RESUMO
Hypoxia-inducible transcription factors (HIFs) directly dictate the expression of multiple RNA species including novel and as yet uncharacterized long noncoding transcripts with unknown function. We used pan-genomic HIF-binding and transcriptomic data to identify a novel long noncoding RNA Noncoding Intergenic Co-Induced transcript (NICI) on chromosome 12p13.31 which is regulated by hypoxia via HIF-1 promoter-binding in multiple cell types. CRISPR/Cas9-mediated deletion of the hypoxia-response element revealed co-regulation of NICI and the neighboring protein-coding gene, solute carrier family 2 member 3 (SLC2A3) which encodes the high-affinity glucose transporter 3 (GLUT3). Knockdown or knockout of NICI attenuated hypoxic induction of SLC2A3, indicating a direct regulatory role of NICI in SLC2A3 expression, which was further evidenced by CRISPR/Cas9-VPR-mediated activation of NICI expression. We also demonstrate that regulation of SLC2A3 is mediated through transcriptional activation rather than posttranscriptional mechanisms because knockout of NICI leads to reduced recruitment of RNA polymerase 2 to the SLC2A3 promoter. Consistent with this we observe NICI-dependent regulation of glucose consumption and cell proliferation. Furthermore, NICI expression is regulated by the von Hippel-Lindau (VHL) tumor suppressor and is highly expressed in clear cell renal cell carcinoma (ccRCC), where SLC2A3 expression is associated with patient prognosis, implying an important role for the HIF/NICI/SLC2A3 axis in this malignancy.
Assuntos
Carcinoma de Células Renais/genética , Transportador de Glucose Tipo 3/genética , RNA Longo não Codificante/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Sistemas CRISPR-Cas/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Inativação de Genes , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Regiões Promotoras Genéticas/genética , RNA Polimerase II/genética , Ativação Transcricional/genética , Hipóxia Tumoral/genéticaRESUMO
The mRNA cap is a structure added to RNA pol II transcripts in eukaryotes, which recruits factors involved in RNA processing, nuclear export and translation initiation. RNA guanine-7 methyltransferase (RNMT)-RNA-activating miniprotein (RAM), the mRNA cap methyltransferase complex, completes the basic functional mRNA cap structure, cap 0, by methylating the cap guanosine. Here, we report that RNMT-RAM co-ordinates mRNA processing with ribosome production. Suppression of RNMT-RAM reduces synthesis of the 45S ribosomal RNA (rRNA) precursor. RNMT-RAM is required for c-Myc expression, a major regulator of RNA pol I, which synthesises 45S rRNA. Constitutive expression of c-Myc restores rRNA synthesis when RNMT-RAM is suppressed, indicating that RNMT-RAM controls rRNA production predominantly by controlling c-Myc expression. We report that RNMT-RAM is recruited to the ribosomal DNA locus, which may contribute to rRNA synthesis in certain contexts.
Assuntos
Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Capuzes de RNA/química , RNA Mensageiro/metabolismo , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Núcleo Celular , Cromatina/química , Cromatina/metabolismo , Células HeLa , Humanos , Metilação , Metiltransferases/genética , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-myc/genética , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Ribossômico/genética , Proteínas de Ligação a RNA/genética , Ribossomos/química , Ribossomos/metabolismo , Transcrição GênicaRESUMO
Neuronal responses during behavior are diverse, ranging from highly reliable 'classical' responses to irregular 'non-classically responsive' firing. While a continuum of response properties is observed across neural systems, little is known about the synaptic origins and contributions of diverse responses to network function, perception, and behavior. To capture the heterogeneous responses measured from auditory cortex of rodents performing a frequency recognition task, we use a novel task-performing spiking recurrent neural network incorporating spike-timing-dependent plasticity. Reliable and irregular units contribute differentially to task performance via output and recurrent connections, respectively. Excitatory plasticity shifts the response distribution while inhibition constrains its diversity. Together both improve task performance with full network engagement. The same local patterns of synaptic inputs predict spiking response properties of network units and auditory cortical neurons from in vivo whole-cell recordings during behavior. Thus, diverse neural responses contribute to network function and emerge from synaptic plasticity rules.
Assuntos
Potenciais de Ação , Córtex Auditivo , Plasticidade Neuronal , Neurônios , Sinapses , Animais , Plasticidade Neuronal/fisiologia , Córtex Auditivo/fisiologia , Córtex Auditivo/citologia , Neurônios/fisiologia , Potenciais de Ação/fisiologia , Sinapses/fisiologia , Ratos , Rede Nervosa/fisiologia , Modelos Neurológicos , Análise e Desempenho de TarefasRESUMO
Defining the initial events in oncogenesis and the cellular responses they entrain, even in advance of morphologic abnormality, is a fundamental challenge in understanding cancer initiation. As a paradigm to address this, we longitudinally studied the changes induced by loss of the tumor suppressor gene von Hippel Lindau (VHL), which ultimately drives clear cell renal cell carcinoma. Vhl inactivation was directly coupled to expression of a tdTomato reporter within a single allele, allowing accurate visualization of affected cells in their native context and retrieval from the kidney for single-cell RNA sequencing. This strategy uncovered cell type-specific responses to Vhl inactivation, defined a proximal tubular cell class with oncogenic potential, and revealed longer term adaptive changes in the renal epithelium and the interstitium. Oncogenic cell tagging also revealed markedly heterogeneous cellular effects including time-limited proliferation and elimination of specific cell types. Overall, this study reports an experimental strategy for understanding oncogenic processes in which cells bearing genetic alterations can be generated in their native context, marked, and analyzed over time. The observed effects of loss of Vhl in kidney cells provide insights into VHL tumor suppressor action and development of renal cell carcinoma. SIGNIFICANCE: Single-cell analysis of heterogeneous and dynamic responses to Vhl inactivation in the kidney suggests that early events shape the cell type specificity of oncogenesis, providing a focus for mechanistic understanding and therapeutic targeting.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Análise de Célula Única , Proteína Supressora de Tumor Von Hippel-Lindau , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Neoplasias Renais/genética , Neoplasias Renais/patologia , Neoplasias Renais/metabolismo , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/metabolismo , Análise de Célula Única/métodos , Animais , Camundongos , Transcriptoma , Humanos , Rim/patologia , Rim/metabolismo , Carcinogênese/genética , Proliferação de Células/genéticaRESUMO
The ability to flexibly respond to sensory cues in dynamic environments is essential to adaptive auditory-guided behaviors. Cortical spiking responses during behavior are highly diverse, ranging from reliable trial-averaged responses to seemingly random firing patterns. While the reliable responses of 'classically responsive' cells have been extensively studied for decades, the contribution of irregular spiking 'non-classically responsive' cells to behavior has remained underexplored despite their prevalence. Here, we show that flexible auditory behavior results from interactions between local auditory cortical circuits comprised of heterogeneous responses and inputs from secondary motor cortex. Strikingly, non-classically responsive neurons in auditory cortex were preferentially recruited during learning, specifically during rapid learning phases when the greatest gains in behavioral performance occur. Population-level decoding revealed that during rapid learning mixed ensembles comprised of both classically and non-classically responsive cells encode significantly more task information than homogenous ensembles of either type and emerge as a functional unit critical for learning. Optogenetically silencing inputs from secondary motor cortex selectively modulated non-classically responsive cells in the auditory cortex and impaired reversal learning by preventing the remapping of a previously learned stimulus-reward association. Top-down inputs orchestrated highly correlated non-classically responsive ensembles in sensory cortex providing a unique task-relevant manifold for learning. Thus, non-classically responsive cells in sensory cortex are preferentially recruited by top-down inputs to enable neural and behavioral flexibility.
RESUMO
Activation of cellular hypoxia pathways, orchestrated by HIF (hypoxia-inducible factor) transcription factors, is a common feature of multiple tumor types, resulting from microenvironment factors and oncogenic mutation. Although they help drive many of the "hallmarks" of cancer and are associated with poor outcome and resistance to therapy, the transcriptional targets of HIF vary considerably depending on the cell type. By integrating 72 genome-wide assays of HIF binding and transcriptional regulation from multiple cancer types, we define a consensus set of 48 HIF target genes that is highly conserved across cancer types and cell lineages. These genes provide an effective marker of HIF activation in bulk and single-cell transcriptomic analyses across a wide range of cancer types and in malignant and stromal cell types. This allows the tissue-orchestrated responses to the hypoxic tumor microenvironment and to oncogenic HIF activation to be deconvoluted at the tumor and single-cell level.
Assuntos
Neoplasias , Humanos , Neoplasias/genética , Fatores de Transcrição/metabolismo , Microambiente Tumoral/genética , Hipóxia Celular/genética , Hipóxia/metabolismoRESUMO
Hypoxia-inducible transcription factors (HIFs) are fundamental to cellular adaptation to low oxygen levels, but it is unclear how they interact with chromatin and activate their target genes. Here, we use genome-wide mutagenesis to identify genes involved in HIF transcriptional activity, and define a requirement for the histone H3 lysine 4 (H3K4) methyltransferase SET1B. SET1B loss leads to a selective reduction in transcriptional activation of HIF target genes, resulting in impaired cell growth, angiogenesis and tumor establishment in SET1B-deficient xenografts. Mechanistically, we show that SET1B accumulates on chromatin in hypoxia, and is recruited to HIF target genes by the HIF complex. The selective induction of H3K4 trimethylation at HIF target loci is both HIF- and SET1B-dependent and, when impaired, correlates with decreased promoter acetylation and gene expression. Together, these findings show SET1B as a determinant of site-specific histone methylation and provide insight into how HIF target genes are differentially regulated.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Hipóxia/genética , Acetilação , Animais , Humanos , Hipóxia/metabolismo , Metilação , Camundongos , Camundongos Knockout , Modelos Animais , Regiões Promotoras Genéticas , Ligação ProteicaRESUMO
Basic mechanisms in gene expression are currently being investigated as targets in cancer therapeutics. One such fundamental process is the addition of the cap to pre-mRNA, which recruits mediators of mRNA processing and translation initiation. Maturation of the cap involves mRNA cap guanosine N-7 methylation, catalysed by RNMT (RNA guanine-7 methyltransferase). In a panel of breast cancer cell lines, we investigated whether all are equivalently dependent on RNMT for proliferation. When cellular RNMT activity was experimentally reduced by 50%, the proliferation rate of non-transformed mammary epithelial cells was unchanged, whereas a subset of breast cancer cell lines exhibited reduced proliferation and increased apoptosis. Most of the cell lines which exhibited enhanced dependency on RNMT harboured oncogenic mutations in PIK3CA, which encodes the p110α subunit of PI3Kα. Conversely, all cell lines insensitive to RNMT depletion expressed wild-type PIK3CA. Expression of oncogenic PIK3CA mutants, which increase PI3K p110α activity, was sufficient to increase dependency on RNMT. Conversely, inhibition of PI3Kα reversed dependency on RNMT, suggesting that PI3Kα signalling is required. Collectively, these findings provide evidence to support RNMT as a therapeutic target in breast cancer and suggest that therapies targeting RNMT would be most valuable in a PIK3CA mutant background.
Assuntos
Neoplasias da Mama/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Metiltransferases/genética , Mutação , Capuzes de RNA , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Humanos , Células MCF-7 , Metiltransferases/metabolismo , RNA Mensageiro/genética , Transdução de Sinais/genética , Transcrição GênicaRESUMO
Emerging evidence suggests that dysregulation of oncogenic pathways requires precise tuning in order for cancer to develop. To test this, we examined the overlap between cis-acting elements of the hypoxia-inducible factor (HIF) pathway and cancer-susceptibility polymorphisms as defined in genome-wide association studies (GWAS). In renal cancer, where HIF is constitutively and un-physiologically activated by mutation of the von Hippel-Lindau tumour suppressor, we observed marked excess overlap, which extended to potential susceptibility polymorphisms that are below the conventional threshold applied in GWAS. In contrast, in other cancers where HIF is upregulated by different mechanisms, including micro-environmental hypoxia, we observed no excess in overlap. Our findings support a 'pathway tuning' model of cancer, whereby precise modulation of multiple outputs of specific, activated pathways is important in oncogenesis. This implies that selective pressures to modulate such pathways operate during cancer development and should focus attempts to identify their nature and consequences.
Assuntos
Carcinogênese/genética , Carcinoma de Células Renais/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Transdução de Sinais/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Fator 1 Induzível por Hipóxia/genética , Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Renais/patologia , Mutação , Polimorfismo de Nucleotídeo Único , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
mRNA cap addition occurs early during RNA Pol II-dependent transcription, facilitating pre-mRNA processing and translation. We report that the mammalian mRNA cap methyltransferase, RNMT-RAM, promotes RNA Pol II transcription independent of mRNA capping and translation. In cells, sublethal suppression of RNMT-RAM reduces RNA Pol II occupancy, net mRNA synthesis, and pre-mRNA levels. Conversely, expression of RNMT-RAM increases transcription independent of cap methyltransferase activity. In isolated nuclei, recombinant RNMT-RAM stimulates transcriptional output; this requires the RAM RNA binding domain. RNMT-RAM interacts with nascent transcripts along their entire length and with transcription-associated factors including the RNA Pol II subunits SPT4, SPT6, and PAFc. Suppression of RNMT-RAM inhibits transcriptional markers including histone H2BK120 ubiquitination, H3K4 and H3K36 methylation, RNA Pol II CTD S5 and S2 phosphorylation, and PAFc recruitment. These findings suggest that multiple interactions among RNMT-RAM, RNA Pol II factors, and RNA along the transcription unit stimulate transcription.
Assuntos
Metiltransferases/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica/fisiologia , Células HEK293 , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Metiltransferases/genética , RNA Polimerase II/genética , Proteínas de Ligação a RNA/genética , Ubiquitinação/fisiologiaRESUMO
c-Myc is a potent driver of many human cancers. Since strategies for directly targeting c-Myc protein have had limited success, upstream regulators and downstream effectors of c-Myc are being investigated as alternatives for therapeutic intervention. c-Myc regulates transcription and formation of the mRNA cap, which is important for transcript maturation and translation. However, the direct mechanism by which c-Myc upregulates mRNA capping is unclear. mRNA cap formation initiates with the linkage of inverted guanosine via a triphosphate bridge to the first transcribed nucleotide, catalysed by mRNA capping enzyme (CE/RNGTT). Here we report that c-Myc increases the recruitment of catalytically active CE to RNA polymerase II and to its target genes. c-Myc-induced target gene expression, cell proliferation and cell transformation is highly dependent on CE, but only when c-Myc is deregulated. Cells retaining normal control of c-Myc expression are insensitive to repression of CE. c-Myc expression is also dependent on CE. Therefore, inhibiting CE provides an attractive route for selective therapeutic targeting of cancer cells which have acquired deregulated c-Myc.