RESUMEN
von Economo neurons (VENs) are bipolar, spindle-shaped neurons restricted to layer 5 of human frontoinsula and anterior cingulate cortex that appear to be selectively vulnerable to neuropsychiatric and neurodegenerative diseases, although little is known about other VEN cellular phenotypes. Single nucleus RNA-sequencing of frontoinsula layer 5 identifies a transcriptomically-defined cell cluster that contained VENs, but also fork cells and a subset of pyramidal neurons. Cross-species alignment of this cell cluster with a well-annotated mouse classification shows strong homology to extratelencephalic (ET) excitatory neurons that project to subcerebral targets. This cluster also shows strong homology to a putative ET cluster in human temporal cortex, but with a strikingly specific regional signature. Together these results suggest that VENs are a regionally distinctive type of ET neuron. Additionally, we describe the first patch clamp recordings of VENs from neurosurgically-resected tissue that show distinctive intrinsic membrane properties relative to neighboring pyramidal neurons.
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Neuronas/fisiología , Lóbulo Temporal/citología , Transcriptoma , Animales , Encéfalo/citología , Encéfalo/fisiología , Electrofisiología/métodos , Perfilación de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Ratones , Neuronas/citología , Células Piramidales/fisiología , Telencéfalo/citología , Lóbulo Temporal/fisiologíaRESUMEN
The era of targeted therapies has seen significant improvements in depth of response, progression-free survival, and overall survival for patients with multiple myeloma. Despite these improvements in clinical outcome, patients inevitably relapse and require further treatment. Drug-resistant dormant myeloma cells that reside in specific niches within the skeleton are considered a basis of disease relapse but remain elusive and difficult to study. Here, we developed a method to sequence the transcriptome of individual dormant myeloma cells from the bones of tumor-bearing mice. Our analyses show that dormant myeloma cells express a distinct transcriptome signature enriched for immune genes and, unexpectedly, genes associated with myeloid cell differentiation. These genes were switched on by coculture with osteoblastic cells. Targeting AXL, a gene highly expressed by dormant cells, using small-molecule inhibitors released cells from dormancy and promoted their proliferation. Analysis of the expression of AXL and coregulated genes in human cohorts showed that healthy human controls and patients with monoclonal gammopathy of uncertain significance expressed higher levels of the dormancy signature genes than patients with multiple myeloma. Furthermore, in patients with multiple myeloma, the expression of this myeloid transcriptome signature translated into a twofold increase in overall survival, indicating that this dormancy signature may be a marker of disease progression. Thus, engagement of myeloma cells with the osteoblastic niche induces expression of a suite of myeloid genes that predicts disease progression and that comprises potential drug targets to eradicate dormant myeloma cells.
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Mieloma Múltiple/genética , Mieloma Múltiple/patología , Recurrencia Local de Neoplasia/genética , Células Madre Neoplásicas/patología , Nicho de Células Madre/genética , Animales , Humanos , Ratones , Recurrencia Local de Neoplasia/patología , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Transcriptoma , Tirosina Quinasa del Receptor AxlRESUMEN
Transcriptomic profiling of complex tissues by single-nucleus RNA-sequencing (snRNA-seq) affords some advantages over single-cell RNA-sequencing (scRNA-seq). snRNA-seq provides less biased cellular coverage, does not appear to suffer cell isolation-based transcriptional artifacts, and can be applied to archived frozen specimens. We used well-matched snRNA-seq and scRNA-seq datasets from mouse visual cortex to compare cell type detection. Although more transcripts are detected in individual whole cells (~11,000 genes) than nuclei (~7,000 genes), we demonstrate that closely related neuronal cell types can be similarly discriminated with both methods if intronic sequences are included in snRNA-seq analysis. We estimate that the nuclear proportion of total cellular mRNA varies from 20% to over 50% for large and small pyramidal neurons, respectively. Together, these results illustrate the high information content of nuclear RNA for characterization of cellular diversity in brain tissues.
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Núcleo Celular/genética , Análisis de la Célula Individual , Transcriptoma/genética , Corteza Visual/metabolismo , Animales , Linaje de la Célula/genética , Linaje de la Célula/fisiología , Perfilación de la Expresión Génica/métodos , Ratones , Neuronas/metabolismo , Análisis de Secuencia de ARN/métodos , Corteza Visual/fisiologíaRESUMEN
The mammary gland consists of cells with gene expression patterns reflecting their cellular origins, function, and spatiotemporal context. However, knowledge of developmental kinetics and mechanisms of lineage specification is lacking. We address this significant knowledge gap by generating a single-cell transcriptome atlas encompassing embryonic, postnatal, and adult mouse mammary development. From these data, we map the chronology of transcriptionally and epigenetically distinct cell states and distinguish fetal mammary stem cells (fMaSCs) from their precursors and progeny. fMaSCs show balanced co-expression of factors associated with discrete adult lineages and a metabolic gene signature that subsides during maturation but reemerges in some human breast cancers and metastases. These data provide a useful resource for illuminating mammary cell heterogeneity, the kinetics of differentiation, and developmental correlates of tumorigenesis.
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Glándulas Mamarias Animales/crecimiento & desarrollo , Animales , Diferenciación Celular/fisiología , Femenino , Humanos , Glándulas Mamarias Animales/citología , Ratones , Células Madre/metabolismo , TranscriptomaRESUMEN
We describe convergent evidence from transcriptomics, morphology, and physiology for a specialized GABAergic neuron subtype in human cortex. Using unbiased single-nucleus RNA sequencing, we identify ten GABAergic interneuron subtypes with combinatorial gene signatures in human cortical layer 1 and characterize a group of human interneurons with anatomical features never described in rodents, having large 'rosehip'-like axonal boutons and compact arborization. These rosehip cells show an immunohistochemical profile (GAD1+CCK+, CNR1-SST-CALB2-PVALB-) matching a single transcriptomically defined cell type whose specific molecular marker signature is not seen in mouse cortex. Rosehip cells in layer 1 make homotypic gap junctions, predominantly target apical dendritic shafts of layer 3 pyramidal neurons, and inhibit backpropagating pyramidal action potentials in microdomains of the dendritic tuft. These cells are therefore positioned for potent local control of distal dendritic computation in cortical pyramidal neurons.
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Corteza Cerebral/metabolismo , Corteza Cerebral/ultraestructura , Neuronas GABAérgicas/metabolismo , Neuronas GABAérgicas/ultraestructura , Transcriptoma , Adulto , Anciano , Axones/ultraestructura , Espinas Dendríticas/metabolismo , Espinas Dendríticas/ultraestructura , Uniones Comunicantes/metabolismo , Uniones Comunicantes/ultraestructura , Biblioteca de Genes , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Células Piramidales/metabolismo , Células Piramidales/ultraestructura , ARN/análisis , ARN/genética , Análisis de Secuencia de ARNRESUMEN
In the version of this article initially published, NIH grant U01 MH106882 to F.H.G. was missing from the Acknowledgments. The error has been corrected in the HTML and PDF versions of the article.
RESUMEN
Cells are fundamental function units of multicellular organisms, with different cell types playing distinct physiological roles in the body. The recent advent of single-cell transcriptional profiling using RNA sequencing is producing 'big data', enabling the identification of novel human cell types at an unprecedented rate. In this review, we summarize recent work characterizing cell types in the human central nervous and immune systems using single-cell and single-nuclei RNA sequencing, and discuss the implications that these discoveries are having on the representation of cell types in the reference Cell Ontology (CL). We propose a method, based on random forest machine learning, for identifying sets of necessary and sufficient marker genes, which can be used to assemble consistent and reproducible cell type definitions for incorporation into the CL. The representation of defined cell type classes and their relationships in the CL using this strategy will make the cell type classes being identified by high-throughput/high-content technologies findable, accessible, interoperable and reusable (FAIR), allowing the CL to serve as a reference knowledgebase of information about the role that distinct cellular phenotypes play in human health and disease.
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Macrodatos , Perfilación de la Expresión Génica/tendencias , Análisis de Secuencia de ARN/tendencias , Análisis de la Célula Individual/tendencias , Linaje de la Célula/genética , Humanos , Transcriptoma/genéticaRESUMEN
Chest pain is a leading reason patients seek medical evaluation. While assays to detect myocyte death are used to diagnose a heart attack (acute myocardial infarction, AMI), there is no biomarker to indicate an impending cardiac event. Transcriptional patterns present in circulating endothelial cells (CEC) may provide a window into the plaque rupture process and identify a proximal biomarker for AMI. Thus, we aimed to identify a transcriptomic signature of AMI present in whole blood, but derived from CECs. Candidate genes indicative of AMI were nominated from microarray of enriched CEC samples, and then verified for detectability and predictive potential via qPCR in whole blood. This signature was validated in an independent cohort. Our findings suggest that a whole blood CEC-derived molecular signature identifies patients with AMI and sets the framework to potentially identify the earlier stages of an impending cardiac event when used in concert with clinical history and other diagnostics where conventional biomarkers indicative of myonecrosis remain undetected.
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Biomarcadores/sangre , Células Endoteliales/patología , Perfilación de la Expresión Génica , Infarto del Miocardio/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto JovenRESUMEN
BACKGROUND: Cancer research to date has largely focused on somatically acquired genetic aberrations. In contrast, the degree to which germline, or inherited, variation contributes to tumorigenesis remains unclear, possibly due to a lack of accessible germline variant data. Here we called germline variants on 9618 cases from The Cancer Genome Atlas (TCGA) database representing 31 cancer types. RESULTS: We identified batch effects affecting loss of function (LOF) variant calls that can be traced back to differences in the way the sequence data were generated both within and across cancer types. Overall, LOF indel calls were more sensitive to technical artifacts than LOF Single Nucleotide Variant (SNV) calls. In particular, whole genome amplification of DNA prior to sequencing led to an artificially increased burden of LOF indel calls, which confounded association analyses relating germline variants to tumor type despite stringent indel filtering strategies. The samples affected by these technical artifacts include all acute myeloid leukemia and practically all ovarian cancer samples. CONCLUSIONS: We demonstrate how technical artifacts induced by whole genome amplification of DNA can lead to false positive germline-tumor type associations and suggest TCGA whole genome amplified samples be used with caution. This study draws attention to the need to be sensitive to problems associated with a lack of uniformity in data generation in TCGA data.
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Artefactos , Bases de Datos Genéticas , Genómica , Mutación de Línea Germinal , Neoplasias/genética , Genoma Humano/genética , Humanos , Mutación con Pérdida de FunciónRESUMEN
Candidate phyla (CP) are broad phylogenetic clusters of organisms that lack cultured representatives. Included in this fraction is the candidate Parcubacteria superphylum. Specific characteristics that have been ascribed to the Parcubacteria include reduced genome size, limited metabolic potential and exclusive reliance on fermentation for energy acquisition. The study of new environmental niches, such as the marine versus terrestrial subsurface, often expands the understanding of the genetic potential of taxonomic groups. For this reason, we analyzed 12 Parcubacteria single amplified genomes (SAGs) from sediment samples collected within the Challenger Deep of the Mariana Trench, obtained during the Deepsea Challenge (DSC) Expedition. Many of these SAGs are closely related to environmental sequences obtained from deep-sea environments based on 16S rRNA gene similarity and BLAST matches to predicted proteins. DSC SAGs encode features not previously identified in Parcubacteria obtained from other habitats. These include adaptation to oxidative stress, polysaccharide modification and genes associated with respiratory nitrate reduction. The DSC SAGs are also distinguished by relative greater abundance of genes for nucleotide and amino acid biosynthesis, repair of alkylated DNA and the synthesis of mechanosensitive ion channels. These results present an expanded view of the Parcubacteria, among members residing in an ultra-deep hadal environment.
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Bacterias/genética , Genoma Bacteriano/genética , Sedimentos Geológicos/microbiología , Análisis de la Célula Individual/métodos , Aminoácidos/biosíntesis , Bacterias/metabolismo , Reparación del ADN/genética , Ecosistema , Ambiente , Tamaño del Genoma/genética , Nitrato Reductasas/genética , Nitratos/metabolismo , Océanos y Mares , Filogenia , Polisacáridos/metabolismo , ARN Ribosómico 16S/genéticaRESUMEN
Next generation sequencing of the RNA content of single cells or single nuclei (sc/nRNA-seq) has become a powerful approach to understand the cellular complexity and diversity of multicellular organisms and environmental ecosystems. However, the fact that the procedure begins with a relatively small amount of starting material, thereby pushing the limits of the laboratory procedures required, dictates that careful approaches for sample quality control (QC) are essential to reduce the impact of technical noise and sample bias in downstream analysis applications. Here we present a preliminary framework for sample level quality control that is based on the collection of a series of quantitative laboratory and data metrics that are used as features for the construction of QC classification models using random forest machine learning approaches. We've applied this initial framework to a dataset comprised of 2272 single nuclei RNA-seq results and determined that ~79% of samples were of high quality. Removal of the poor quality samples from downstream analysis was found to improve the cell type clustering results. In addition, this approach identified quantitative features related to the proportion of unique or duplicate reads and the proportion of reads remaining after quality trimming as useful features for pass/fail classification. The construction and use of classification models for the identification of poor quality samples provides for an objective and scalable approach to sc/nRNA-seq quality control.
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Secuenciación de Nucleótidos de Alto Rendimiento/estadística & datos numéricos , Neocórtex/citología , Neocórtex/metabolismo , ARN Nuclear/genética , Análisis de Secuencia de ARN/estadística & datos numéricos , Autopsia , Sesgo , Núcleo Celular/genética , Biología Computacional , Bases de Datos de Ácidos Nucleicos , Árboles de Decisión , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Humanos , Aprendizaje Automático , Control de Calidad , Análisis de Secuencia de ARN/normas , Análisis de la Célula Individual , Programas InformáticosRESUMEN
BACKGROUND: A fundamental characteristic of multicellular organisms is the specialization of functional cell types through the process of differentiation. These specialized cell types not only characterize the normal functioning of different organs and tissues, they can also be used as cellular biomarkers of a variety of different disease states and therapeutic/vaccine responses. In order to serve as a reference for cell type representation, the Cell Ontology has been developed to provide a standard nomenclature of defined cell types for comparative analysis and biomarker discovery. Historically, these cell types have been defined based on unique cellular shapes and structures, anatomic locations, and marker protein expression. However, we are now experiencing a revolution in cellular characterization resulting from the application of new high-throughput, high-content cytometry and sequencing technologies. The resulting explosion in the number of distinct cell types being identified is challenging the current paradigm for cell type definition in the Cell Ontology. RESULTS: In this paper, we provide examples of state-of-the-art cellular biomarker characterization using high-content cytometry and single cell RNA sequencing, and present strategies for standardized cell type representations based on the data outputs from these cutting-edge technologies, including "context annotations" in the form of standardized experiment metadata about the specimen source analyzed and marker genes that serve as the most useful features in machine learning-based cell type classification models. We also propose a statistical strategy for comparing new experiment data to these standardized cell type representations. CONCLUSION: The advent of high-throughput/high-content single cell technologies is leading to an explosion in the number of distinct cell types being identified. It will be critical for the bioinformatics community to develop and adopt data standard conventions that will be compatible with these new technologies and support the data representation needs of the research community. The proposals enumerated here will serve as a useful starting point to address these challenges.
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Ontologías Biológicas , Biomarcadores/metabolismo , Células/clasificación , Células/metabolismo , Biología Computacional/métodos , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , HumanosRESUMEN
The healthy human brain is a mosaic of varied genomes. Long interspersed element-1 (LINE-1 or L1) retrotransposition is known to create mosaicism by inserting L1 sequences into new locations of somatic cell genomes. Using a machine learning-based, single-cell sequencing approach, we discovered that somatic L1-associated variants (SLAVs) are composed of two classes: L1 retrotransposition insertions and retrotransposition-independent L1-associated variants. We demonstrate that a subset of SLAVs comprises somatic deletions generated by L1 endonuclease cutting activity. Retrotransposition-independent rearrangements in inherited L1s resulted in the deletion of proximal genomic regions. These rearrangements were resolved by microhomology-mediated repair, which suggests that L1-associated genomic regions are hotspots for somatic copy number variants in the brain and therefore a heritable genetic contributor to somatic mosaicism. We demonstrate that SLAVs are present in crucial neural genes, such as DLG2 (also called PSD93), and affect 44-63% of cells of the cells in the healthy brain.
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Encéfalo/metabolismo , Elementos de Nucleótido Esparcido Largo/genética , Neuronas/metabolismo , Células Cultivadas , Dosificación de Gen , Estudio de Asociación del Genoma Completo/métodos , Genómica/métodos , Humanos , Eliminación de SecuenciaRESUMEN
Single-cell sequencing methods have emerged as powerful tools for identification of heterogeneous cell types within defined brain regions. Application of single-cell techniques to study the transcriptome of activated neurons can offer insight into molecular dynamics associated with differential neuronal responses to a given experience. Through evaluation of common whole-cell and single-nuclei RNA-sequencing (snRNA-seq) methods, here we show that snRNA-seq faithfully recapitulates transcriptional patterns associated with experience-driven induction of activity, including immediate early genes (IEGs) such as Fos, Arc and Egr1. SnRNA-seq of mouse dentate granule cells reveals large-scale changes in the activated neuronal transcriptome after brief novel environment exposure, including induction of MAPK pathway genes. In addition, we observe a continuum of activation states, revealing a pseudotemporal pattern of activation from gene expression alone. In summary, snRNA-seq of activated neurons enables the examination of gene expression beyond IEGs, allowing for novel insights into neuronal activation patterns in vivo.
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Neuronas/metabolismo , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Transcriptoma , Animales , Núcleo Celular/genética , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Giro Dentado/citología , Giro Dentado/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Ontología de Genes , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Inmunohistoquímica , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
A protocol is described for sequencing the transcriptome of a cell nucleus. Nuclei are isolated from specimens and sorted by FACS, cDNA libraries are constructed and RNA-seq is performed, followed by data analysis. Some steps follow published methods (Smart-seq2 for cDNA synthesis and Nextera XT barcoded library preparation) and are not described in detail here. Previous single-cell approaches for RNA-seq from tissues include cell dissociation using protease treatment at 30 °C, which is known to alter the transcriptome. We isolate nuclei at 4 °C from tissue homogenates, which cause minimal damage. Nuclear transcriptomes can be obtained from postmortem human brain tissue stored at -80 °C, making brain archives accessible for RNA-seq from individual neurons. The method also allows investigation of biological features unique to nuclei, such as enrichment of certain transcripts and precursors of some noncoding RNAs. By following this procedure, it takes about 4 d to construct cDNA libraries that are ready for sequencing.
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Encéfalo/citología , Nucléolo Celular/genética , Neuronas/metabolismo , ARN/genética , Análisis de Secuencia de ARN/métodos , Transcriptoma , Autopsia/métodos , Fraccionamiento Celular/métodos , ADN Complementario/genética , Perfilación de la Expresión Génica/métodos , Biblioteca de Genes , Humanos , Análisis de la Célula Individual/métodos , Conservación de TejidoRESUMEN
Hadal ecosystems are found at a depth of 6,000 m below sea level and below, occupying less than 1% of the total area of the ocean. The microbial communities and metabolic potential in these ecosystems are largely uncharacterized. Here, we present four single amplified genomes (SAGs) obtained from 8,219 m below the sea surface within the hadal ecosystem of the Puerto Rico Trench (PRT). These SAGs are derived from members of deep-sea clades, including the Thaumarchaeota and SAR11 clade, and two are related to previously isolated piezophilic (high-pressure-adapted) microorganisms. In order to identify genes that might play a role in adaptation to deep-sea environments, comparative analyses were performed with genomes from closely related shallow-water microbes. The archaeal SAG possesses genes associated with mixotrophy, including lipoylation and the glycine cleavage pathway. The SAR11 SAG encodes glycolytic enzymes previously reported to be missing from this abundant and cosmopolitan group. The other SAGs, which are related to piezophilic isolates, possess genes that may supplement energy demands through the oxidation of hydrogen or the reduction of nitrous oxide. We found evidence for potential trench-specific gene distributions, as several SAG genes were observed only in a PRT metagenome and not in shallower deep-sea metagenomes. These results illustrate new ecotype features that might perform important roles in the adaptation of microorganisms to life in hadal environments.
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Archaea/clasificación , Archaea/genética , Genoma Arqueal/genética , Metagenoma/genética , Agua de Mar/microbiología , Aclimatación , Archaea/aislamiento & purificación , Secuencia de Bases , ADN de Archaea/genética , Ecosistema , Metabolismo Energético/fisiología , Ácidos Grasos/metabolismo , Lípidos/biosíntesis , Datos de Secuencia Molecular , Océanos y Mares , Puerto Rico , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Azufre/metabolismo , Microbiología del AguaRESUMEN
Dioxygenases of the TET (Ten-Eleven Translocation) family produce oxidized methylcytosines, intermediates in DNA demethylation, as well as new epigenetic marks. Here we show data suggesting that TET proteins maintain the consistency of gene transcription. Embryos lacking Tet1 and Tet3 (Tet1/3 DKO) displayed a strong loss of 5-hydroxymethylcytosine (5hmC) and a concurrent increase in 5-methylcytosine (5mC) at the eight-cell stage. Single cells from eight-cell embryos and individual embryonic day 3.5 blastocysts showed unexpectedly variable gene expression compared with controls, and this variability correlated in blastocysts with variably increased 5mC/5hmC in gene bodies and repetitive elements. Despite the variability, genes encoding regulators of cholesterol biosynthesis were reproducibly down-regulated in Tet1/3 DKO blastocysts, resulting in a characteristic phenotype of holoprosencephaly in the few embryos that survived to later stages. Thus, TET enzymes and DNA cytosine modifications could directly or indirectly modulate transcriptional noise, resulting in the selective susceptibility of certain intracellular pathways to regulation by TET proteins.