RESUMEN
A fundamental feature of cellular growth is that total protein and RNA amounts increase with cell size to keep concentrations approximately constant. A key component of this is that global transcription rates increase in larger cells. Here, we identify RNA polymerase II (RNAPII) as the limiting factor scaling mRNA transcription with cell size in budding yeast, as transcription is highly sensitive to the dosage of RNAPII but not to other components of the transcriptional machinery. Our experiments support a dynamic equilibrium model where global RNAPII transcription at a given size is set by the mass action recruitment kinetics of unengaged nucleoplasmic RNAPII to the genome. However, this only drives a sub-linear increase in transcription with size, which is then partially compensated for by a decrease in mRNA decay rates as cells enlarge. Thus, limiting RNAPII and feedback on mRNA stability work in concert to scale mRNA amounts with cell size.
Asunto(s)
Tamaño de la Célula , ARN Polimerasa II , Transcripción Genética , Retroalimentación , ARN Polimerasa II/metabolismo , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Cells communicate with each other via receptor-ligand interactions. Here, we describe lentiviral-mediated cell entry by engineered receptor-ligand interaction (ENTER) to display ligand proteins, deliver payloads, and record receptor specificity. We optimize ENTER to decode interactions between T cell receptor (TCR)-MHC peptides, antibody-antigen, and other receptor-ligand pairs. A viral presentation strategy allows ENTER to capture interactions between B cell receptor and any antigen. We engineer ENTER to deliver genetic payloads to antigen-specific T or B cells to selectively modulate cellular behavior in mixed populations. Single-cell readout of ENTER by RNA sequencing (ENTER-seq) enables multiplexed enumeration of antigen specificities, TCR clonality, cell type, and states of individual T cells. ENTER-seq of CMV-seropositive patient blood samples reveals the viral epitopes that drive effector memory T cell differentiation and inter-clonal vs. intra-clonal phenotypic diversity targeting the same epitope. ENTER technology enables systematic discovery of receptor specificity, linkage to cell fates, and antigen-specific cargo delivery.
Asunto(s)
Receptores de Antígenos de Linfocitos T , Internalización del Virus , Humanos , Biología , Epítopos , Ligandos , Péptidos , Receptores de Antígenos de Linfocitos T/metabolismo , Análisis de la Célula Individual , GenómicaRESUMEN
To define the multi-cellular epigenomic and transcriptional landscape of cardiac cellular development, we generated single-cell chromatin accessibility maps of human fetal heart tissues. We identified eight major differentiation trajectories involving primary cardiac cell types, each associated with dynamic transcription factor (TF) activity signatures. We contrasted regulatory landscapes of iPSC-derived cardiac cell types and their in vivo counterparts, which enabled optimization of in vitro differentiation of epicardial cells. Further, we interpreted sequence based deep learning models of cell-type-resolved chromatin accessibility profiles to decipher underlying TF motif lexicons. De novo mutations predicted to affect chromatin accessibility in arterial endothelium were enriched in congenital heart disease (CHD) cases vs. controls. In vitro studies in iPSCs validated the functional impact of identified variation on the predicted developmental cell types. This work thus defines the cell-type-resolved cis-regulatory sequence determinants of heart development and identifies disruption of cell type-specific regulatory elements in CHD.
Asunto(s)
Cromatina , Cardiopatías Congénitas , Humanos , Cromatina/genética , Cardiopatías Congénitas/genética , Corazón , Mutación , Análisis de la Célula IndividualRESUMEN
Adaptive immunity relies on specialized effector functions elicited by lymphocytes, yet how antigen recognition activates appropriate effector responses through nonspecific signaling intermediates is unclear. Here we examined the role of chromatin priming in specifying the functional outputs of effector T cells and found that most of the cis-regulatory landscape active in effector T cells was poised early in development before the expression of the T cell antigen receptor. We identified two principal mechanisms underpinning this poised landscape: the recruitment of the nucleosome remodeler mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) by the transcription factors RUNX1 and PU.1 to establish chromatin accessibility at T effector loci; and a 'relay' whereby the transcription factor BCL11B succeeded PU.1 to maintain occupancy of the chromatin remodeling complex mSWI/SNF together with RUNX1, after PU.1 silencing during lineage commitment. These mechanisms define modes by which T cells acquire the potential to elicit specialized effector functions early in their ontogeny and underscore the importance of integrating extrinsic cues to the developmentally specified intrinsic program.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal , Proteínas Proto-Oncogénicas , Proteínas Represoras , Transactivadores , Factores de Transcripción , Proteínas Supresoras de Tumor , Proteínas Proto-Oncogénicas/metabolismo , Animales , Transactivadores/metabolismo , Transactivadores/genética , Ratones , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Ratones Endogámicos C57BL , Proteínas Cromosómicas no Histona/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Ratones Noqueados , Ensamble y Desensamble de Cromatina , Diferenciación Celular/inmunologíaRESUMEN
Genetic perturbations of cortical development can lead to neurodevelopmental disease, including autism spectrum disorder (ASD). To identify genomic regions crucial to corticogenesis, we mapped the activity of gene-regulatory elements generating a single-cell atlas of gene expression and chromatin accessibility both independently and jointly. This revealed waves of gene regulation by key transcription factors (TFs) across a nearly continuous differentiation trajectory, distinguished the expression programs of glial lineages, and identified lineage-determining TFs that exhibited strong correlation between linked gene-regulatory elements and expression levels. These highly connected genes adopted an active chromatin state in early differentiating cells, consistent with lineage commitment. Base-pair-resolution neural network models identified strong cell-type-specific enrichment of noncoding mutations predicted to be disruptive in a cohort of ASD individuals and identified frequently disrupted TF binding sites. This approach illustrates how cell-type-specific mapping can provide insights into the programs governing human development and disease.
Asunto(s)
Corteza Cerebral/embriología , Cromatina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Análisis de la Célula Individual , Astrocitos/citología , Diferenciación Celular , Linaje de la Célula/genética , Análisis por Conglomerados , Aprendizaje Profundo , Epigénesis Genética , Lógica Difusa , Glutamatos/metabolismo , Humanos , Mutación/genética , Neuronas/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genéticaRESUMEN
The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a GGGGCC repeat expansion in the C9orf72 gene. We developed a platform to interrogate the chromatin accessibility landscape and transcriptional program within neurons during degeneration. We provide evidence that neurons expressing the dipeptide repeat protein poly(proline-arginine), translated from the C9orf72 repeat expansion, activate a highly specific transcriptional program, exemplified by a single transcription factor, p53. Ablating p53 in mice completely rescued neurons from degeneration and markedly increased survival in a C9orf72 mouse model. p53 reduction also rescued axonal degeneration caused by poly(glycine-arginine), increased survival of C9orf72 ALS/FTD-patient-induced pluripotent stem cell (iPSC)-derived motor neurons, and mitigated neurodegeneration in a C9orf72 fly model. We show that p53 activates a downstream transcriptional program, including Puma, which drives neurodegeneration. These data demonstrate a neurodegenerative mechanism dynamically regulated through transcription-factor-binding events and provide a framework to apply chromatin accessibility and transcription program profiles to neurodegeneration.
Asunto(s)
Proteína C9orf72/metabolismo , Expansión de las Repeticiones de ADN/genética , Degeneración Nerviosa/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Axones/metabolismo , Proteína C9orf72/genética , Muerte Celular , Células Cultivadas , Corteza Cerebral/patología , Cromatina/metabolismo , Daño del ADN , Modelos Animales de Enfermedad , Drosophila , Ratones Endogámicos C57BL , Degeneración Nerviosa/patología , Estabilidad Proteica , Transcripción Genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Immune aging combines cellular defects in adaptive immunity with the activation of pathways causing a low-inflammatory state. Here we examined the influence of age on the kinetic changes in the epigenomic and transcriptional landscape induced by T cell receptor (TCR) stimulation in naive CD4+ T cells. Despite attenuated TCR signaling in older adults, TCR activation accelerated remodeling of the epigenome and induced transcription factor networks favoring effector cell differentiation. We identified increased phosphorylation of STAT5, at least in part due to aberrant IL-2 receptor and lower HELIOS expression, as upstream regulators. Human HELIOS-deficient, naive CD4+ T cells, when transferred into human-synovium-mouse chimeras, infiltrated tissues more efficiently. Inhibition of IL-2 or STAT5 activity in T cell responses of older adults restored the epigenetic response pattern to the one seen in young adults. In summary, reduced HELIOS expression in non-regulatory naive CD4+ T cells in older adults directs T cell fate decisions toward inflammatory effector cells that infiltrate tissue.
Asunto(s)
Envejecimiento , Linfocitos T CD4-Positivos , Factor de Transcripción Ikaros , Anciano , Animales , Humanos , Ratones , Adulto Joven , Envejecimiento/inmunología , Envejecimiento/patología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Ensamble y Desensamble de Cromatina , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T , Factor de Transcripción STAT5 , Factor de Transcripción Ikaros/metabolismoRESUMEN
Adult mesenchymal stem cells, including preadipocytes, possess a cellular sensory organelle called the primary cilium. Ciliated preadipocytes abundantly populate perivascular compartments in fat and are activated by a high-fat diet. Here, we sought to understand whether preadipocytes use their cilia to sense and respond to external cues to remodel white adipose tissue. Abolishing preadipocyte cilia in mice severely impairs white adipose tissue expansion. We discover that TULP3-dependent ciliary localization of the omega-3 fatty acid receptor FFAR4/GPR120 promotes adipogenesis. FFAR4 agonists and ω-3 fatty acids, but not saturated fatty acids, trigger mitosis and adipogenesis by rapidly activating cAMP production inside cilia. Ciliary cAMP activates EPAC signaling, CTCF-dependent chromatin remodeling, and transcriptional activation of PPARγ and CEBPα to initiate adipogenesis. We propose that dietary ω-3 fatty acids selectively drive expansion of adipocyte numbers to produce new fat cells and store saturated fatty acids, enabling homeostasis of healthy fat tissue.
Asunto(s)
Adipogénesis , Cilios/metabolismo , Ácidos Grasos Omega-3/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Adipogénesis/efectos de los fármacos , Tejido Adiposo Blanco/metabolismo , Animales , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Factor de Unión a CCCTC/metabolismo , Cromatina/metabolismo , Cilios/efectos de los fármacos , AMP Cíclico/metabolismo , Ácidos Docosahexaenoicos/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , PPAR gamma/metabolismoRESUMEN
Here, we present Perturb-ATAC, a method that combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by assay of transposase-accessible chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in â¼4,300 single cells, encompassing more than 63 genotype-phenotype relationships. Perturb-ATAC in human B lymphocytes uncovered regulators of chromatin accessibility, TF occupancy, and nucleosome positioning and identified a hierarchy of TFs that govern B cell state, variation, and disease-associated cis-regulatory elements. Perturb-ATAC in primary human epidermal cells revealed three sequential modules of cis-elements that specify keratinocyte fate. Combinatorial deletion of all pairs of these TFs uncovered their epistatic relationships and highlighted genomic co-localization as a basis for synergistic interactions. Thus, Perturb-ATAC is a powerful strategy to dissect gene regulatory networks in development and disease.
Asunto(s)
Epigenómica/métodos , Redes Reguladoras de Genes/genética , Análisis de la Célula Individual/métodos , Cromatina/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/fisiología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/fisiología , Redes Reguladoras de Genes/fisiología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Análisis de Secuencia de ADN/métodos , Factores de Transcripción/metabolismoRESUMEN
RNAs fold into defined tertiary structures to function in critical biological processes. While quantitative models can predict RNA secondary structure stability, we are still unable to predict the thermodynamic stability of RNA tertiary structure. Here, we probe conformational preferences of diverse RNA two-way junctions to develop a predictive model for the formation of RNA tertiary structure. We quantitatively measured tertiary assembly energetics of >1,000 of RNA junctions inserted in multiple structural scaffolds to generate a "thermodynamic fingerprint" for each junction. Thermodynamic fingerprints enabled comparison of junction conformational preferences, revealing principles for how sequence influences 3-dimensional conformations. Utilizing fingerprints of junctions with known crystal structures, we generated ensembles for related junctions that predicted their thermodynamic effects on assembly formation. This work reveals sequence-structure-energetic relationships in RNA, demonstrates the capacity for diverse compensation strategies within tertiary structures, and provides a path to quantitative modeling of RNA folding energetics based on "ensemble modularity."
Asunto(s)
ARN/metabolismo , Disparidad de Par Base , Biblioteca de Genes , Conformación de Ácido Nucleico , Fotoblanqueo , ARN/química , Pliegue del ARN , Estabilidad del ARN , TermodinámicaRESUMEN
Human hematopoiesis involves cellular differentiation of multipotent cells into progressively more lineage-restricted states. While the chromatin accessibility landscape of this process has been explored in defined populations, single-cell regulatory variation has been hidden by ensemble averaging. We collected single-cell chromatin accessibility profiles across 10 populations of immunophenotypically defined human hematopoietic cell types and constructed a chromatin accessibility landscape of human hematopoiesis to characterize differentiation trajectories. We find variation consistent with lineage bias toward different developmental branches in multipotent cell types. We observe heterogeneity within common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) and develop a strategy to partition GMPs along their differentiation trajectory. Furthermore, we integrated single-cell RNA sequencing (scRNA-seq) data to associate transcription factors to chromatin accessibility changes and regulatory elements to target genes through correlations of expression and regulatory element accessibility. Overall, this work provides a framework for integrative exploration of complex regulatory dynamics in a primary human tissue at single-cell resolution.
Asunto(s)
Cromatina/química , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Análisis de la Célula Individual , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular , Linaje de la Célula , Epigénesis Genética , Epigenómica , Hematopoyesis , Trasplante de Células Madre Hematopoyéticas , Humanos , Células Progenitoras Mieloides/citología , Análisis de Componente Principal , Secuencias Reguladoras de Ácidos Nucleicos , Análisis de Secuencia de ARN , TranscriptomaRESUMEN
Metastases are the main cause of cancer deaths, but the mechanisms underlying metastatic progression remain poorly understood. We isolated pure populations of cancer cells from primary tumors and metastases from a genetically engineered mouse model of human small cell lung cancer (SCLC) to investigate the mechanisms that drive the metastatic spread of this lethal cancer. Genome-wide characterization of chromatin accessibility revealed the opening of large numbers of distal regulatory elements across the genome during metastatic progression. These changes correlate with copy number amplification of the Nfib locus, and differentially accessible sites were highly enriched for Nfib transcription factor binding sites. Nfib is necessary and sufficient to increase chromatin accessibility at a large subset of the intergenic regions. Nfib promotes pro-metastatic neuronal gene expression programs and drives the metastatic ability of SCLC cells. The identification of widespread chromatin changes during SCLC progression reveals an unexpected global reprogramming during metastatic progression.
Asunto(s)
Neoplasias Pulmonares/patología , Factores de Transcripción NFI/metabolismo , Metástasis de la Neoplasia/patología , Carcinoma Pulmonar de Células Pequeñas/patología , Secuencias de Aminoácidos , Animales , Línea Celular Tumoral , Células Cultivadas , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Factores de Transcripción NFI/genética , Regiones Promotoras Genéticas , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Regulación hacia ArribaRESUMEN
Aire mediates the expression of tissue-specific antigens in thymic epithelial cells to promote tolerance against self-reactive T lymphocytes. However, the mechanism that allows expression of tissue-specific genes at levels that prevent harm is unknown. Here we show that Brg1 generates accessibility at tissue-specific loci to impose central tolerance. We found that Aire has an intrinsic repressive function that restricts chromatin accessibility and opposes Brg1 across the genome. Aire exerted this repressive influence within minutes after recruitment to chromatin and restrained the amplitude of active transcription. Disease-causing mutations that impair Aire-induced activation also impair the protein's repressive function, which indicates dual roles for Aire. Together, Brg1 and Aire fine-tune the expression of tissue-specific genes at levels that prevent toxicity yet promote immune tolerance.
Asunto(s)
Tolerancia Central/inmunología , ADN Helicasas/inmunología , Regulación de la Expresión Génica/inmunología , Proteínas Nucleares/inmunología , Timo/inmunología , Factores de Transcripción/inmunología , Animales , Cromatina , Ratones , Ratones Transgénicos , Proteína AIRERESUMEN
Argonautes are nucleic acid-guided proteins that perform numerous cellular functions across all domains of life. Little is known about how distinct evolutionary pressures have shaped each Argonaute's biophysical properties. We applied high-throughput biochemistry to characterize how Thermus thermophilus Argonaute (TtAgo), a DNA-guided DNA endonuclease, finds, binds, and cleaves its targets. We found that TtAgo uses biophysical adaptations similar to those of eukaryotic Argonautes for rapid association but requires more extensive complementarity to achieve high-affinity target binding. Using these data, we constructed models for TtAgo association rates and equilibrium binding affinities that estimate the nucleic acid- and protein-mediated components of the target interaction energies. Finally, we showed that TtAgo cleavage rates vary widely based on the DNA guide, suggesting that only a subset of guides cleaves targets on physiologically relevant timescales.
Asunto(s)
Proteínas Argonautas , Thermus thermophilus , Proteínas Argonautas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN/genética , Endonucleasas/metabolismo , Thermus thermophilus/genéticaRESUMEN
RNAs are central to fundamental biological processes in all known organisms. The set of possible intramolecular interactions of RNA nucleotides defines the range of alternative structural conformations of a specific RNA that can coexist, and these structures enable functional catalytic properties of RNAs and/or their productive intermolecular interactions with other RNAs or proteins. However, the immense combinatorial space of potential RNA sequences has precluded predictive mapping between RNA sequence and molecular structure and function. Recent advances in high-throughput approaches in vitro have enabled quantitative thermodynamic and kinetic measurements of RNA-RNA and RNA-protein interactions, across hundreds of thousands of sequence variations. In this Review, we explore these techniques, how they can be used to understand RNA function and how they might form the foundations of an accurate model to predict the structure and function of an RNA directly from its nucleotide sequence. The experimental techniques and modelling frameworks discussed here are also highly relevant for the sampling of sequence-structure-function space of DNAs and proteins.
Asunto(s)
ADN , ARN , Conformación de Ácido Nucleico , Secuencia de Bases , ARN/metabolismo , Análisis de Secuencia de ARN/métodosRESUMEN
The intestine is a complex organ that promotes digestion, extracts nutrients, participates in immune surveillance, maintains critical symbiotic relationships with microbiota and affects overall health1. The intesting has a length of over nine metres, along which there are differences in structure and function2. The localization of individual cell types, cell type development trajectories and detailed cell transcriptional programs probably drive these differences in function. Here, to better understand these differences, we evaluated the organization of single cells using multiplexed imaging and single-nucleus RNA and open chromatin assays across eight different intestinal sites from nine donors. Through systematic analyses, we find cell compositions that differ substantially across regions of the intestine and demonstrate the complexity of epithelial subtypes, and find that the same cell types are organized into distinct neighbourhoods and communities, highlighting distinct immunological niches that are present in the intestine. We also map gene regulatory differences in these cells that are suggestive of a regulatory differentiation cascade, and associate intestinal disease heritability with specific cell types. These results describe the complexity of the cell composition, regulation and organization for this organ, and serve as an important reference map for understanding human biology and disease.
Asunto(s)
Intestinos , Análisis de la Célula Individual , Humanos , Diferenciación Celular/genética , Cromatina/genética , Células Epiteliales/citología , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Mucosa Intestinal/citología , Intestinos/citología , Intestinos/inmunología , Análisis de Expresión Génica de una Sola CélulaRESUMEN
Mammalian SWI/SNF (BAF) chromatin remodelers play dosage-sensitive roles in many human malignancies and neurologic disorders. The gene encoding the BAF subunit actin-like 6a (ACTL6A) is amplified early in the development of many squamous cell carcinomas (SCCs), but its oncogenic role remains unclear. Here we demonstrate that ACTL6A overexpression leads to its stoichiometric assembly into BAF complexes and drives their interaction and engagement with specific regulatory regions in the genome. In normal epithelial cells, ACTL6A was substoichiometric to other BAF subunits. However, increased ACTL6A levels by ectopic expression or in SCC cells led to near saturation of ACTL6A within BAF complexes. Increased ACTL6A occupancy enhanced polycomb opposition genome-wide to activate SCC genes and facilitated the co-dependent loading of BAF and TEAD-YAP complexes on chromatin. Both mechanisms appeared to be critical and function as a molecular AND gate for SCC initiation and maintenance, thereby explaining the specificity of the role of ACTL6A amplification in SCCs.
Asunto(s)
Actinas/metabolismo , Carcinoma de Células Escamosas/metabolismo , Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Actinas/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Epigénesis Genética , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Proteínas del Grupo Polycomb/genética , Unión Proteica , Factores de Transcripción de Dominio TEA/genética , Factores de Transcripción de Dominio TEA/metabolismo , Proteínas Señalizadoras YAP/genética , Proteínas Señalizadoras YAP/metabolismoRESUMEN
Biosynthesis scales with cell size such that protein concentrations generally remain constant as cells grow. As an exception, synthesis of the cell-cycle inhibitor Whi5 "sub-scales" with cell size so that its concentration is lower in larger cells to promote cell-cycle entry. Here, we find that transcriptional control uncouples Whi5 synthesis from cell size, and we identify histones as the major class of sub-scaling transcripts besides WHI5 by screening for similar genes. Histone synthesis is thereby matched to genome content rather than cell size. Such sub-scaling proteins are challenged by asymmetric cell division because proteins are typically partitioned in proportion to newborn cell volume. To avoid this fate, Whi5 uses chromatin-binding to partition similar protein amounts to each newborn cell regardless of cell size. Disrupting both Whi5 synthesis and chromatin-based partitioning weakens G1 size control. Thus, specific transcriptional and partitioning mechanisms determine protein sub-scaling to control cell size.
Asunto(s)
Cromatina/química , Regulación Fúngica de la Expresión Génica , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Transcripción Genética , Ciclo Celular , Cromatina/metabolismo , Biología Computacional , Histonas/química , Homeostasis , Hibridación Fluorescente in Situ , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Análisis de Regresión , Proteínas Represoras , Proteínas de Saccharomyces cerevisiaeRESUMEN
Combination therapy with PD-1 blockade and IL-2 is highly effective during chronic lymphocytic choriomeningitis virus infection1. Here we examine the underlying basis for this synergy. We show that PD-1 + IL-2 combination therapy, in contrast to PD-1 monotherapy, substantially changes the differentiation program of the PD-1+TCF1+ stem-like CD8+ T cells and results in the generation of transcriptionally and epigenetically distinct effector CD8+ T cells that resemble highly functional effector CD8+ T cells seen after an acute viral infection. The generation of these qualitatively superior CD8+ T cells that mediate viral control underlies the synergy between PD-1 and IL-2. Our results show that the PD-1+TCF1+ stem-like CD8+ T cells, also referred to as precursors of exhausted CD8+ T cells, are not fate-locked into the exhaustion program and their differentiation trajectory can be changed by IL-2 signals. These virus-specific effector CD8+ T cells emerging from the stem-like CD8+ T cells after combination therapy expressed increased levels of the high-affinity IL-2 trimeric (CD25-CD122-CD132) receptor. This was not seen after PD-1 blockade alone. Finally, we show that CD25 engagement with IL-2 has an important role in the observed synergy between IL-2 cytokine and PD-1 blockade. Either blocking CD25 with an antibody or using a mutated version of IL-2 that does not bind to CD25 but still binds to CD122 and CD132 almost completely abrogated the synergistic effects observed after PD-1 + IL-2 combination therapy. There is considerable interest in PD-1 + IL-2 combination therapy for patients with cancer2,3, and our fundamental studies defining the underlying mechanisms of how IL-2 synergizes with PD-1 blockade should inform these human translational studies.