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1.
Mol Cell ; 82(5): 1035-1052.e9, 2022 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-35182477

RESUMEN

The nucleus is highly compartmentalized through the formation of distinct classes of membraneless domains. However, the composition and function of many of these structures are not well understood. Using APEX2-mediated proximity labeling and RNA sequencing, we surveyed human transcripts associated with nuclear speckles, several additional domains, and the lamina. Remarkably, speckles and lamina are associated with distinct classes of retained introns enriched in genes that function in RNA processing, translation, and the cell cycle, among other processes. In contrast to the lamina-proximal introns, retained introns associated with speckles are relatively short, GC-rich, and enriched for functional sites of RNA-binding proteins that are concentrated in these domains. They are also highly differentially regulated across diverse cellular contexts, including the cell cycle. Thus, our study provides a resource of nuclear domain-associated transcripts and further reveals speckles and lamina as hubs of distinct populations of retained introns linked to gene regulation and cell cycle progression.


Asunto(s)
Núcleo Celular , Proteínas de Unión al ARN , Núcleo Celular/genética , Núcleo Celular/metabolismo , Regulación de la Expresión Génica , Humanos , Intrones/genética , Empalme del ARN , Proteínas de Unión al ARN/genética
2.
Mol Cell ; 69(6): 1039-1045.e3, 2018 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-29526697

RESUMEN

Imaging (fluorescence in situ hybridization [FISH]) and genome-wide chromosome conformation capture (Hi-C) are two major approaches to the study of higher-order genome organization in the nucleus. Intra-chromosomal and inter-chromosomal interactions (referred to as non-homologous chromosomal contacts [NHCCs]) have been observed by several FISH-based studies, but locus-specific NHCCs have not been detected by Hi-C. Due to crosslinking, neither of these approaches assesses spatiotemporal properties. Toward resolving the discrepancies between imaging and Hi-C, we sought to understand the spatiotemporal properties of NHCCs in living cells by CRISPR/Cas9 live-cell imaging (CLING). In mammalian cells, we find that NHCCs are stable and occur as frequently as intra-chromosomal interactions, but NHCCs occur at farther spatial distance that could explain their lack of detection in Hi-C. By revealing the spatiotemporal properties in living cells, our study provides fundamental insights into the biology of NHCCs.


Asunto(s)
Cromosomas Humanos/genética , Microscopía Confocal/métodos , Imagen de Lapso de Tiempo/métodos , Animales , Sistemas CRISPR-Cas , Línea Celular , Cromosomas Humanos/metabolismo , Femenino , Edición Génica/métodos , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Cinética , Masculino , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Conformación de Ácido Nucleico , Conformación Proteica , Epitelio Pigmentado de la Retina/metabolismo
3.
Circulation ; 146(23): 1758-1778, 2022 12 06.
Artículo en Inglés | MEDLINE | ID: mdl-36259389

RESUMEN

BACKGROUND: Phosphodiesterase 3A (PDE3A) gain-of-function mutations cause hypertension with brachydactyly (HTNB) and lead to stroke. Increased peripheral vascular resistance, rather than salt retention, is responsible. It is surprising that the few patients with HTNB examined so far did not develop cardiac hypertrophy or heart failure. We hypothesized that, in the heart, PDE3A mutations could be protective. METHODS: We studied new patients. CRISPR-Cas9-engineered rat HTNB models were phenotyped by telemetric blood pressure measurements, echocardiography, microcomputed tomography, RNA-sequencing, and single nuclei RNA-sequencing. Human induced pluripotent stem cells carrying PDE3A mutations were established, differentiated to cardiomyocytes, and analyzed by Ca2+ imaging. We used Förster resonance energy transfer and biochemical assays. RESULTS: We identified a new PDE3A mutation in a family with HTNB. It maps to exon 13 encoding the enzyme's catalytic domain. All hitherto identified HTNB PDE3A mutations cluster in exon 4 encoding a region N-terminally from the catalytic domain of the enzyme. The mutations were recapitulated in rat models. Both exon 4 and 13 mutations led to aberrant phosphorylation, hyperactivity, and increased PDE3A enzyme self-assembly. The left ventricles of our patients with HTNB and the rat models were normal despite preexisting hypertension. A catecholamine challenge elicited cardiac hypertrophy in HTNB rats only to the level of wild-type rats and improved the contractility of the mutant hearts, compared with wild-type rats. The ß-adrenergic system, phosphodiesterase activity, and cAMP levels in the mutant hearts resembled wild-type hearts, whereas phospholamban phosphorylation was decreased in the mutants. In our induced pluripotent stem cell cardiomyocyte models, the PDE3A mutations caused adaptive changes of Ca2+ cycling. RNA-sequencing and single nuclei RNA-sequencing identified differences in mRNA expression between wild-type and mutants, affecting, among others, metabolism and protein folding. CONCLUSIONS: Although in vascular smooth muscle, PDE3A mutations cause hypertension, they confer protection against hypertension-induced cardiac damage in hearts. Nonselective PDE3A inhibition is a final, short-term option in heart failure treatment to increase cardiac cAMP and improve contractility. Our data argue that mimicking the effect of PDE3A mutations in the heart rather than nonselective PDE3 inhibition is cardioprotective in the long term. Our findings could facilitate the search for new treatments to prevent hypertension-induced cardiac damage.


Asunto(s)
Insuficiencia Cardíaca , Hipertensión , Células Madre Pluripotentes Inducidas , Humanos , Ratas , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Microtomografía por Rayos X , Células Madre Pluripotentes Inducidas/metabolismo , Hipertensión/complicaciones , Hipertensión/genética , Miocitos Cardíacos/metabolismo , Cardiomegalia , ARN
4.
EMBO J ; 37(6)2018 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-29335281

RESUMEN

In the post-genomic era, thousands of putative noncoding regulatory regions have been identified, such as enhancers, promoters, long noncoding RNAs (lncRNAs), and a cadre of small peptides. These ever-growing catalogs require high-throughput assays to test their functionality at scale. Massively parallel reporter assays have greatly enhanced the understanding of noncoding DNA elements en masse Here, we present a massively parallel RNA assay (MPRNA) that can assay 10,000 or more RNA segments for RNA-based functionality. We applied MPRNA to identify RNA-based nuclear localization domains harbored in lncRNAs. We examined a pool of 11,969 oligos densely tiling 38 human lncRNAs that were fused to a cytosolic transcript. After cell fractionation and barcode sequencing, we identified 109 unique RNA regions that significantly enriched this cytosolic transcript in the nucleus including a cytosine-rich motif. These nuclear enrichment sequences are highly conserved and over-represented in global nuclear fractionation sequencing. Importantly, many of these regions were independently validated by single-molecule RNA fluorescence in situ hybridization. Overall, we demonstrate the utility of MPRNA for future investigation of RNA-based functionalities.


Asunto(s)
ARN Largo no Codificante/genética , Núcleo Celular/genética , Células HeLa , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hibridación Fluorescente in Situ , Análisis de Secuencia de ARN
5.
EMBO J ; 37(15)2018 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-29921581

RESUMEN

Chromosomes occupy distinct interphase territories in the three-dimensional nucleus. However, how these chromosome territories are arranged relative to one another is poorly understood. Here, we investigated the inter-chromosomal interactions between chromosomes 2q, 12, and 17 in human mesenchymal stem cells (MSCs) and MSC-derived cell types by DNA-FISH We compared our findings in normal karyotypes with a three-generation family harboring a 2q37-deletion syndrome, featuring a heterozygous partial deletion of histone deacetylase 4 (HDAC4) on chr2q37. In normal karyotypes, we detected stable, recurring arrangements and interactions between the three chromosomal territories with a tissue-specific interaction bias at certain loci. These inter-chromosomal interactions were confirmed by Hi-C. Interestingly, the disease-related HDAC4 deletion resulted in displaced inter-chromosomal arrangements and altered interactions between the deletion-affected chromosome 2 and chromosome 12 and/or 17 in 2q37-deletion syndrome patients. Our findings provide evidence for a direct link between a structural chromosomal aberration and altered interphase architecture that results in a nuclear configuration, supporting a possible molecular pathogenesis.


Asunto(s)
Cromosomas Humanos Par 12/genética , Cromosomas Humanos Par 17/genética , Cromosomas Humanos Par 2/genética , Eliminación de Gen , Histona Desacetilasas/genética , Proteínas Represoras/genética , Translocación Genética/genética , Núcleo Celular/genética , Deleción Cromosómica , Humanos , Hibridación Fluorescente in Situ , Interfase/genética , Células Madre Mesenquimatosas/citología
6.
Genome Res ; 29(3): 344-355, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30683753

RESUMEN

Transcription initiates at both coding and noncoding genomic elements, including mRNA and long noncoding RNA (lncRNA) core promoters and enhancer RNAs (eRNAs). However, each class has a different expression profile with lncRNAs and eRNAs being the most tissue specific. How these complex differences in expression profiles and tissue specificities are encoded in a single DNA sequence remains unresolved. Here, we address this question using computational approaches and massively parallel reporter assays (MPRA) surveying hundreds of promoters and enhancers. We find that both divergent lncRNA and mRNA core promoters have higher capacities to drive transcription than nondivergent lncRNA and mRNA core promoters, respectively. Conversely, intergenic lncRNAs (lincRNAs) and eRNAs have lower capacities to drive transcription and are more tissue specific than divergent genes. This higher tissue specificity is strongly associated with having less complex transcription factor (TF) motif profiles at the core promoter. We experimentally validated these findings by testing both engineered single-nucleotide deletions and human single-nucleotide polymorphisms (SNPs) in MPRA. In both cases, we observe that single nucleotides associated with many motifs are important drivers of promoter activity. Thus, we suggest that high TF motif density serves as a robust mechanism to increase promoter activity at the expense of tissue specificity. Moreover, we find that 22% of common SNPs in core promoter regions have significant regulatory effects. Collectively, our findings show that high TF motif density provides redundancy and increases promoter activity at the expense of tissue specificity, suggesting that specificity of expression may be regulated by simplicity of motif usage.


Asunto(s)
Regiones Promotoras Genéticas , ARN Largo no Codificante/genética , Genoma Humano , Humanos , Especificidad de Órganos , Polimorfismo de Nucleótido Simple
7.
Circulation ; 142(2): 133-149, 2020 07 14.
Artículo en Inglés | MEDLINE | ID: mdl-32524868

RESUMEN

BACKGROUND: High blood pressure is the primary risk factor for cardiovascular death worldwide. Autosomal dominant hypertension with brachydactyly clinically resembles salt-resistant essential hypertension and causes death by stroke before 50 years of age. We recently implicated the gene encoding phosphodiesterase 3A (PDE3A); however, in vivo modeling of the genetic defect and thus showing an involvement of mutant PDE3A is lacking. METHODS: We used genetic mapping, sequencing, transgenic technology, CRISPR-Cas9 gene editing, immunoblotting, and fluorescence resonance energy transfer. We identified new patients, performed extensive animal phenotyping, and explored new signaling pathways. RESULTS: We describe a novel mutation within a 15 base pair (bp) region of the PDE3A gene and define this segment as a mutational hotspot in hypertension with brachydactyly. The mutations cause an increase in enzyme activity. A CRISPR/Cas9-generated rat model, with a 9-bp deletion within the hotspot analogous to a human deletion, recapitulates hypertension with brachydactyly. In mice, mutant transgenic PDE3A overexpression in smooth muscle cells confirmed that mutant PDE3A causes hypertension. The mutant PDE3A enzymes display consistent changes in their phosphorylation and an increased interaction with the 14-3-3θ adaptor protein. This aberrant signaling is associated with an increase in vascular smooth muscle cell proliferation and changes in vessel morphology and function. CONCLUSIONS: The mutated PDE3A gene drives mechanisms that increase peripheral vascular resistance causing hypertension. We present 2 new animal models that will serve to elucidate the underlying mechanisms further. Our findings could facilitate the search for new antihypertensive treatments.


Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Hipertensión/genética , Mutación , Alelos , Sustitución de Aminoácidos , Animales , Animales Modificados Genéticamente , Presión Arterial , Biomarcadores/sangre , Biomarcadores/orina , Braquidactilia/diagnóstico , Braquidactilia/genética , Sistemas CRISPR-Cas , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Activación Enzimática , Marcación de Gen , Estudios de Asociación Genética/métodos , Genotipo , Inmunohistoquímica , Isoenzimas , Masculino , Linaje , Fenotipo , Radiografía , Ratas , Sistema Renina-Angiotensina/genética
10.
Nat Struct Mol Biol ; 2024 Apr 17.
Artículo en Inglés | MEDLINE | ID: mdl-38632359

RESUMEN

Current models suggest that DNA double-strand breaks (DSBs) can move to the nuclear periphery for repair. It is unclear to what extent human DSBs display such repositioning. Here we show that the human nuclear envelope localizes to DSBs in a manner depending on DNA damage response (DDR) kinases and cytoplasmic microtubules acetylated by α-tubulin acetyltransferase-1 (ATAT1). These factors collaborate with the linker of nucleoskeleton and cytoskeleton complex (LINC), nuclear pore complex (NPC) protein NUP153, nuclear lamina and kinesins KIF5B and KIF13B to generate DSB-capturing nuclear envelope tubules (dsbNETs). dsbNETs are partly supported by nuclear actin filaments and the circadian factor PER1 and reversed by kinesin KIFC3. Although dsbNETs promote repair and survival, they are also co-opted during poly(ADP-ribose) polymerase (PARP) inhibition to restrain BRCA1-deficient breast cancer cells and are hyper-induced in cells expressing the aging-linked lamin A mutant progerin. In summary, our results advance understanding of nuclear structure-function relationships, uncover a nuclear-cytoplasmic DDR and identify dsbNETs as critical factors in genome organization and stability.

11.
STAR Protoc ; 4(4): 102731, 2023 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-37980569

RESUMEN

Association studies describe genetic associations between noncoding variants and disease susceptibility; however, they do not provide functional insight into the underlying molecular mechanisms of these variants. We present a protocol to assay the regulatory potential of thousands of noncoding variants using massively parallel reporter assays. We describe steps for oligo design, generating a plasmid pool, and extracting tag-seq libraries from cells to quantify the tested sequences. For complete details on the use and execution of this protocol, please refer to Oliveros and Delfosse et al.1.


Asunto(s)
Plásmidos , Plásmidos/genética
12.
Cell Genom ; 3(7): 100330, 2023 Jul 12.
Artículo en Inglés | MEDLINE | ID: mdl-37492106

RESUMEN

High blood pressure (BP) is the major risk factor for cardiovascular disease. Genome-wide association studies have identified genetic variants for BP, but functional insights into causality and related molecular mechanisms lag behind. We functionally characterize 4,608 genetic variants in linkage with 135 BP loci in vascular smooth muscle cells and cardiomyocytes by massively parallel reporter assays. High densities of regulatory variants at BP loci (i.e., ULK4, MAP4, CFDP1, PDE5A) indicate that multiple variants drive genetic association. Regulatory variants are enriched in repeats, alter cardiovascular-related transcription factor motifs, and spatially converge with genes controlling specific cardiovascular pathways. Using heuristic scoring, we define likely causal variants, and CRISPR prime editing finally determines causal variants for KCNK9, SFXN2, and PCGF6, which are candidates for developing high BP. Our systems-level approach provides a catalog of functionally relevant variants and their genomic architecture in two trait-relevant cell lines for a better understanding of BP gene regulation.

13.
Hum Mol Genet ; 19(5): 848-60, 2010 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-20015959

RESUMEN

Parathyroid hormone-like hormone (PTHLH) is an important chondrogenic regulator; however, the gene has not been directly linked to human disease. We studied a family with autosomal-dominant Brachydactyly Type E (BDE) and identified a t(8;12)(q13;p11.2) translocation with breakpoints (BPs) upstream of PTHLH on chromosome 12p11.2 and a disrupted KCNB2 on 8q13. We sequenced the BPs and identified a highly conserved Activator protein 1 (AP-1) motif on 12p11.2, together with a C-ets-1 motif translocated from 8q13. AP-1 and C-ets-1 bound in vitro and in vivo at the derivative chromosome 8 breakpoint [der(8) BP], but were differently enriched between the wild-type and BP allele. We differentiated fibroblasts from BDE patients into chondrogenic cells and found that PTHLH and its targets, ADAMTS-7 and ADAMTS-12 were downregulated along with impaired chondrogenic differentiation. We next used human and murine chondrocytes and observed that the AP-1 motif stimulated, whereas der(8) BP or C-ets-1 decreased, PTHLH promoter activity. These results are the first to identify a cis-directed PTHLH downregulation as primary cause of human chondrodysplasia.


Asunto(s)
Cromosomas Humanos Par 12/genética , Cromosomas Humanos Par 8/genética , Regulación hacia Abajo , Dedos/anomalías , Proteína Relacionada con la Hormona Paratiroidea/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Dedos del Pie/anomalías , Translocación Genética , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS7 , Animales , Deformidades Congénitas del Pie/genética , Deformidades Congénitas de la Mano/genética , Humanos , Ratones
14.
Cancers (Basel) ; 14(19)2022 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-36230862

RESUMEN

MicroRNA (miRNA) alterations significantly impact the formation and progression of human cancers. miRNAs interact with messenger RNAs (mRNAs) to facilitate degradation or translational repression. Thus, identifying miRNA-mRNA regulatory modules in cohorts of primary tumor tissues are fundamental for understanding the biology of tumor heterogeneity and precise diagnosis and treatment. We established a multitask learning sparse regularized factor regression (MSRFR) method to determine key tissue- and cohort-specific miRNA-mRNA regulatory modules from expression profiles of tumors. MSRFR simultaneously models the sparse relationship between miRNAs and mRNAs and extracts tissue- and cohort-specific miRNA-mRNA regulatory modules separately. We tested the model's ability to determine cohort-specific regulatory modules of multiple cancer cohorts from the same tissue and their underlying tissue-specific regulatory modules by extracting similarities between cancer cohorts (i.e., blood, kidney, and lung). We also detected tissue-specific and cohort-specific signatures in the corresponding regulatory modules by comparing our findings from various other tissues. We show that MSRFR effectively determines cancer-related miRNAs in cohort-specific regulatory modules, distinguishes tissue- and cohort-specific regulatory modules from each other, and extracts tissue-specific information from different cohorts of disease-related tissue. Our findings indicate that the MSRFR model can support current efforts in precision medicine to define tumor-specific miRNA-mRNA signatures.

15.
JCI Insight ; 7(10)2022 05 23.
Artículo en Inglés | MEDLINE | ID: mdl-35603789

RESUMEN

The eukaryotic CDC45/MCM2-7/GINS (CMG) helicase unwinds the DNA double helix during DNA replication. The GINS subcomplex is required for helicase activity and is, therefore, essential for DNA replication and cell viability. Here, we report the identification of 7 individuals from 5 unrelated families presenting with a Meier-Gorlin syndrome-like (MGS-like) phenotype associated with hypomorphic variants of GINS3, a gene not previously associated with this syndrome. We found that MGS-associated GINS3 variants affecting aspartic acid 24 (D24) compromised cell proliferation and caused accumulation of cells in S phase. These variants shortened the protein half-life, altered key protein interactions at the replisome, and negatively influenced DNA replication fork progression. Yeast expressing MGS-associated variants of PSF3 (the yeast GINS3 ortholog) also displayed impaired growth, S phase progression defects, and decreased Psf3 protein stability. We further showed that mouse embryos homozygous for a D24 variant presented intrauterine growth retardation and did not survive to birth, and that fibroblasts derived from these embryos displayed accelerated cellular senescence. Taken together, our findings implicate GINS3 in the pathogenesis of MGS and support the notion that hypomorphic variants identified in this gene impaired cell and organismal growth by compromising DNA replication.


Asunto(s)
Micrognatismo , Saccharomyces cerevisiae , Animales , Proteínas Cromosómicas no Histona , Microtia Congénita , Replicación del ADN/genética , Trastornos del Crecimiento , Humanos , Ratones , Micrognatismo/genética , Proteínas de Mantenimiento de Minicromosoma/genética , Rótula/anomalías
16.
NPJ Genom Med ; 7(1): 18, 2022 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-35288587

RESUMEN

Cardiomyopathy (CMP) is a heritable disorder. Over 50% of cases are gene-elusive on clinical gene panel testing. The contribution of variants in non-coding DNA elements that result in cryptic splicing and regulate gene expression has not been explored. We analyzed whole-genome sequencing (WGS) data in a discovery cohort of 209 pediatric CMP patients and 1953 independent replication genomes and exomes. We searched for protein-coding variants, and non-coding variants predicted to affect the function or expression of genes. Thirty-nine percent of cases harbored pathogenic coding variants in known CMP genes, and 5% harbored high-risk loss-of-function (LoF) variants in additional candidate CMP genes. Fifteen percent harbored high-risk regulatory variants in promoters and enhancers of CMP genes (odds ratio 2.25, p = 6.70 × 10-7 versus controls). Genes involved in α-dystroglycan glycosylation (FKTN, DTNA) and desmosomal signaling (DSC2, DSG2) were most highly enriched for regulatory variants (odds ratio 6.7-58.1). Functional effects were confirmed in patient myocardium and reporter assays in human cardiomyocytes, and in zebrafish CRISPR knockouts. We provide strong evidence for the genomic contribution of functionally active variants in new genes and in regulatory elements of known CMP genes to early onset CMP.

17.
Science ; 378(6615): 68-78, 2022 10 07.
Artículo en Inglés | MEDLINE | ID: mdl-36201590

RESUMEN

Establishing causal links between inherited polymorphisms and cancer risk is challenging. Here, we focus on the single-nucleotide polymorphism rs55705857, which confers a sixfold greater risk of isocitrate dehydrogenase (IDH)-mutant low-grade glioma (LGG). We reveal that rs55705857 itself is the causal variant and is associated with molecular pathways that drive LGG. Mechanistically, we show that rs55705857 resides within a brain-specific enhancer, where the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. Mutating the orthologous mouse rs55705857 locus accelerated tumor development in an Idh1R132H-driven LGG mouse model from 472 to 172 days and increased penetrance from 30% to 75%. Our work reveals mechanisms of the heritable predisposition to lethal glioma in ~40% of LGG patients.


Asunto(s)
Neoplasias Encefálicas , Cromosomas Humanos Par 8 , Glioma , Isocitrato Deshidrogenasa , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Cromosomas Humanos Par 8/genética , Glioma/genética , Glioma/patología , Humanos , Isocitrato Deshidrogenasa/genética , Ratones , Mutación , Polimorfismo de Nucleótido Simple
18.
PLoS One ; 16(11): e0252848, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34731163

RESUMEN

Although many long non-coding RNAs (lncRNAs) exhibit lineage-specific expression, the vast majority remain functionally uncharacterized in the context of development. Here, we report the first described human embryonic stem cell (hESC) lines to repress (CRISPRi) or activate (CRISPRa) transcription during differentiation into all three germ layers, facilitating the modulation of lncRNA expression during early development. We performed an unbiased, genome-wide CRISPRi screen targeting thousands of lncRNA loci expressed during endoderm differentiation. While dozens of lncRNA loci were required for proper differentiation, most differentially expressed lncRNAs were not, supporting the necessity for functional screening instead of relying solely on gene expression analyses. In parallel, we developed a clustering approach to infer mechanisms of action of lncRNA hits based on a variety of genomic features. We subsequently identified and validated FOXD3-AS1 as a functional lncRNA essential for pluripotency and differentiation. Taken together, the cell lines and methodology described herein can be adapted to discover and characterize novel regulators of differentiation into any lineage.


Asunto(s)
Diferenciación Celular/genética , Sistemas CRISPR-Cas , Factores de Transcripción Forkhead/genética , Humanos , Interferencia de ARN , ARN Largo no Codificante
19.
Front Mol Neurosci ; 14: 664912, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34025350

RESUMEN

In early development, the environment triggers mnemonic epigenomic programs resulting in memory and learning experiences to confer cognitive phenotypes into adulthood. To uncover how environmental stimulation impacts the epigenome and genome organization, we used the paradigm of environmental enrichment (EE) in young mice constantly receiving novel stimulation. We profiled epigenome and chromatin architecture in whole cortex and sorted neurons by deep-sequencing techniques. Specifically, we studied chromatin accessibility, gene and protein regulation, and 3D genome conformation, combined with predicted enhancer and chromatin interactions. We identified increased chromatin accessibility, transcription factor binding including CTCF-mediated insulation, differential occupancy of H3K36me3 and H3K79me2, and changes in transcriptional programs required for neuronal development. EE stimuli led to local genome re-organization by inducing increased contacts between chromosomes 7 and 17 (inter-chromosomal). Our findings support the notion that EE-induced learning and memory processes are directly associated with the epigenome and genome organization.

20.
Genome Biol ; 21(1): 210, 2020 08 20.
Artículo en Inglés | MEDLINE | ID: mdl-32819422

RESUMEN

BACKGROUND: Gene expression differences between species are driven by both cis and trans effects. Whereas cis effects are caused by genetic variants located on the same DNA molecule as the target gene, trans effects are due to genetic variants that affect diffusible elements. Previous studies have mostly assessed the impact of cis and trans effects at the gene level. However, how cis and trans effects differentially impact regulatory elements such as enhancers and promoters remains poorly understood. Here, we use massively parallel reporter assays to directly measure the transcriptional outputs of thousands of individual regulatory elements in embryonic stem cells and measure cis and trans effects between human and mouse. RESULTS: Our approach reveals that cis effects are widespread across transcribed regulatory elements, and the strongest cis effects are associated with the disruption of motifs recognized by strong transcriptional activators. Conversely, we find that trans effects are rare but stronger in enhancers than promoters and are associated with a subset of transcription factors that are differentially expressed between human and mouse. While we find that cis-trans compensation is common within promoters, we do not see evidence of widespread cis-trans compensation at enhancers. Cis-trans compensation is inversely correlated with enhancer redundancy, suggesting that such compensation may often occur across multiple enhancers. CONCLUSIONS: Our results highlight differences in the mode of evolution between promoters and enhancers in complex mammalian genomes and indicate that studying the evolution of individual regulatory elements is pivotal to understand the tempo and mode of gene expression evolution.


Asunto(s)
Elementos de Facilitación Genéticos , Evolución Molecular , Regulación del Desarrollo de la Expresión Génica , Regiones Promotoras Genéticas , Animales , Secuencia Conservada , Genes Reporteros , Humanos , Ratones , Elementos Reguladores de la Transcripción , Factores de Transcripción
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