RESUMEN

The macula and fovea comprise a highly sensitive visual detection tissue that is susceptible to common disease processes like age-related macular degeneration (AMD). Our understanding of the molecular determinants of high acuity vision remains unclear, as few model organisms possess a human-like fovea. We explore transcription factor networks and receptor-ligand interactions to elucidate tissue interactions in the macula and peripheral retina and concomitant changes in the underlying retinal pigment epithelium (RPE)/choroid. Poly-A selected, 100 bp paired-end RNA-sequencing (RNA-seq) was performed across the macular/foveal, perimacular, and temporal peripheral regions of the neural retina and RPE/choroid tissues of four adult Rhesus macaque eyes to characterize region- and tissue-specific gene expression. RNA-seq reads were mapped to both the macaque and human genomes for maximum alignment and analyzed for differential expression and Gene Ontology (GO) enrichment. Comparison of the neural retina and RPE/choroid tissues indicated distinct, contiguously changing gene expression profiles from fovea through perimacula to periphery. Top GO enrichment of differentially expressed genes in the RPE/choroid included cell junction organization and epithelial cell development. Expression of transcriptional regulators and various disease-associated genes show distinct location-specific preference and retina-RPE/choroid tissue-tissue interactions. Regional gene expression changes in the macaque retina and RPE/choroid is greater than that found in previously published transcriptome analysis of the human retina and RPE/choroid. Further, conservation of human macula-specific transcription factor profiles and gene expression in macaque tissues suggest a conservation of programs required for retina and RPE/choroid function and disease susceptibility.

RESUMEN

BACKGROUND: The development of highly scalable single-cell transcriptome technology has resulted in the creation of thousands of datasets, >30 in the retina alone. Analyzing the transcriptomes between different projects is highly desirable because this would allow for better assessment of which biological effects are consistent across independent studies. However it is difficult to compare and contrast data across different projects because there are substantial batch effects from computational processing, single-cell technology utilized, and the natural biological variation. While many single-cell transcriptome-specific batch correction methods purport to remove the technical noise, it is difficult to ascertain which method functions best. RESULTS: We developed a lightweight R package (scPOP, single-cell Pick Optimal Parameters) that brings in batch integration methods and uses a simple heuristic to balance batch merging and cell type/cluster purity. We use this package along with a Snakefile-based workflow system to demonstrate how to optimally merge 766,615 cells from 33 retina datsets and 3 species to create a massive ocular single-cell transcriptome meta-atlas. CONCLUSIONS: This provides a model for how to efficiently create meta-atlases for tissues and cells of interest.

Asunto(s)

Transcriptoma

RESUMEN

How homeodomain proteins gain sufficient specificity to control different cell fates has been a long-standing problem in developmental biology. The conserved Gsx homeodomain proteins regulate specific aspects of neural development in animals from flies to mammals, and yet they belong to a large transcription factor family that bind nearly identical DNA sequences in vitro. Here, we show that the mouse and fly Gsx factors unexpectedly gain DNA binding specificity by forming cooperative homodimers on precisely spaced and oriented DNA sites. High-resolution genomic binding assays revealed that Gsx2 binds both monomer and homodimer sites in the developing mouse ventral telencephalon. Importantly, reporter assays showed that Gsx2 mediates opposing outcomes in a DNA binding site-dependent manner: Monomer Gsx2 binding represses transcription, whereas homodimer binding stimulates gene expression. In Drosophila, the Gsx homolog, Ind, similarly represses or stimulates transcription in a site-dependent manner via an autoregulatory enhancer containing a combination of monomer and homodimer sites. Integrating these findings, we test a model showing how the homodimer to monomer site ratio and the Gsx protein levels defines gene up-regulation versus down-regulation. Altogether, these data serve as a new paradigm for how cooperative homeodomain transcription factor binding can increase target specificity and alter regulatory outcomes.

Asunto(s)

Proteínas de Drosophila/metabolismo , Drosophila/embriología , Drosophila/genética , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/metabolismo , Animales , Proteínas de Drosophila/genética , Genoma/genética , Estudio de Asociación del Genoma Completo , Proteínas de Homeodominio/genética , Ratones , Unión Proteica , Telencéfalo/embriología

RESUMEN

BACKGROUND: The MAPK/ERK signaling pathway is an essential regulator of numerous cell processes that are crucial for normal development as well as cancer progression. While much is known regarding MAPK/ERK signal conveyance from the cell membrane to the nucleus, the transcriptional and epigenetic mechanisms that govern gene expression downstream of MAPK signaling are not fully elucidated. RESULTS: This study employed an integrated epigenome analysis approach to interrogate the effects of MAPK/ERK pathway inhibition on the global transcriptome, the active chromatin landscape, and protein-DNA interactions in 501mel melanoma cells. Treatment of these cells with the small-molecule MEK inhibitor AZD6244 induces hyperpigmentation, widespread gene expression changes including alteration of genes linked to pigmentation, and extensive epigenomic reprogramming of transcriptionally distinct regulatory regions associated with the active chromatin mark H3K27ac. Regulatory regions with differentially acetylated H3K27ac regions following AZD6244 treatment are enriched in transcription factor binding motifs of ETV/ETS and ATF family members as well as the lineage-determining factors MITF and SOX10. H3K27ac-dense enhancer clusters known as super-enhancers show similar transcription factor motif enrichment, and furthermore, these super-enhancers are associated with genes encoding MITF, SOX10, and ETV/ETS proteins. Along with genome-wide resetting of the active enhancer landscape, MEK inhibition also results in widespread SOX10 recruitment throughout the genome, including increased SOX10 binding density at H3K27ac-marked enhancers. Importantly, these MEK inhibitor-responsive enhancers marked by H3K27ac and occupied by SOX10 are located near melanocyte lineage-specific and pigmentation genes and overlap numerous human SNPs associated with pigmentation and melanoma phenotypes, highlighting the variants located within these regions for prioritization in future studies. CONCLUSIONS: These results reveal the epigenetic reprogramming underlying the re-activation of melanocyte pigmentation and developmental transcriptional programs in 501mel cells in response to MEK inhibition and suggest extensive involvement of a MEK-SOX10 axis in the regulation of these processes. The dynamic chromatin changes identified here provide a rich genomic resource for further analyses of the molecular mechanisms governing the MAPK pathway in pigmentation- and melanocyte-associated diseases.

Asunto(s)

Cromatina/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Factores de Transcripción SOXE/metabolismo , Bencimidazoles/farmacología , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Código de Histonas , Histonas/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , Melanoma/metabolismo , Melanoma/patología , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Mutación , Pigmentación/efectos de los fármacos , Unión Proteica , Proteínas Proto-Oncogénicas B-raf/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Factores de Transcripción SOXE/antagonistas & inhibidores , Factores de Transcripción SOXE/genética

RESUMEN

The human eye is built from several specialized tissues which direct, capture and pre-process information to provide vision. The gene expression of the different eye tissues has been extensively profiled with RNA-seq across numerous studies. Large consortium projects have also used RNA-seq to study gene expression patterning across many different human tissues, minus the eye. There has not been an integrated study of expression patterns from multiple eye tissues compared with other human body tissues. We have collated all publicly available healthy human eye RNA-seq datasets as well as dozens of other tissues. We use this fully integrated dataset to probe the biological processes and pan expression relationships between the cornea, retina, retinal pigment epithelium (RPE)-choroid complex, and the rest of the human tissues with differential expression, clustering and gene ontology term enrichment tools. We also leverage our large collection of retina and RPE-choroid tissues to build the first human weighted gene correlation networks and use them to highlight known biological pathways and eye gene disease enrichment. We also have integrated publicly available single-cell RNA-seq data from mouse retina into our framework for validation and discovery. Finally, we make all these data, analyses and visualizations available via a powerful interactive web application (https://eyeintegration.nei.nih.gov/).

Asunto(s)

Ojo/metabolismo , Regulación de la Expresión Génica/genética , Especificidad de Órganos/genética , Retina/metabolismo , Animales , Coroides/metabolismo , Córnea/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Epitelio Pigmentado de la Retina/metabolismo

RESUMEN

Melanocyte stem cells (McSCs) and mouse models of hair graying serve as useful systems to uncover mechanisms involved in stem cell self-renewal and the maintenance of regenerating tissues. Interested in assessing genetic variants that influence McSC maintenance, we found previously that heterozygosity for the melanogenesis associated transcription factor, Mitf, exacerbates McSC differentiation and hair graying in mice that are predisposed for this phenotype. Based on transcriptome and molecular analyses of Mitfmi-vga9/+ mice, we report a novel role for MITF in the regulation of systemic innate immune gene expression. We also demonstrate that the viral mimic poly(I:C) is sufficient to expose genetic susceptibility to hair graying. These observations point to a critical suppressor of innate immunity, the consequences of innate immune dysregulation on pigmentation, both of which may have implications in the autoimmune, depigmenting disease, vitiligo.

Asunto(s)

Células Madre Adultas , Color del Cabello/inmunología , Inmunidad Innata , Melanocitos , Factor de Transcripción Asociado a Microftalmía/fisiología , Animales , Femenino , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Color del Cabello/genética , Interferón Tipo I/metabolismo , Ratones , Ratones Transgénicos , Poli I-C

RESUMEN

Stem cells display a fundamentally different mechanism of proliferation control when compared to somatic cells. Uncovering these mechanisms would maximize the impact in drug discovery with a higher translational applicability. The unbiased approach used in phenotype-based drug discovery (PDD) programs can offer a unique opportunity to identify such novel biological phenomenon. Here, we describe an integrated phenotypic screening approach, employing a combination of in vitro and in vivo PDD models to identify a small molecule increasing stem cell proliferation. We demonstrate that a combination of both in vitro and in vivo screening models improves hit identification and reproducibility of effects across various PDD models. Using cell viability and colony size phenotype measurement we characterize the structure activity relationship of the lead molecule, and identify that the small molecule inhibits phosphorylation of ERK2 and promotes stem cell proliferation. This study demonstrates a PDD approach that employs combinatorial models to identify compounds promoting stem cell proliferation.

RESUMEN

Hypoxia and HIF1α signaling direct tissue-specific gene responses regulating tumor progression, invasion, and metastasis. By integrating HIF1α knockdown and hypoxia-induced gene expression changes, this study identifies a melanocyte-specific, HIF1α-dependent/hypoxia-responsive gene expression signature. Integration of these gene expression changes with HIF1α ChIP-Seq analysis identifies 81 HIF1α direct target genes in melanocytes. The expression levels for 10 of the HIF1α direct targets - GAPDH, PKM, PPAT, DARS, DTWD1, SEH1L, ZNF292, RLF, AGTRAP, and GPC6 - are significantly correlated with reduced time of disease-free status in melanoma by logistic regression (P-value = 0.0013) and ROC curve analysis (AUC = 0.826, P-value < 0.0001). This HIF1α-regulated profile defines a melanocyte-specific response under hypoxia, and demonstrates the role of HIF1α as an invasive cell state gatekeeper in regulating cellular metabolism, chromatin and transcriptional regulation, vascularization, and invasion.

Asunto(s)

Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Melanocitos/metabolismo , Melanocitos/patología , Melanoma/genética , Melanoma/patología , Animales , Secuencia de Bases , Hipoxia de la Célula , Línea Celular Tumoral , Análisis por Conglomerados , Supervivencia sin Enfermedad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Motivos de Nucleótidos/genética , Pronóstico , Reproducibilidad de los Resultados

RESUMEN

The cellular cytoskeleton forms the primary basis through which a cell governs the changes in size, shape, migration, proliferation, and forms the primary means through which the cells respond to their environment. Indeed, cell and tissue morphologies are used routinely not only to grade tumors but also in various high-content screening methods with an aim to identify new small molecules with therapeutic potential. This study examines the expression of various cytoskeleton regulators in glioblastoma multiforme (GBM). GBM is a very aggressive disease with a low life expectancy even after chemo- and radiotherapy. Cancer cells of GBM are notorious for their invasiveness, ability to develop resistance to chemo- and radiotherapy, and to form secondary site tumors. This study aims to gain insight into cytoskeleton regulators in GBM cells and to understand the effect of various oncology drugs, including temozolomide, on cytoskeleton regulators. We compare the expression of various cytoskeleton regulators in GBM-derived tumor and normal tissue, CD133-postive and -negative cells from GBM and neural cells, and GBM stem-like and differentiated cells. In addition, the correlation between the expression of cytoskeleton regulators with the clinical outcome was examined to identify genes associated with longer patient survival. This was followed by a small molecule screening with US Food and Drug Administration (FDA)-approved oncology drugs, and its effect on cellular cytoskeleton was compared to treatment with temozolomide. This study identifies various groups of cytoskeletal regulators that have an important effect on patient survival and tumor development. Importantly, this work highlights the advantage of using cytoskeleton regulators as biomarkers for assessing prognosis and treatment design for GBM.

RESUMEN

The eleven-nineteen lysine-rich leukemia protein (ELL) is a key regulator of RNA polymerase II mediated transcription. ELL facilitates RNA polymerase II transcription pause site entry and release by dynamically interacting with p300 and the positive transcription elongation factor b (P-TEFb). In this study, we investigated the role of ELL during the HTLV-1 Tax oncogene induced transactivation. We show that ectopic expression of Tax enhances ELL incorporation into p300 and P-TEFb containing transcriptional complexes and the subsequent recruitment of these complexes to target genes in vivo. Depletion of ELL abrogates Tax induced transactivation of the immediate early genes Fos, Egr2 and NF-kB, suggesting that ELL is an essential cellular cofactor of the Tax oncogene. Thus, our study identifies a novel mechanism of ELL-dependent transactivation of immediate early genes by Tax and provides the rational for further defining the genome-wide targets of Tax and ELL.

Asunto(s)

Proteína p300 Asociada a E1A/genética , Productos del Gen tax/genética , Virus Linfotrópico T Tipo 1 Humano/genética , Factor B de Elongación Transcripcional Positiva/genética , Activación Transcripcional , Factores de Elongación Transcripcional/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Proteína p300 Asociada a E1A/metabolismo , Canales de Potasio Éter-A-Go-Go/genética , Canales de Potasio Éter-A-Go-Go/metabolismo , Regulación de la Expresión Génica , Productos del Gen tax/metabolismo , Células HEK293 , Interacciones Huésped-Patógeno , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Células Jurkat , FN-kappa B/genética , FN-kappa B/metabolismo , Factor B de Elongación Transcripcional Positiva/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Factores de Elongación Transcripcional/antagonistas & inhibidores , Factores de Elongación Transcripcional/metabolismo

RESUMEN

SOX10 is required for melanocyte development and maintenance, and has been linked to melanoma initiation and progression. However, the molecular mechanisms by which SOX10 guides the appropriate gene expression programs necessary to promote the melanocyte lineage are not fully understood. Here we employ genetic and epigenomic analysis approaches to uncover novel genomic targets and previously unappreciated molecular roles of SOX10 in melanocytes. Through global analysis of SOX10-binding sites and epigenetic characteristics of chromatin states, we uncover an extensive catalog of SOX10 targets genome-wide. Our findings reveal that SOX10 predominantly engages 'open' chromatin regions and binds to distal regulatory elements, including novel and previously known melanocyte enhancers. Integrated chromatin occupancy and transcriptome analysis suggest a role for SOX10 in both transcriptional activation and repression to regulate functionally distinct classes of genes. We demonstrate that distinct epigenetic signatures and cis-regulatory sequence motifs predicted to bind putative co-regulatory transcription factors define SOX10-activated and SOX10-repressed target genes. Collectively, these findings uncover a central role of SOX10 as a global regulator of gene expression in the melanocyte lineage by targeting diverse regulatory pathways.

Asunto(s)

Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Melanocitos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Factores de Transcripción SOXE/metabolismo , Animales , Sitios de Unión , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Epigenómica/métodos , Melanocitos/citología , Ratones , Factores de Transcripción SOXE/química , Factores de Transcripción SOXE/genética

Asunto(s)

Resistencia a Antineoplásicos/genética , Elementos de Facilitación Genéticos , Regulación Neoplásica de la Expresión Génica , Genoma Humano , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Humanos

RESUMEN

Transcription is a multi-stage process that coordinates several steps within the transcription cycle including chromatin reorganization, RNA polymerase II recruitment, initiation, promoter clearance and elongation. Recent advances have identified the super elongation complex, containing the eleven-nineteen lysine-rich leukaemia (ELL) protein, as a key regulator of transcriptional elongation. Here we show that ELL has a diverse and kinetically distinct role before its assembly into the super elongation complex by stabilizing Pol II recruitment/initiation and entry into the pause site. Loss of ELL destabilizes the pre-initiation complexes and results in disruption of early elongation and promoter proximal chromatin structure before recruitment of AFF4 and other super elongation complex components. These changes result in significantly reduced transcriptional activation of rapidly induced genes. Thus, ELL has an early and essential role during rapid high-amplitude gene expression that is required for both Pol II pause site entry and release.

Asunto(s)

ARN Polimerasa II/química , Factores de Elongación Transcripcional/metabolismo , Sitios de Unión , Cromatina/química , Inmunoprecipitación de Cromatina , Regulación de la Expresión Génica , Células HEK293 , Histonas/química , Humanos , Células Jurkat , Cinética , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Regiones Promotoras Genéticas , Interferencia de ARN , Transcripción Genética

RESUMEN

The BRCA1 gene product plays numerous roles in regulating genome integrity. Its role in assembling supermolecular complexes in response to DNA damage has been extensively studied; however, much less is understood about its role as a transcriptional coregulator. Loss or mutation is associated with hereditary breast and ovarian cancers, whereas altered expression occurs frequently in sporadic forms of breast cancer, suggesting that the control of BRCA1 transcription might be important to tumorigenesis. Here, we provide evidence of a striking linkage between the roles for BRCA1 as a transcriptional coregulator with control of its expression via an autoregulatory transcriptional loop. BRCA1 assembles with complexes containing E2F-1 and RB to form a repressive multicomponent transcriptional complex that inhibits BRCA1 promoter transcription. This complex is disrupted by genotoxic stress, resulting in the displacement of BRCA1 protein from the BRCA1 promoter and subsequent upregulation of BRCA1 transcription. Cells depleted of BRCA1 respond by upregulating BRCA1 transcripts, whereas cells overexpressing BRCA1 respond by downregulating BRCA1 transcripts. Tandem chromatin immmunoprecipitation studies show that BRCA1 is regulated by a dynamic coregulatory complex containing BRCA1, E2F1, and Rb at the BRCA1 promoter that is disrupted by DNA-damaging agents to increase its transcription. These results define a novel transcriptional mechanism of autoregulated homeostasis of BRCA1 that selectively titrates its levels to maintain genome integrity in response to genotoxic insult.

Asunto(s)

Proteína BRCA1/biosíntesis , Regulación Neoplásica de la Expresión Génica , Genes BRCA1 , Regiones Promotoras Genéticas , Proteína BRCA1/genética , Línea Celular Tumoral , Regulación hacia Abajo , Homeostasis , Humanos , Células Jurkat , Masculino , Transcripción Genética