RESUMEN
Master regulators, such as the hematopoietic transcription factor (TF) GATA1, play an essential role in orchestrating lineage commitment and differentiation. However, the precise mechanisms by which such TFs regulate transcription through interactions with specific cis-regulatory elements remain incompletely understood. Here, we describe a form of congenital hemolytic anemia caused by missense mutations in an intrinsically disordered region of GATA1, with a poorly understood role in transcriptional regulation. Through integrative functional approaches, we demonstrate that these mutations perturb GATA1 transcriptional activity by partially impairing nuclear localization and selectively altering precise chromatin occupancy by GATA1. These alterations in chromatin occupancy and concordant chromatin accessibility changes alter faithful gene expression, with failure to both effectively silence and activate select genes necessary for effective terminal red cell production. We demonstrate how disease-causing mutations can reveal regulatory mechanisms that enable the faithful genomic targeting of master TFs during cellular differentiation.
Asunto(s)
Anemia , Factor de Transcripción GATA1 , Diferenciación Celular/genética , Cromatina/genética , Inmunoprecipitación de Cromatina , Eritropoyesis/genética , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , HumanosRESUMEN
We are only just beginning to catalog the vast diversity of cell types in the cerebral cortex. Such categorization is a first step toward understanding how diversification relates to function. All cortical projection neurons arise from a uniform pool of progenitor cells that lines the ventricles of the forebrain. It is still unclear how these progenitor cells generate the more than 50 unique types of mature cortical projection neurons defined by their distinct gene-expression profiles. Moreover, exactly how and when neurons diversify their function during development is unknown. Here we relate gene expression and chromatin accessibility of two subclasses of projection neurons with divergent morphological and functional features as they develop in the mouse brain between embryonic day 13 and postnatal day 5 in order to identify transcriptional networks that diversify neuron cell fate. We compare these gene-expression profiles with published profiles of single cells isolated from similar populations and establish that layer-defined cell classes encompass cell subtypes and developmental trajectories identified using single-cell sequencing. Given the depth of our sequencing, we identify groups of transcription factors with particularly dense subclass-specific regulation and subclass-enriched transcription factor binding motifs. We also describe transcription factor-adjacent long noncoding RNAs that define each subclass and validate the function of Myt1l in balancing the ratio of the two subclasses in vitro. Our multidimensional approach supports an evolving model of progressive restriction of cell fate competence through inherited transcriptional identities.
Asunto(s)
Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Análisis de la Célula Individual , Factores de Transcripción/genética , Animales , Diferenciación Celular/genética , Corteza Cerebral/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Ratones , RNA-Seq/métodosRESUMEN
Giant cell arteritis (GCA) is a common medium to large vessel vasculitis of the elderly that can lead to permanent vision loss. Neuroimaging in GCA may reveal optic nerve sheath enhancement, which is a cardinal finding of optic perineuritis (OPN). The clinical manifestations of GCA can mimic that of other ocular disorders including amiodarone-associated optic neuropathy (AAON). We report a case of biopsy-proven GCA in a patient initially suspected to have AAON. This patient presented with bilateral optic disc oedema in conjunction with subacute predominately monocular vision loss and enhancement of the corresponding optic nerve sheath on neuroimaging. Clinicians should recognise that clinical and neuroimaging findings of GCA can mimic a variety of ocular and orbital pathologies including idiopathic OPN and AAON.
RESUMEN
The >800 human G protein-coupled receptors (GPCRs) are responsible for transducing diverse chemical stimuli to alter cell state- and are the largest class of drug targets. Their myriad structural conformations and various modes of signaling make it challenging to understand their structure and function. Here, we developed a platform to characterize large libraries of GPCR variants in human cell lines with a barcoded transcriptional reporter of G protein signal transduction. We tested 7800 of 7828 possible single amino acid substitutions to the beta-2 adrenergic receptor (ß2AR) at four concentrations of the agonist isoproterenol. We identified residues specifically important for ß2AR signaling, mutations in the human population that are potentially loss of function, and residues that modulate basal activity. Using unsupervised learning, we identify residues critical for signaling, including all major structural motifs and molecular interfaces. We also find a previously uncharacterized structural latch spanning the first two extracellular loops that is highly conserved across Class A GPCRs and is conformationally rigid in both the inactive and active states of the receptor. More broadly, by linking deep mutational scanning with engineered transcriptional reporters, we establish a generalizable method for exploring pharmacogenomics, structure and function across broad classes of drug receptors.