thought to have a crucial role in the evolution of genome complexity.


Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Variaciones en el Número de Copia de ADN/genética , Enfermedad/genética , Duplicación de Gen/genética , Animales , ADN/genética , Facies , Femenino , Fibroblastos , Dedos/anomalías , Deformidades Congénitas del Pie/genética , Expresión Génica , Genómica , Deformidades Congénitas de la Mano/genética , Masculino , Ratones , Fenotipo , Factor de Transcripción SOX9/genética
8.
Proc Natl Acad Sci U S A ; 116(25): 12390-12399, 2019 06 18.
Artículo en Inglés | MEDLINE | ID: mdl-31147463

RESUMEN

Long-range gene regulation involves physical proximity between enhancers and promoters to generate precise patterns of gene expression in space and time. However, in some cases, proximity coincides with gene activation, whereas, in others, preformed topologies already exist before activation. In this study, we investigate the preformed configuration underlying the regulation of the Shh gene by its unique limb enhancer, the ZRS, in vivo during mouse development. Abrogating the constitutive transcription covering the ZRS region led to a shift within the Shh-ZRS contacts and a moderate reduction in Shh transcription. Deletion of the CTCF binding sites around the ZRS resulted in the loss of the Shh-ZRS preformed interaction and a 50% decrease in Shh expression but no phenotype, suggesting an additional, CTCF-independent mechanism of promoter-enhancer communication. This residual activity, however, was diminished by combining the loss of CTCF binding with a hypomorphic ZRS allele, resulting in severe Shh loss of function and digit agenesis. Our results indicate that the preformed chromatin structure of the Shh locus is sustained by multiple components and acts to reinforce enhancer-promoter communication for robust transcription.


Asunto(s)
Cromatina/metabolismo , Extremidades/embriología , Proteínas Hedgehog/genética , Transcripción Genética , Animales , Sitios de Unión , Factor de Unión a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Regulación hacia Abajo , Elementos de Facilitación Genéticos , Proteínas de la Membrana/genética , Ratones , Regiones Promotoras Genéticas , Cohesinas
9.
Hum Genet ; 140(10): 1459-1469, 2021 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-34436670

RESUMEN

During human organogenesis, lung development is a timely and tightly regulated developmental process under the control of a large number of signaling molecules. Understanding how genetic variants can disturb normal lung development causing different lung malformations is a major goal for dissecting molecular mechanisms during embryogenesis. Here, through exome sequencing (ES), array CGH, genome sequencing (GS) and Hi-C, we aimed at elucidating the molecular basis of bilateral isolated lung agenesis in three fetuses born to a non-consanguineous family. We detected a complex genomic rearrangement containing duplicated, triplicated and deleted fragments involving the SHH locus in fetuses presenting complete agenesis of both lungs and near-complete agenesis of the trachea, diagnosed by ultrasound screening and confirmed at autopsy following termination. The rearrangement did not include SHH itself, but several regulatory elements for lung development, such as MACS1, a major SHH lung enhancer, and the neighboring genes MNX1 and NOM1. The rearrangement incorporated parts of two topologically associating domains (TADs) including their boundaries. Hi-C of cells from one of the affected fetuses showed the formation of two novel TADs each containing SHH enhancers and the MNX1 and NOM1 genes. Hi-C together with GS indicate that the new 3D conformation is likely causative for this condition by an inappropriate activation of MNX1 included in the neo-TADs by MACS1 enhancer, further highlighting the importance of the 3D chromatin conformation in human disease.


Asunto(s)
Anomalías Múltiples/genética , Evolución Molecular , Enfermedades Pulmonares/genética , Pulmón/anomalías , Pulmón/crecimiento & desarrollo , Pulmón/ultraestructura , Organogénesis/genética , Adulto , Cadáver , Femenino , Feto , Variación Genética , Genoma Humano , Humanos , Masculino , Embarazo
10.
Genome Res ; 27(2): 223-233, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-27923844

RESUMEN

Complex regulatory landscapes control the pleiotropic transcriptional activities of developmental genes. For most genes, the number, location, and dynamics of their associated regulatory elements are unknown. In this work, we characterized the three-dimensional chromatin microarchitecture and regulatory landscape of 446 limb-associated gene loci in mouse using Capture-C, ChIP-seq, and RNA-seq in forelimb, hindlimb at three developmental stages, and midbrain. The fine mapping of chromatin interactions revealed a strong preference for functional genomic regions such as repressed or active domains. By combining chromatin marks and interaction peaks, we annotated more than 1000 putative limb enhancers and their associated genes. Moreover, the analysis of chromatin interactions revealed two regimes of chromatin folding, one producing interactions stable across tissues and stages and another one associated with tissue and/or stage-specific interactions. Whereas stable interactions associate strongly with CTCF/RAD21 binding, the intensity of variable interactions correlates with changes in underlying chromatin modifications, specifically at the viewpoint and at the interaction site. In conclusion, this comprehensive data set provides a resource for the characterization of hundreds of limb-associated regulatory landscapes and a framework to interpret the chromatin folding dynamics observed during embryogenesis.


Asunto(s)
Cromatina/genética , Elementos de Facilitación Genéticos , Factores de Transcripción/genética , Activación Transcripcional/genética , Animales , Sitios de Unión , Inmunoprecipitación de Cromatina , Extremidades/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Histonas/genética , Ratones , Regiones Promotoras Genéticas
11.
Nucleic Acids Res ; 46(6): 2868-2882, 2018 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-29385519

RESUMEN

Genomic binding of transcription factors, like the glucocorticoid receptor (GR), is linked to the regulation of genes. However, as we show here, GR binding is a poor predictor of GR-dependent gene regulation even when taking the 3D organization of the genome into account. To connect GR binding sites to the regulation of genes in the endogenous genomic context, we turned to genome editing. By deleting GR binding sites, individually or in combination, we uncovered how cooperative interactions between binding sites contribute to the regulation of genes. Specifically, for the GR target gene GILZ, we show that the simultaneous presence of a cluster of GR binding sites is required for the activity of an individual enhancer and that the GR-dependent regulation of GILZ depends on multiple GR-bound enhancers. Further, by deleting GR binding sites that are shared between different cell types, we show how cell type-specific genome organization and enhancer-blocking can result in cell type-specific wiring of promoter-enhancer contacts. This rewiring allows an individual GR binding site shared between different cell types to direct the expression of distinct transcripts and thereby contributes to the cell type-specific consequences of glucocorticoid signaling.


Asunto(s)
Elementos de Facilitación Genéticos/genética , Genoma/genética , Genómica/métodos , Receptores de Glucocorticoides/metabolismo , Factores de Transcripción/metabolismo , Células A549 , Animales , Secuencia de Bases , Sitios de Unión/genética , Línea Celular Tumoral , Dexametasona/farmacología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Humanos , Unión Proteica
12.
Nucleic Acids Res ; 45(4): 1805-1819, 2017 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-27903902

RESUMEN

The genomic loci bound by the glucocorticoid receptor (GR), a hormone-activated transcription factor, show little overlap between cell types. To study the role of chromatin and sequence in specifying where GR binds, we used Bayesian modeling within the universe of accessible chromatin. Taken together, our results uncovered that although GR preferentially binds accessible chromatin, its binding is biased against accessible chromatin located at promoter regions. This bias can only be explained partially by the presence of fewer GR recognition sequences, arguing for the existence of additional mechanisms that interfere with GR binding at promoters. Therefore, we tested the role of H3K9ac, the chromatin feature with the strongest negative association with GR binding, but found that this correlation does not reflect a causative link. Finally, we find a higher percentage of promoter-proximal GR binding for genes regulated by GR across cell types than for cell type-specific target genes. Given that GR almost exclusively binds accessible chromatin, we propose that cell type-specific regulation by GR preferentially occurs via distal enhancers, whose chromatin accessibility is typically cell type-specific, whereas ubiquitous target gene regulation is more likely to result from binding to promoter regions, which are often accessible regardless of cell type examined.


Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/genética , Cromatina/metabolismo , Regulación de la Expresión Génica , Receptores de Glucocorticoides/metabolismo , Animales , Secuencia de Bases , Teorema de Bayes , Sitios de Unión , Línea Celular , Inmunoprecipitación de Cromatina , Biología Computacional/métodos , Estudio de Asociación del Genoma Completo , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Motivos de Nucleótidos , Especificidad de Órganos/genética , Regiones Promotoras Genéticas , Unión Proteica , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismo
14.
Biophys J ; 107(9): 2141-50, 2014 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-25418099

RESUMEN

Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ?10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell.


Asunto(s)
Cromatina/química , Nucleosomas/química , Ensamble y Desensamble de Cromatina , Simulación por Computador , ADN/química , Histonas/química , Modelos Genéticos , Modelos Moleculares , Método de Montecarlo , Conformación de Ácido Nucleico , Docilidad
15.
Bioinformatics ; 29(19): 2380-6, 2013 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-23846748

RESUMEN

MOTIVATION: Recent experimental advancements allow determining positions of nucleosomes for complete genomes. However, the resulting nucleosome occupancy maps are averages of heterogeneous cell populations. Accordingly, they represent a snapshot of a dynamic ensemble at a single time point with an overlay of many configurations from different cells. To study the organization of nucleosomes along the genome and to understand the mechanisms of nucleosome translocation, it is necessary to retrieve features of specific conformations from the population average. RESULTS: Here, we present a method for identifying non-overlapping nucleosome configurations that combines binary-variable analysis and a Monte Carlo approach with a simulated annealing scheme. In this manner, we obtain specific nucleosome configurations and optimized solutions for the complex positioning patterns from experimental data. We apply the method to compare nucleosome positioning at transcription factor binding sites in different mouse cell types. Our method can model nucleosome translocations at regulatory genomic elements and generate configurations for simulations of the spatial folding of the nucleosome chain. AVAILABILITY: Source code, precompiled binaries, test data and a web-based test installation are freely available at http://bioinformatics.fh-stralsund.de/nucpos/


Asunto(s)
Método de Montecarlo , Nucleosomas/química , Animales , Sitios de Unión , Diferenciación Celular , Ratones , Nucleosomas/metabolismo , Unión Proteica/genética , Factores de Transcripción/química , Factores de Transcripción/metabolismo
16.
Cells ; 12(15)2023 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-37566080

RESUMEN

In this study, we delve into the impact of genotoxic anticancer drug treatment on the chromatin structure of human cells, with a particular focus on the effects of doxorubicin. Using Hi-C, ChIP-seq, and RNA-seq, we explore the changes in chromatin architecture brought about by doxorubicin and ICRF193. Our results indicate that physiologically relevant doses of doxorubicin lead to a local reduction in Hi-C interactions in certain genomic regions that contain active promoters, with changes in chromatin architecture occurring independently of Top2 inhibition, cell cycle arrest, and differential gene expression. Inside the regions with decreased interactions, we detected redistribution of RAD21 around the peaks of H3K27 acetylation. Our study also revealed a common structural pattern in the regions with altered architecture, characterized by two large domains separated from each other. Additionally, doxorubicin was found to increase CTCF binding in H3K27 acetylated regions. Furthermore, we discovered that Top2-dependent chemotherapy causes changes in the distance decay of Hi-C contacts, which are driven by direct and indirect inhibitors. Our proposed model suggests that doxorubicin-induced DSBs cause cohesin redistribution, which leads to increased insulation on actively transcribed TAD boundaries. Our findings underscore the significant impact of genotoxic anticancer treatment on the chromatin structure of the human genome.


Asunto(s)
Cromatina , Cromosomas , Humanos , Factor de Unión a CCCTC/genética , Sitios de Unión , Cromosomas/metabolismo , Doxorrubicina/farmacología
17.
Nat Commun ; 14(1): 1475, 2023 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-36928426

RESUMEN

Split-Hand/Foot Malformation type 3 (SHFM3) is a congenital limb malformation associated with tandem duplications at the LBX1/FGF8 locus. Yet, the disease patho-mechanism remains unsolved. Here we investigate the functional consequences of SHFM3-associated rearrangements on chromatin conformation and gene expression in vivo in transgenic mice. We show that the Lbx1/Fgf8 locus consists of two separate, but interacting, regulatory domains. Re-engineering of a SHFM3-associated duplication and a newly reported inversion in mice results in restructuring of the chromatin architecture. This leads to ectopic activation of the Lbx1 and Btrc genes in the apical ectodermal ridge (AER) in an Fgf8-like pattern induced by AER-specific enhancers of Fgf8. We provide evidence that the SHFM3 phenotype is the result of a combinatorial effect on gene misexpression in the developing limb. Our results reveal insights into the molecular mechanism underlying SHFM3 and provide conceptual framework for how genomic rearrangements can cause gene misexpression and disease.


Asunto(s)
Factor 8 de Crecimiento de Fibroblastos , Reordenamiento Génico , Deformidades Congénitas de las Extremidades , Animales , Ratones , Expresión Génica , Proteínas de Homeodominio/genética , Deformidades Congénitas de las Extremidades/genética , Fenotipo , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/genética
18.
Blood Adv ; 7(21): 6520-6531, 2023 11 14.
Artículo en Inglés | MEDLINE | ID: mdl-37582288

RESUMEN

Acute myeloid leukemia with complex karyotype (CK-AML) is associated with poor prognosis, which is only in part explained by underlying TP53 mutations. Especially in the presence of complex chromosomal rearrangements, such as chromothripsis, the outcome of CK-AML is dismal. However, this degree of complexity of genomic rearrangements contributes to the leukemogenic phenotype and treatment resistance of CK-AML remains largely unknown. Applying an integrative workflow for the detection of structural variants (SVs) based on Oxford Nanopore (ONT) genomic DNA long-read sequencing (gDNA-LRS) and high-throughput chromosome confirmation capture (Hi-C) in a well-defined cohort of CK-AML identified regions with an extreme density of SVs. These rearrangements consisted to a large degree of focal amplifications enriched in the proximity of mammalian-wide interspersed repeat elements, which often result in oncogenic fusion transcripts, such as USP7::MVD, or the deregulation of oncogenic driver genes as confirmed by RNA-seq and ONT direct complementary DNA sequencing. We termed this novel phenomenon chromocataclysm. Thus, our integrative SV detection workflow combing gDNA-LRS and Hi-C enables to unravel complex genomic rearrangements at a very high resolution in regions hard to analyze by conventional sequencing technology, thereby providing an important tool to identify novel important drivers underlying cancer with complex karyotypic changes.


Asunto(s)
Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Cariotipo Anormal , Aberraciones Cromosómicas , Mutación , Genómica , Peptidasa Específica de Ubiquitina 7/genética
19.
Nat Commun ; 14(1): 2034, 2023 04 11.
Artículo en Inglés | MEDLINE | ID: mdl-37041138

RESUMEN

Heterotopic ossification is a disorder caused by abnormal mineralization of soft tissues in which signaling pathways such as BMP, TGFß and WNT are known key players in driving ectopic bone formation. Identifying novel genes and pathways related to the mineralization process are important steps for future gene therapy in bone disorders. In this study, we detect an inter-chromosomal insertional duplication in a female proband disrupting a topologically associating domain and causing an ultra-rare progressive form of heterotopic ossification. This structural variant lead to enhancer hijacking and misexpression of ARHGAP36 in fibroblasts, validated here by orthogonal in vitro studies. In addition, ARHGAP36 overexpression inhibits TGFß, and activates hedgehog signaling and genes/proteins related to extracellular matrix production. Our work on the genetic cause of this heterotopic ossification case has revealed that ARHGAP36 plays a role in bone formation and metabolism, outlining first details of this gene contributing to bone-formation and -disease.


Asunto(s)
Proteínas Hedgehog , Osificación Heterotópica , Femenino , Humanos , Tejido Conectivo/metabolismo , Proteínas Hedgehog/metabolismo , Osificación Heterotópica/metabolismo , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta
20.
Biophys J ; 103(2): 323-30, 2012 Jul 18.
Artículo en Inglés | MEDLINE | ID: mdl-22853910

RESUMEN

The wormlike-chain (WLC) model is widely used to describe the energetics of DNA bending. Motivated by recent experiments, alternative, so-called subelastic chain models were proposed that predict a lower elastic energy of highly bent DNA conformations. Until now, no unambiguous verification of these models has been obtained because probing the elasticity of DNA on short length scales remains challenging. Here we investigate the limits of the WLC model using coarse-grained Monte Carlo simulations to model the supercoiling of linear DNA molecules under tension. At a critical supercoiling density, the DNA extension decreases abruptly due to the sudden formation of a plectonemic structure. This buckling transition is caused by the large energy required to form the tightly bent end-loop of the plectoneme and should therefore provide a sensitive benchmark for model evaluation. Although simulations based on the WLC energetics could quantitatively reproduce the buckling measured in magnetic tweezers experiments, the buckling almost disappears for the tested linear subelastic chain model. Thus, our data support the validity of a harmonic bending potential even for small bending radii down to 3.5 nm.


Asunto(s)
ADN Superhelicoidal/química , ADN/química , Elasticidad , Modelos Moleculares , Estrés Mecánico , Simulación por Computador
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