RESUMEN
Exposure to stressful life events increases the risk for psychiatric disorders. Mechanistic insight into the genetic factors moderating the impact of stress can increase our understanding of disease processes. Here, we test 3,662 single nucleotide polymorphisms (SNPs) from preselected expression quantitative trait loci in massively parallel reporter assays to identify genetic variants that modulate the activity of regulatory elements sensitive to glucocorticoids, important mediators of the stress response. Of the tested SNP sequences, 547 were located in glucocorticoid-responsive regulatory elements of which 233 showed allele-dependent activity. Transcripts regulated by these functional variants were enriched for those differentially expressed in psychiatric disorders in the postmortem brain. Phenome-wide Mendelian randomization analysis in 4,439 phenotypes revealed potentially causal associations specifically in neurobehavioral traits, including major depression and other psychiatric disorders. Finally, a functional gene score derived from these variants was significantly associated with differences in the physiological stress response, suggesting that these variants may alter disease risk by moderating the individual set point of the stress response.
Asunto(s)
Glucocorticoides , Trastornos Mentales , Humanos , Ensayos Analíticos de Alto Rendimiento , Secuencias Reguladoras de Ácidos Nucleicos , Sitios de Carácter Cuantitativo , Trastornos Mentales/genética , Polimorfismo de Nucleótido Simple , Estudio de Asociación del Genoma Completo , Predisposición Genética a la EnfermedadRESUMEN
The glucocorticoid (GR) and androgen (AR) receptors execute unique functions in vivo, yet have nearly identical DNA binding specificities. To identify mechanisms that facilitate functional diversification among these transcription factor paralogs, we studied them in an equivalent cellular context. Analysis of chromatin and sequence suggest that divergent binding, and corresponding gene regulation, are driven by different abilities of AR and GR to interact with relatively inaccessible chromatin. Divergent genomic binding patterns can also be the result of subtle differences in DNA binding preference between AR and GR. Furthermore, the sequence composition of large regions (>10 kb) surrounding selectively occupied binding sites differs significantly, indicating a role for the sequence environment in guiding AR and GR to distinct binding sites. The comparison of binding sites that are shared shows that the specificity paradox can also be resolved by differences in the events that occur downstream of receptor binding. Specifically, shared binding sites display receptor-specific enhancer activity, cofactor recruitment and changes in histone modifications. Genomic deletion of shared binding sites demonstrates their contribution to directing receptor-specific gene regulation. Together, these data suggest that differences in genomic occupancy as well as divergence in the events that occur downstream of receptor binding direct functional diversification among transcription factor paralogs.
Asunto(s)
Cromatina/metabolismo , ADN/metabolismo , Receptores Androgénicos/metabolismo , Receptores de Glucocorticoides/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión , Línea Celular Tumoral , Regulación de la Expresión Génica , Humanos , Unión ProteicaRESUMEN
The binding of transcription factors to short recognition sequences plays a pivotal role in controlling the expression of genes. The sequence and shape characteristics of binding sites influence DNA binding specificity and have also been implicated in modulating the activity of transcription factors downstream of binding. To quantitatively assess the transcriptional activity of tens of thousands of designed synthetic sites in parallel, we developed a synthetic version of STARR-seq (synSTARR-seq). We used the approach to systematically analyze how variations in the recognition sequence of the glucocorticoid receptor (GR) affect transcriptional regulation. Our approach resulted in the identification of a novel highly active functional GR binding sequence and revealed that sequence variation both within and flanking GR's core binding site can modulate GR activity without apparent changes in DNA binding affinity. Notably, we found that the sequence composition of variants with similar activity profiles was highly diverse. In contrast, groups of variants with similar activity profiles showed specific DNA shape characteristics indicating that DNA shape may be a better predictor of activity than DNA sequence. Finally, using single cell experiments with individual enhancer variants, we obtained clues indicating that the architecture of the response element can independently tune expression mean and cell-to cell variability in gene expression (noise). Together, our studies establish synSTARR as a powerful method to systematically study how DNA sequence and shape modulate transcriptional output and noise.
Asunto(s)
ADN/genética , Análisis de Secuencia de ADN/métodos , Transcripción Genética , Sitios de Unión/genética , ADN/química , ADN/metabolismo , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Genes Reporteros , Genes Sintéticos , Variación Genética , Humanos , Conformación de Ácido Nucleico , Conformación Proteica , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Elementos de Respuesta , Análisis de Secuencia de ADN/estadística & datos numéricos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
One snapshot of the peer-review process for "Somatic XIST activation and features of X chromosome inactivation in male human cancers" (Sadagopan et al., 2022).
Asunto(s)
Neoplasias , ARN Largo no Codificante , Masculino , Humanos , Inactivación del Cromosoma X/genética , ARN no Traducido/genética , ARN Largo no Codificante/genética , Factores de Transcripción/genética , Neoplasias/genéticaRESUMEN
Glucocorticoids are stress hormones that elicit cellular responses by binding to the glucocorticoid receptor, a ligand-activated transcription factor. The exposure of cells to this hormone induces wide-spread changes in the chromatin landscape and gene expression. Previous studies have suggested that some of these changes are reversible whereas others persist even when the hormone is no longer around. However, when we examined chromatin accessibility in human airway epithelial cells after hormone washout, we found that the hormone-induced changes were universally reversed after 1 d. Moreover, priming of cells by a previous exposure to hormone, in general, did not alter the transcriptional response to a subsequent encounter of the same cue except for one gene, ZBTB16, that displays transcriptional memory manifesting itself as a more robust transcriptional response upon repeated hormone stimulation. Single-cell analysis revealed that the more robust response is driven by a higher probability of primed cells to activate ZBTB16 and by a subset of cells that express the gene at levels that are higher than the induction levels observed for naïve cells.
Asunto(s)
Cromatina , Glucocorticoides/metabolismo , Transducción de Señal , Transcripción Genética/genética , Células A549 , Animales , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Humanos , Ratas , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiología , Análisis de la Célula IndividualRESUMEN
The glucocorticoid receptor (GR), a hormone-activated transcription factor, binds to a myriad of genomic binding sites yet seems to regulate a much smaller number of genes. Genome-wide analysis of GR binding and gene regulation has shown that the likelihood of GR-dependent regulation increases with decreased distance of its binding to the transcriptional start site of a gene. To test if we can adopt this knowledge to expand the repertoire of GR target genes, we used CRISPR/Cas-mediated homology-directed repair to add a single GR-binding site directly upstream of the transcriptional start site of each of four genes. To our surprise, we found that the addition of a single GR-binding site can be enough to convert a gene into a GR target. The gain of GR-dependent regulation was observed for two of four genes analyzed and coincided with acquired GR binding at the introduced binding site. However, the gene-specific gain of GR-dependent regulation could not be explained by obvious differences in chromatin accessibility between converted genes and their non-converted counterparts. Furthermore, by introducing GR-binding sequences with different nucleotide compositions, we show that activation can be facilitated by distinct sequences without obvious differences in activity between the GR-binding sequence variants we tested. The approach to use genome engineering to build genomic response elements facilitates the generation of cell lines with tailored repertoires of GR-responsive genes and a framework to test and refine our understanding of the cis-regulatory logic of gene regulation by testing if engineered response elements behave as predicted.
Asunto(s)
Edición Génica/métodos , Receptores de Glucocorticoides/genética , Elementos de Respuesta/genética , Animales , Sitios de Unión/genética , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Regulación de la Expresión Génica , Humanos , Unión Proteica/genética , RNA-Seq , Ratas , Elementos Reguladores de la Transcripción/genética , Sitio de Iniciación de la Transcripción , Transcripción Genética/genética , Activación Transcripcional/genética , TransfecciónRESUMEN
The transcription factor GATA2 is required for expansion and differentiation of hematopoietic stem cells (HSCs). In mesenchymal stem cells (MSCs), GATA2 blocks adipogenesis, but its biological relevance and underlying genomic events are unknown. We report a dual function of GATA2 in bone homeostasis. GATA2 in MSCs binds near genes involved in skeletal system development and colocalizes with motifs for FOX and HOX transcription factors, known regulators of skeletal development. Ectopic GATA2 blocks osteoblastogenesis by interfering with SMAD1/5/8 activation. MSC-specific deletion of GATA2 in mice increases the numbers and differentiation capacity of bone-derived precursors, resulting in elevated bone formation. Surprisingly, MSC-specific GATA2 deficiency impairs the trabecularization and mechanical strength of bone, involving reduced MSC expression of the osteoclast inhibitor osteoprotegerin and increased osteoclast numbers. Thus, GATA2 affects bone turnover via MSC-autonomous and indirect effects. By regulating bone trabecularization, GATA2 expression in the osteogenic lineage may contribute to the anatomical and cellular microenvironment of the HSC niche required for hematopoiesis.