RESUMO
Long non-coding RNA (lncRNA) function is mediated by the process of transcription or through transcript-dependent associations with proteins or nucleic acids to control gene regulatory networks. Many lncRNAs are transcribed in the ventricular-subventricular zone (V-SVZ), a postnatal neural stem cell niche. lncRNAs in the V-SVZ are implicated in neurodevelopmental disorders, cancer, and brain disease, but their functions are poorly understood. V-SVZ neurogenesis capacity declines with age due to stem cell depletion and resistance to neural stem cell activation. Here we analyzed V-SVZ transcriptomics by pooling current single-cell RNA-seq data. They showed consistent lncRNA expression during stem cell activation, lineage progression, and aging. In conjunction with epigenetic and genetic data, we predicted V-SVZ lncRNAs that regulate stem cell activation and differentiation. Some of the lncRNAs validate known epigenetic mechanisms, but most remain uninvestigated. Our analysis points to several lncRNAs that likely participate in key aspects of V-SVZ stem cell activation and neurogenesis in health and disease.
Assuntos
Células-Tronco Neurais , RNA Longo não Codificante , Ventrículos Laterais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Transcriptoma , Células-Tronco Neurais/metabolismo , Diferenciação Celular/genética , Neurogênese/genéticaRESUMO
Central nervous system-expressed long non-coding RNAs (lncRNAs) are often located in the genome close to protein coding genes involved in transcriptional control. Such lncRNA-protein coding gene pairs are frequently temporally and spatially co-expressed in the nervous system and are predicted to act together to regulate neuronal development and function. Although some of these lncRNAs also bind and modulate the activity of the encoded transcription factors, the regulatory mechanisms controlling co-expression of neighbouring lncRNA-protein coding genes remain unclear. Here, we used high resolution NG Capture-C to map the cis-regulatory interaction landscape of the key neuro-developmental Paupar-Pax6 lncRNA-mRNA locus. The results define chromatin architecture changes associated with high Paupar-Pax6 expression in neurons and identify both promoter selective as well as shared cis-regulatory-promoter interactions involved in regulating Paupar-Pax6 co-expression. We discovered that the TCF7L2 transcription factor, a regulator of chromatin architecture and major effector of the Wnt signalling pathway, binds to a subset of these candidate cis-regulatory elements to coordinate Paupar and Pax6 co-expression. We describe distinct roles for Paupar in Pax6 expression control and show that the Paupar DNA locus contains a TCF7L2 bound transcriptional silencer whilst the Paupar transcript can act as an activator of Pax6. Our work provides important insights into the chromatin interactions, signalling pathways and transcription factors controlling co-expression of adjacent lncRNAs and protein coding genes in the brain.
Assuntos
RNA Longo não Codificante , Cromatina/genética , Neurônios/metabolismo , Fator de Transcrição PAX6/genética , Fator de Transcrição PAX6/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fatores de Transcrição/genéticaRESUMO
In many tumors, cells transition reversibly between slow-proliferating tumor-initiating cells (TIC) and their differentiated, faster-growing progeny. Yet, how transcriptional regulation of cell-cycle and self-renewal genes is orchestrated during these conversions remains unclear. In this study, we show that as breast TIC form, a decrease in cell-cycle gene expression and increase in self-renewal gene expression are coregulated by SOX2 and EZH2, which colocalize at CpG islands. This pattern was negatively controlled by a novel long noncoding RNA (lncRNA) that we named Stem Cell Inhibitory RNA Transcript (SCIRT), which was markedly upregulated in tumorspheres but colocalized with and counteracted EZH2 and SOX2 during cell-cycle and self-renewal regulation to restrain tumorigenesis. SCIRT specifically interacted with EZH2 to increase EZH2 affinity to FOXM1 without binding the latter. In this manner, SCIRT induced transcription at cell-cycle gene promoters by recruiting FOXM1 through EZH2 to antagonize EZH2-mediated effects at target genes. Conversely, on stemness genes, FOXM1 was absent and SCIRT antagonized EZH2 and SOX2 activity, balancing toward repression. These data suggest that the interaction of an lncRNA with EZH2 can alter the affinity of EZH2 for its protein-binding partners to regulate cancer cell state transitions. SIGNIFICANCE: These findings show that a novel lncRNA SCIRT counteracts breast tumorigenesis by opposing transcriptional networks associated with cell cycle and self-renewal.See related commentary by Pardini and Dragomir, p. 535.
Assuntos
Neoplasias da Mama , RNA Longo não Codificante , Neoplasias da Mama/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , RNA Longo não Codificante/genéticaRESUMO
The MITF and SOX10 transcription factors regulate the expression of genes important for melanoma proliferation, invasion and metastasis. Despite growing evidence of the contribution of long noncoding RNAs (lncRNAs) in cancer, including melanoma, their functions within MITF-SOX10 transcriptional programmes remain poorly investigated. Here we identify 245 candidate melanoma associated lncRNAs whose loci are co-occupied by MITF-SOX10 and that are enriched at active enhancer-like regions. Our work suggests that one of these, Disrupted In Renal Carcinoma 3 (DIRC3), may be a clinically important MITF-SOX10 regulated tumour suppressor. DIRC3 depletion in human melanoma cells leads to increased anchorage-independent growth, a hallmark of malignant transformation, whilst melanoma patients classified by low DIRC3 expression have decreased survival. DIRC3 is a nuclear lncRNA that activates expression of its neighbouring IGFBP5 tumour suppressor through modulating chromatin structure and suppressing SOX10 binding to putative regulatory elements within the DIRC3 locus. In turn, DIRC3 dependent regulation of IGFBP5 impacts the expression of genes involved in cancer associated processes and is needed for DIRC3 control of anchorage-independent growth. Our work indicates that lncRNA components of MITF-SOX10 networks are an important new class of melanoma regulators and candidate therapeutic targets that can act not only as downstream mediators of MITF-SOX10 function but as feedback regulators of MITF-SOX10 activity.
Assuntos
Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Melanoma/genética , Fator de Transcrição Associado à Microftalmia/genética , RNA Longo não Codificante/genética , Fatores de Transcrição SOXE/genética , Linhagem Celular Tumoral , Núcleo Celular/genética , Proliferação de Células , Regulação para Baixo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Prognóstico , Análise de Sequência de RNA , Análise de SobrevidaRESUMO
Many long non-coding RNAs (lncRNAs) are expressed during central nervous system (CNS) development, yet their in vivo roles and mechanisms of action remain poorly understood. Paupar, a CNS-expressed lncRNA, controls neuroblastoma cell growth by binding and modulating the activity of transcriptional regulatory elements in a genome-wide manner. We show here that the Paupar lncRNA directly binds KAP1, an essential epigenetic regulatory protein, and thereby regulates the expression of shared target genes important for proliferation and neuronal differentiation. Paupar promotes KAP1 chromatin occupancy and H3K9me3 deposition at a subset of distal targets, through the formation of a ribonucleoprotein complex containing Paupar, KAP1 and the PAX6 transcription factor. Paupar-KAP1 genome-wide co-occupancy reveals a fourfold enrichment of overlap between Paupar and KAP1 bound sequences, the majority of which also appear to associate with PAX6. Furthermore, both Paupar and Kap1 loss-of-function in vivo disrupt olfactory bulb neurogenesis. These observations provide important conceptual insights into the trans-acting modes of lncRNA-mediated epigenetic regulation and the mechanisms of KAP1 genomic recruitment, and identify Paupar and Kap1 as regulators of neurogenesis in vivo.
Assuntos
Cromatina/genética , Células-Tronco Neurais/citologia , Neuroblastoma/patologia , Neurogênese , Bulbo Olfatório/citologia , RNA Longo não Codificante/metabolismo , Proteína 28 com Motivo Tripartido/metabolismo , Animais , Animais Recém-Nascidos , Ciclo Celular , Proliferação de Células , Células Cultivadas , Epigênese Genética , Genômica , Camundongos , Células-Tronco Neurais/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Bulbo Olfatório/metabolismo , Fator de Transcrição PAX6/genética , Fator de Transcrição PAX6/metabolismo , RNA Longo não Codificante/genética , Elementos Reguladores de Transcrição , Proteína 28 com Motivo Tripartido/genéticaRESUMO
Capture Hybridization Analysis of RNA Targets (CHART) has recently been developed to map the genome-wide binding profile of chromatin-associated RNAs. This protocol uses a small number of 22-28 nucleotide biotinylated antisense oligonucleotides, complementary to regions of the target RNA that are accessible for hybridization, to purify RNAs from a cross-linked chromatin extract. RNA-chromatin complexes are next immobilized on beads, washed, and specifically eluted using RNase H. Associated genomic DNA is then sequenced using high-throughput sequencing technologies and mapped to the genome to identify RNA-chromatin associations on a large scale. CHART-based strategies can be applied to determine the nature and extent of long noncoding RNA (long ncRNA) association with chromatin genome-wide and identify direct long ncRNA transcriptional targets.
Assuntos
Cromatina/metabolismo , Hibridização de Ácido Nucleico/métodos , RNA Longo não Codificante/genética , Animais , Sítios de Ligação , Cromatina/química , Cromatina/genética , Reagentes de Ligações Cruzadas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Oligonucleotídeos Antissenso/metabolismo , RNA Longo não Codificante/metabolismo , Ribonuclease H/metabolismo , Análise de Sequência de DNARESUMO
Malignant melanoma is a highly aggressive form of skin cancer, the incidence of which is rising rapidly. Although MAPK-targeting therapies and immune checkpoint blockade are emerging as attractive therapeutic approaches, their utility is limited to only a subset of patients who often acquire resistance. A better understanding of the aetiologies and genetic underpinnings of melanoma is therefore critical for the development of adjuvant or alternative therapeutic strategies aimed at increasing the proportion of responders and improving treatment efficacy. A key step in identifying novel therapeutic targets may be the shift in focus from the protein-coding components to the non-coding portion of the genome. The latter, representing about 98% of the genome, serves as a template for the transcription of many thousands of long non-coding RNAs (lncRNAs). Intriguingly, lncRNA loci are frequently mutated or altered in a variety of cancers, including melanoma, and there is growing evidence that lncRNAs can function as cancer-causing oncogenes or tumour suppressors. In this review, we summarize recent data highlighting the importance of lncRNAs in the biology of melanoma and their potential utility as biomarkers and therapeutic targets.
Assuntos
Biomarcadores Tumorais/análise , Regulação Neoplásica da Expressão Gênica , Melanoma/genética , RNA Longo não Codificante/genética , Neoplasias Cutâneas/genética , Animais , Humanos , Melanoma/diagnóstico , Melanoma/terapia , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/terapia , Melanoma Maligno CutâneoRESUMO
The process of transcription is highly stochastic leading to cell-to-cell variations and noise in gene expression levels. However, key essential genes have to be precisely expressed at the correct amount and time to ensure proper cellular development and function. Studies in yeast and bacterial systems have shown that gene expression noise decreases as mean expression levels increase, a relationship that is controlled by promoter DNA sequence. However, the function of distal cis-regulatory modules (CRMs), an evolutionary novelty of metazoans, in controlling transcriptional robustness and variability is poorly understood. In this study, we used live cell imaging of transfected reporters combined with a mathematical modelling and statistical inference scheme to quantify the function of conserved Msx1 CRMs and promoters in modulating single-cell real-time transcription rates in C2C12 mouse myoblasts. The results show that the mean expression-noise relationship is solely promoter controlled for this key pluripotency regulator. In addition, we demonstrate that CRMs modulate single-cell basal promoter rate distributions in a graded manner across a population of cells. This extends the rheostatic model of CRM action to provide a more detailed understanding of CRM function at single-cell resolution. We also identify a novel CRM transcriptional filter function that acts to reduce intracellular variability in transcription rates and show that this can be phylogenetically separable from rate modulating CRM activities. These results are important for understanding how the expression of key vertebrate developmental transcription factors is precisely controlled both within and between individual cells.
Assuntos
Regulação da Expressão Gênica , Fator de Transcrição MSX1/genética , Regiões Promotoras Genéticas , Elementos Reguladores de Transcrição , Transcrição Gênica , Animais , Sequência de Bases , Linhagem Celular , Sequência Conservada , Fator de Transcrição MSX1/biossíntese , Camundongos , Modelos Genéticos , Análise de Célula ÚnicaRESUMO
Many intergenic long noncoding RNA (lncRNA) loci regulate the expression of adjacent protein coding genes. Less clear is whether intergenic lncRNAs commonly regulate transcription by modulating chromatin at genomically distant loci. Here, we report both genomically local and distal RNA-dependent roles of Dali, a conserved central nervous system expressed intergenic lncRNA. Dali is transcribed downstream of the Pou3f3 transcription factor gene and its depletion disrupts the differentiation of neuroblastoma cells. Locally, Dali transcript regulates transcription of the Pou3f3 locus. Distally, it preferentially targets active promoters and regulates expression of neural differentiation genes, in part through physical association with the POU3F3 protein. Dali interacts with the DNMT1 DNA methyltransferase in mouse and human and regulates DNA methylation status of CpG island-associated promoters in trans. These results demonstrate, for the first time, that a single intergenic lncRNA controls the activity and methylation of genomically distal regulatory elements to modulate large-scale transcriptional programmes.
Assuntos
Diferenciação Celular/genética , Epigênese Genética , Neurônios/citologia , Neurônios/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Linhagem da Célula/genética , Cromatina/metabolismo , Sequência Conservada/genética , Metilação de DNA/genética , Técnicas de Silenciamento de Genes , Loci Gênicos , Humanos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Ligação Proteica/genética , RNA Longo não Codificante/genética , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
What causes the tissue-specific pathology of diseases resulting from mutations in housekeeping genes? Specifically, in spinocerebellar ataxia type 7 (SCA7), a neurodegenerative disorder caused by a CAG-repeat expansion in ATXN7 (which encodes an essential component of the mammalian transcription coactivation complex, STAGA), the factors underlying the characteristic progressive cerebellar and retinal degeneration in patients were unknown. We found that STAGA is required for the transcription initiation of miR-124, which in turn mediates the post-transcriptional cross-talk between lnc-SCA7, a conserved long noncoding RNA, and ATXN7 mRNA. In SCA7, mutations in ATXN7 disrupt these regulatory interactions and result in a neuron-specific increase in ATXN7 expression. Strikingly, in mice this increase is most prominent in the SCA7 disease-relevant tissues, namely the retina and cerebellum. Our results illustrate how noncoding RNA-mediated feedback regulation of a ubiquitously expressed housekeeping gene may contribute to specific neurodegeneration.
Assuntos
Cerebelo/metabolismo , MicroRNAs/genética , Proteínas do Tecido Nervoso/genética , RNA Longo não Codificante/genética , Retina/metabolismo , Ataxias Espinocerebelares/genética , Animais , Ataxina-7 , Linhagem Celular Tumoral , Cerebelo/patologia , Retroalimentação Fisiológica , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , MicroRNAs/metabolismo , Mutação , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neurônios/patologia , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retina/patologia , Transdução de Sinais , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/patologia , Iniciação da Transcrição GenéticaRESUMO
Several nuclear localised intergenic long noncoding RNAs (lncRNAs) have been ascribed regulatory roles in transcriptional control and their number is growing rapidly. Initially, these transcripts were shown to function locally, near their sites of synthesis, by regulating the expression of neighbouring genes. More recently, lncRNAs have been demonstrated to interact with chromatin at several thousand different locations across multiple chromosomes and to modulate large-scale gene expression programs. Although the molecular mechanisms involved in targeting lncRNAs to distal binding sites remain poorly understood, the spatial organisation of the genome may have a role in specifying lncRNA function. Recent advances indicate that intergenic lncRNAs may exert more widespread effects on gene regulation than previously anticipated.
Assuntos
Núcleo Celular/genética , Regulação da Expressão Gênica , RNA Longo não Codificante/metabolismo , Transcrição Gênica , Animais , Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , HumanosRESUMO
Although some long noncoding RNAs (lncRNAs) have been shown to regulate gene expression in cis, it remains unclear whether lncRNAs can directly regulate transcription in trans by interacting with chromatin genome-wide independently of their sites of synthesis. Here, we describe the genomically local and more distal functions of Paupar, a vertebrate-conserved and central nervous system-expressed lncRNA transcribed from a locus upstream of the gene encoding the PAX6 transcription factor. Knockdown of Paupar disrupts the normal cell cycle profile of neuroblastoma cells and induces neural differentiation. Paupar acts in a transcript-dependent manner both locally, to regulate Pax6, as well as distally by binding and regulating genes on multiple chromosomes, in part through physical association with PAX6 protein. Paupar binding sites are enriched near promoters and can function as transcriptional regulatory elements whose activity is modulated by Paupar transcript levels. Our findings demonstrate that a lncRNA can function in trans at transcriptional regulatory elements distinct from its site of synthesis to control large-scale transcriptional programmes.
Assuntos
Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Fatores de Transcrição Box Pareados/genética , RNA Longo não Codificante/fisiologia , Proteínas Repressoras/genética , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Cromatina/metabolismo , Sequência Conservada , Proteínas do Olho/biossíntese , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Genes cdc , Estudo de Associação Genômica Ampla , Proteínas de Homeodomínio/biossíntese , Camundongos , Proteínas do Tecido Nervoso/genética , Neuroblastoma/patologia , Neurogênese , Neurônios/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/biossíntese , Ligação Proteica , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/farmacologia , Elementos Reguladores de Transcrição , Proteínas Repressoras/biossíntese , Transcrição Gênica , TransfecçãoRESUMO
MOTIVATION: cis-regulatory DNA sequence elements, such as enhancers and silencers, function to control the spatial and temporal expression of their target genes. Although the overall levels of gene expression in large cell populations seem to be precisely controlled, transcription of individual genes in single cells is extremely variable in real time. It is, therefore, important to understand how these cis-regulatory elements function to dynamically control transcription at single-cell resolution. Recently, statistical methods have been proposed to back calculate the rates involved in mRNA transcription using parameter estimation of a mathematical model of transcription and translation. However, a major complication in these approaches is that some of the parameters, particularly those corresponding to the gene copy number and transcription rate, cannot be distinguished; therefore, these methods cannot be used when the copy number is unknown. RESULTS: Here, we develop a hierarchical Bayesian model to estimate biokinetic parameters from live cell enhancer-promoter reporter measurements performed on a population of single cells. This allows us to investigate transcriptional dynamics when the copy number is variable across the population. We validate our method using synthetic data and then apply it to quantify the function of two known developmental enhancers in real time and in single cells. AVAILABILITY: Supporting information is submitted with the article.
Assuntos
Algoritmos , Dosagem de Genes , Modelos Genéticos , Transcrição Gênica , Animais , Teorema de Bayes , Linhagem Celular , Elementos Facilitadores Genéticos , Fator de Transcrição MSX1/metabolismo , Camundongos , Regiões Promotoras Genéticas , Análise de Célula ÚnicaRESUMO
The expression profile of a gene is controlled by DNA sequences called cis-regulatory modules (CRMs). CRMs can function over large genomic distances and can be located many kilobases away from their target promoters. hes1 is a key developmental gene that is overexpressed in certain cancers and is a primary target of NOTCH signaling. Despite this, analysis of hes1 transcriptional control has been limited solely to its promoter. Here, we identify seven conserved DNA sequence blocks, representing the hes1 promoter and six novel CRMs, within 57 kb upstream of the mouse hes1 gene. We identify 12 binding sites for the RBP-Jκ NOTCH effector and a single M-CAT motif within these regions. We validate RBP-Jκ and TEAD family occupancy in cells in culture and test the response of each of these CRMs to active NOTCH. We show that two regions, CRM5 and CRM7, function as enhancers, and four can repress transcription. A pair of RBP-Jκ motifs arranged in a tail-tail configuration in CRM5 and the M-CAT motif in CRM7 are necessary for enhancer function. Furthermore, these enhancers are occupied by transcriptional co-activators and loop onto the hes1 promoter within the endogenous hes1 locus. This work demonstrates the power of combining computational genomics and experimental methodologies to identify novel CRMs and characterize their function.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica/genética , Proteínas de Homeodomínio/genética , Mioblastos/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Linhagem Celular , Biologia Computacional , Sequência Conservada/genética , Elementos Facilitadores Genéticos/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Camundongos , Mioblastos/citologia , Motivos de Nucleotídeos/genética , Regiões Promotoras Genéticas/genética , Receptores Notch/metabolismo , Transdução de Sinais , Fatores de Transcrição HES-1 , Sítio de Iniciação de TranscriçãoRESUMO
The extraction of fluorescence time course data is a major bottleneck in high-throughput live-cell microscopy. Here we present an extendible framework based on the open-source image analysis software ImageJ, which aims in particular at analyzing the expression of fluorescent reporters through cell divisions. The ability to track individual cell lineages is essential for the analysis of gene regulatory factors involved in the control of cell fate and identity decisions. In our approach, cell nuclei are identified using Hoechst, and a characteristic drop in Hoechst fluorescence helps to detect dividing cells. We first compare the efficiency and accuracy of different segmentation methods and then present a statistical scoring algorithm for cell tracking, which draws on the combination of various features, such as nuclear intensity, area or shape, and importantly, dynamic changes thereof. Principal component analysis is used to determine the most significant features, and a global parameter search is performed to determine the weighting of individual features. Our algorithm has been optimized to cope with large cell movements, and we were able to semi-automatically extract cell trajectories across three cell generations. Based on the MTrackJ plugin for ImageJ, we have developed tools to efficiently validate tracks and manually correct them by connecting broken trajectories and reassigning falsely connected cell positions. A gold standard consisting of two time-series with 15,000 validated positions will be released as a valuable resource for benchmarking. We demonstrate how our method can be applied to analyze fluorescence distributions generated from mouse stem cells transfected with reporter constructs containing transcriptional control elements of the Msx1 gene, a regulator of pluripotency, in mother and daughter cells. Furthermore, we show by tracking zebrafish PAC2 cells expressing FUCCI cell cycle markers, our framework can be easily adapted to different cell types and fluorescent markers.
Assuntos
Linhagem da Célula , Genes Reporter , Ensaios de Triagem em Larga Escala/métodos , Microscopia/métodos , Animais , Biomarcadores/metabolismo , Divisão Celular , Linhagem Celular , Núcleo Celular/metabolismo , Rastreamento de Células , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Imageamento Tridimensional , Camundongos , Fatores de Tempo , Peixe-Zebra/metabolismoRESUMO
Tbx2 is a member of a large family of transcription factors defined by homology to the T-box DNA-binding domain. Tbx2 plays a key role in embryonic development, and in cancer through its capacity to suppress senescence and promote invasiveness. Despite its importance, little is known of how Tbx2 is regulated or how it achieves target gene specificity. Here we show that Tbx2 specifically associates with active hypophosphorylated retinoblastoma protein (Rb1), a known regulator of many transcription factors involved in cell cycle progression and cellular differentiation, but not with the Rb1-related proteins p107 or p130. The interaction with Rb1 maps to a domain immediately carboxy-terminal to the T-box and enhances Tbx2 DNA binding and transcriptional repression. Microarray analysis of melanoma cells expressing inducible dominant-negative Tbx2, comprising the T-box and either an intact or mutated Rb1 interaction domain, shows that Tbx2 regulates the expression of many genes involved in cell cycle control and that a mutation which disrupts the Rb1-Tbx2 interaction also affects Tbx2 target gene selectivity. Taken together, the data show that Rb1 is an important determinant of Tbx2 functional specificity.
Assuntos
Proteína do Retinoblastoma/metabolismo , Proteínas com Domínio T/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Linhagem Celular , DNA/metabolismo , Humanos , Melanoma/metabolismo , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteínas Repressoras/metabolismo , Proteína do Retinoblastoma/química , Proteínas com Domínio T/química , Transcrição GênicaRESUMO
The genomic instructions used to regulate development are encoded within a set of functional DNA elements called cis-regulatory modules (CRMs). These elements determine the precise patterns of temporal and spatial gene expression. Here we summarize recent progress made towards cataloguing and characterizing the complete repertoire of CRMs. We describe CRMs as genomic information processing devices containing clusters of transcription factor binding sites and we position CRMs as nodes within large gene regulatory networks. We define CRM architecture and describe how these genomic elements process the information they encode to their target genes. Furthermore, we present an overview describing high-throughput techniques to identify CRMs genome wide and experimental methodologies to validate their function on a large scale. This review emphasizes the advantages and power of a systems biology approach which integrates computational and experimental technologies to further our understanding of CRM function.
Assuntos
Redes Reguladoras de Genes , Biologia de Sistemas/métodos , Animais , Biotecnologia , Variação Genética , Humanos , Modelos GenéticosRESUMO
The INK4a and ARF genes found at the CDKN2A locus are key effectors of cellular senescence that is believed to act as a powerful anticancer mechanism. Accordingly, mutations in these genes are present in a wide variety of spontaneous human cancers and CDKN2A germ line mutations are found in familial melanoma. The TBX2 gene encoding a key developmental transcription factor is amplified in pancreatic cancer cell lines and preferentially amplified and overexpressed in BRCA1 and BRCA2 mutated breast tumors. Overexpression of Tbx2 and the related factor Tbx3, which is also overexpressed in breast cancer and melanomas, can suppress senescence in defined experimental systems through repression of ARF expression. However, it is not known how Tbx2 mediates its repressive effect nor whether endogenous Tbx2 or Tbx3 perform a similar antisenescence function in transformed cells. This is a particularly important question because the loss of CDKN2A in many human cancers would, in principle, bypass the requirement for Tbx2/3-mediated repression of ARF in suppressing senescence. We show here that Tbx2 is overexpressed in melanoma cell lines and that Tbx2 targets histone deacetylase 1 to the p21Cip1 (CDKN1A) initiator. Strikingly, expression of an inducible dominant-negative Tbx2 (dnTbx2) leads to displacement of histone deacetylase 1, up-regulation of p21(Cip1) expression, and the induction of replicative senescence in CDKN2A-null B16 melanoma cells. In human melanoma cells, expression of dnTbx2 leads to severely reduced growth and induction of senescence-associated heterochromatin foci. The results suggest that the activity of endogenous Tbx2 is critically required to maintain proliferation and suppress senescence in melanomas.