RESUMO
Elongin is an RNA polymerase II (RNAPII)-associated factor that has been shown to stimulate transcriptional elongation in vitro. The Elongin complex is thought to be required for transcriptional induction in response to cellular stimuli and to ubiquitinate RNAPII in response to DNA damage. Yet, the impact of the Elongin complex on transcription in vivo has not been well studied. Here, we performed comprehensive studies of the role of Elongin A, the largest subunit of the Elongin complex, on RNAPII transcription genome-wide. Our results suggest that Elongin A localizes to actively transcribed regions and potential enhancers, and the level of recruitment correlated with transcription levels. We also identified a large group of factors involved in transcription as Elongin A-associated factors. In addition, we found that loss of Elongin A leads to dramatically reduced levels of serine2-phosphorylated, but not total, RNAPII, and cells depleted of Elongin A show stronger promoter RNAPII pausing, suggesting that Elongin A may be involved in the release of paused RNAPII. Our RNA-seq studies suggest that loss of Elongin A did not alter global transcription, and unlike prior in vitro studies, we did not observe a dramatic impact on RNAPII elongation rates in our cell-based nascent RNA-seq experiments upon Elongin A depletion. Taken together, our studies provide the first comprehensive analysis of the role of Elongin A in regulating transcription in vivo. Our studies also revealed that unlike prior in vitro findings, depletion of Elongin A has little impact on global transcription profiles and transcription elongation in vivo.
Assuntos
Cromatina/metabolismo , Elonguina/genética , RNA Polimerase II/genética , RNA Mensageiro/genética , Elongação da Transcrição Genética , Linhagem Celular Tumoral , Cromatina/química , Biologia Computacional/métodos , Elonguina/antagonistas & inibidores , Elonguina/metabolismo , Elementos Facilitadores Genéticos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Fosforilação , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Análise de Sequência de RNA , Serina/metabolismo , Transdução de SinaisRESUMO
We performed a high-throughput whole-genome RNAi screen to identify novel inhibitors of ciliogenesis in normal and basal breast cancer cells. Our screen uncovered a previously undisclosed, extensive network of genes linking integrin signaling and cellular adhesion to the extracellular matrix (ECM) with inhibition of ciliation in both normal and cancer cells. Surprisingly, a cohort of genes encoding ECM proteins was also identified. We characterized several ciliation inhibitory genes and showed that their silencing was accompanied by altered cytoskeletal organization and induction of ciliation, which restricts cell growth and migration in normal and breast cancer cells. Conversely, supplying an integrin ligand, vitronectin, to the ECM rescued the enhanced ciliation observed on silencing this gene. Aberrant ciliation could also be suppressed through hyperactivation of the YAP/TAZ pathway, indicating a potential mechanistic basis for our findings. Our findings suggest an unanticipated reciprocal relationship between ciliation and cellular adhesion to the ECM and provide a resource that could vastly expand our understanding of controls involving "outside-in" and "inside-out" signaling that restrain cilium assembly.
Assuntos
Cílios/genética , Testes Genéticos , Genoma Humano , Organogênese/genética , Transdução de Sinais/genética , Actinas/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Matriz Extracelular/metabolismo , Feminino , Adesões Focais/metabolismo , Inativação Gênica , Estudos de Associação Genética , Humanos , Integrinas/metabolismo , Ligantes , RNA Interferente Pequeno/metabolismo , Supressão GenéticaRESUMO
The primary cilium is an antenna-like organelle assembled on most types of quiescent and differentiated mammalian cells. This immotile structure is essential for interpreting extracellular signals that regulate growth, development and homeostasis. As such, ciliary defects produce a spectrum of human diseases, termed ciliopathies, and deregulation of this important organelle also plays key roles during tumor formation and progression. Recent studies have begun to clarify the key mechanisms that regulate ciliary assembly and disassembly in both normal and tumor cells, highlighting new possibilities for therapeutic intervention. Here, we review these exciting new findings, discussing the molecular factors involved in cilium formation and removal, the intrinsic and extrinsic control of cilium assembly and disassembly, and the relevance of these processes to mammalian cell growth and disease.
Assuntos
Cílios/metabolismo , Ciliopatias/genética , Neoplasias/patologia , Animais , Centríolos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Crescimento e Desenvolvimento/genética , Humanos , Neoplasias/genética , Transdução de SinaisRESUMO
A long-standing mystery in the centrosome field pertains to the origin of asymmetry within the organelle. The removal of daughter centriole-specific/enriched proteins (DCPs) and acquisition of distal appendages on the future mother centriole are two important steps in the generation of asymmetry. We find that DCPs are recruited sequentially, and their removal is abolished in cells lacking Talpid3 or C2CD3. We show that removal of certain DCPs constitutes another level of control for distal appendage (DA) assembly. Remarkably, we also find that Talpid3 forms a distal centriolar multi-functional hub that coordinates the removal of specific DCPs, DA assembly, and recruitment of ciliary vesicles through distinct regions mutated in ciliopathies. Finally, we show that Talpid3, C2CD3, and OFD1 differentially regulate the assembly of sub-distal appendages, the CEP350/FOP/CEP19 module, centriolar satellites, and actin networks. Our work extends the spatial and functional understanding of proteins that control organelle maturation and asymmetry, ciliogenesis, and human disease.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas/metabolismo , Anormalidades Múltiplas/genética , Proteínas de Ciclo Celular/genética , Cerebelo/anormalidades , Ciliopatias/genética , Anormalidades do Olho/genética , Células HEK293 , Humanos , Doenças Renais Císticas/genética , Retina/anormalidadesRESUMO
Pluripotent stem cells (PSC) hold great promise for the treatment of human skeletal muscle diseases. However, it remains challenging to convert PSC to skeletal muscle cells, and the mechanisms by which the master regulatory transcription factor, Pax7, promotes muscle stem (satellite) cell identity are not yet understood. We have taken advantage of PSC-derived skeletal muscle precursor cells (iPax7), wherein the induced expression of Pax7 robustly initiates the muscle program and enables the in vitro generation of precursors that seed the satellite cell compartment upon transplantation. Remarkably, we found that chromatin accessibility in myogenic precursors pre-figures subsequent activation of myogenic differentiation genes. We also found that Pax7 binding is generally restricted to euchromatic regions and excluded from H3K27 tri-methylated regions in muscle cells, suggesting that recruitment of this factor is circumscribed by chromatin state. Further, we show that Pax7 binding induces dramatic, localized remodeling of chromatin characterized by the acquisition of histone marks associated with enhancer activity and induction of chromatin accessibility in both muscle precursors and lineage-committed myoblasts. Conversely, removal of Pax7 leads to rapid reversal of these features on a subset of enhancers. Interestingly, another cluster of Pax7 binding sites is associated with a durably accessible and remodeled chromatin state after removal of Pax7, and persistent enhancer accessibility is associated with subsequent, proximal binding by the muscle regulatory factors, MyoD1 and myogenin. Our studies provide new insights into the epigenetic landscape of skeletal muscle stem cells and precursors and the role of Pax7 in satellite cell specification.
Assuntos
Cromatina/metabolismo , Células Musculares/metabolismo , Desenvolvimento Muscular/fisiologia , Fator de Transcrição PAX7/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Linhagem Celular , Camundongos , Músculo Esquelético/metabolismo , Proteína MyoD/metabolismo , Miogenina/metabolismo , Fator de Transcrição PAX7/genética , Células Satélites de Músculo Esquelético/metabolismoRESUMO
Defective primary ciliogenesis or cilium stability forms the basis of human ciliopathies, including Joubert syndrome (JS), with defective cerebellar vermis development. We performed a high-content genome-wide small interfering RNA (siRNA) screen to identify genes regulating ciliogenesis as candidates for JS. We analyzed results with a supervised-learning approach, using SYSCILIA gold standard, Cildb3.0, a centriole siRNA screen and the GTex project, identifying 591 likely candidates. Intersection of this data with whole exome results from 145 individuals with unexplained JS identified six families with predominantly compound heterozygous mutations in KIAA0586. A c.428del base deletion in 0.1% of the general population was found in trans with a second mutation in an additional set of 9 of 163 unexplained JS patients. KIAA0586 is an orthologue of chick Talpid3, required for ciliogenesis and Sonic hedgehog signaling. Our results uncover a relatively high frequency cause for JS and contribute a list of candidates for future gene discoveries in ciliopathies.
Assuntos
Proteínas de Ciclo Celular/genética , Cerebelo/anormalidades , Predisposição Genética para Doença , Proteínas Mutantes/genética , Retina/anormalidades , Anormalidades Múltiplas/genética , Anormalidades do Olho/genética , Frequência do Gene , Testes Genéticos , Estudo de Associação Genômica Ampla , Heterozigoto , Humanos , Doenças Renais Císticas/genética , RNA Interferente Pequeno/genéticaRESUMO
Monomethylation of lysine 4 on histone H3 (H3K4me1) is a well-established feature of enhancers and promoters, although its function is unknown. Here, we uncover roles for H3K4me1 in diverse cell types. Remarkably, we find that MLL3/4 provokes monomethylation of promoter regions and the conditional repression of muscle and inflammatory response genes in myoblasts. During myogenesis, muscle genes are activated, lose MLL3 occupancy, and become H3K4-trimethylated through an alternative COMPASS complex. Monomethylation-mediated repression was not restricted to skeletal muscle. Together with H3K27me3 and H4K20me1, H3K4me1 was associated with transcriptional silencing in embryonic fibroblasts, macrophages, and human embryonic stem cells (ESCs). On promoters of active genes, we find that H3K4me1 spatially demarcates the recruitment of factors that interact with H3K4me3, including ING1, which, in turn, recruits Sin3A. Our findings point to a unique role for H3K4 monomethylation in establishing boundaries that restrict the recruitment of chromatin-modifying enzymes to defined regions within promoters.
Assuntos
Cromatina , Regulação da Expressão Gênica no Desenvolvimento , Histonas/genética , Desenvolvimento Muscular/genética , Mioblastos/metabolismo , Animais , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Fibroblastos/citologia , Fibroblastos/metabolismo , Genoma , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Humanos , Proteína 1 Inibidora do Crescimento , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Mioblastos/citologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Complexo Correpressor Histona Desacetilase e Sin3 , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Cilia are hair-like protrusions found at the surface of most eukaryotic cells. They can be divided into two types, motile and non-motile. Motile cilia are found in a restricted number of cell types, are generally present in large numbers, and beat in a coordinated fashion to generate fluid flow or locomotion. Non-motile or primary cilia, on the other hand, are detected in many different cell types, appear once per cell, and primarily function to transmit signals from the extracellular milieu to the cell nucleus. Defects in cilia formation, function, or maintenance are known to cause a bewildering set of human diseases, or ciliopathies, typified by retinal degeneration, renal failure and cystic kidneys, obesity, liver dysfunction, and neurological disorders. A common denominator between motile and primary cilia is their structural similarity, as both types of cilia are composed of an axoneme, the ciliary backbone that is made up of microtubules emanating from a mother centriole/basal body anchored to the cell membrane, surrounded by a ciliary membrane continuous with the plasma membrane. This structural similarity is indicative of a universal mechanism of cilia assembly involving a common set of molecular players and a sophisticated, highly regulated series of molecular events. In this review, we will mainly focus on recent advances in our understanding of the regulatory mechanisms underlying cilia assembly, with special attention paid to the centriolar protein, CP110, its interacting partner Cep290, and the various downstream molecular players and events leading to intraflagellar transport (IFT), a process that mediates the bidirectional movement of protein cargos along the axoneme and that is essential for cilia formation and maintenance.
RESUMO
The role of centrioles changes as a function of the cell cycle. Centrioles promote formation of spindle poles in mitosis and act as basal bodies to assemble primary cilia in interphase. Stringent regulations govern conversion between these two states. Although the molecular mechanisms have not been fully elucidated, recent findings have begun to shed light on pathways that regulate the conversion of centrioles to basal bodies and vice versa. Emerging studies also provide insights into how defects in the balance between centrosome and cilia function could promote ciliopathies and cancer.
Assuntos
Centríolos/ultraestrutura , Cílios/metabolismo , Microtúbulos/metabolismo , Animais , Ciclo Celular , Centrossomo/metabolismo , Fase G1 , Humanos , Doenças Renais Císticas/patologia , Microtúbulos/ultraestrutura , Mitose , Modelos Biológicos , Modelos Genéticos , Neoplasias/metabolismo , Neoplasias Pancreáticas/patologiaRESUMO
We have identified the E3 ligase Traf7 as a direct MyoD1 target and show that cell cycle exit-an early event in muscle differentiation-is linked to decreased Traf7 expression. Depletion of Traf7 accelerates myogenesis, in part through downregulation of nuclear factor-κB (NF-κB) activity. We used a proteomic screen to identify NEMO, the NF-κB essential modulator, as a Traf7-interacting protein. Finally, we show that ubiquitylation of NF-κB essential modulator is regulated exclusively by Traf7 activity in myoblasts. Our results suggest a new mechanism by which MyoD1 function is coupled to NF-κB activity through Traf7, regulating the balance between cell cycle progression and differentiation during myogenesis.
Assuntos
Desenvolvimento Muscular/genética , Proteína MyoD/metabolismo , NF-kappa B/metabolismo , Transcrição Gênica , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Animais , Ciclo Celular/genética , Diferenciação Celular/genética , Ciclina D1/metabolismo , Regulação da Expressão Gênica , Quinase I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Mioblastos/citologia , Mioblastos/metabolismo , Fosforilação , Ligação Proteica , Proteína do Retinoblastoma/metabolismo , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/deficiência , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Centrosomes duplicate only once per cell cycle, but the controls that govern this process are largely unknown. We have identified Cep76, a centriolar protein that interacts with CP110. Cep76 is expressed at low levels in G1 and is induced in S and G2 phase, during which point centrioles have already commenced duplication. Interestingly, depletion of Cep76 drives the accumulation of centriolar intermediates in certain types of cancer cells. Enforced Cep76 expression specifically inhibits centriole amplification in cells undergoing multiple rounds of duplication without preventing the formation of extra procentrioles from a parental template. Furthermore, elevated levels of Cep76 do not affect normal centriole duplication. Thus, Cep76 helps limit duplication to once per cell cycle. Our findings also point to mechanistic differences between normal duplication and aberrant centriole amplification, as well as distinctions between diverse modes of amplification.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Centríolos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Fase G2 , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Fase SRESUMO
Polycomb group proteins are critical to maintaining gene repression established during Drosophila development. Part of this group forms the PRC2 complex containing Ez that catalyzes di- and trimethylation of histone H3 lysine 27 (H3K37me2/3), marks repressive to transcription. We report that the mammalian homologs Ezh1 and Ezh2 form similar PRC2 complexes but exhibit contrasting repressive roles. While PRC2-Ezh2 catalyzes H3K27me2/3 and its knockdown affects global H3K27me2/3 levels, PRC2-Ezh1 performs this function weakly. In accordance, Ezh1 knockdown was ineffectual on global H3K27me2/3 levels. Instead, PRC2-Ezh1 directly and robustly represses transcription from chromatinized templates and compacts chromatin in the absence of the methyltransferase cofactor SAM, as evidenced by electron microscopy. Ezh1 targets a subset of Ezh2 genes, yet Ezh1 is more abundant in nonproliferative adult organs while Ezh2 expression is tightly associated with proliferation, as evidenced when analyzing aging mouse kidney. These results might reflect subfunctionalization of a PcG protein during evolution.
Assuntos
Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Fatores de Transcrição/metabolismo , Animais , Baculoviridae/genética , Linhagem Celular , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/isolamento & purificação , Cromatina/ultraestrutura , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Proteína Potenciadora do Homólogo 2 de Zeste , Genes Reporter , Células HeLa , Histona-Lisina N-Metiltransferase/genética , Humanos , Imuno-Histoquímica , Células Jurkat , Rim/citologia , Luciferases/metabolismo , Camundongos , Mutação , Células NIH 3T3 , Complexo Repressor Polycomb 2 , Regiões Promotoras Genéticas , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , TransfecçãoRESUMO
Characterization of the transcriptional regulatory network of the normal cell cycle is essential for understanding the perturbations that lead to cancer. However, the complete set of cycling genes in primary cells has not yet been identified. Here, we report the results of genome-wide expression profiling experiments on synchronized primary human foreskin fibroblasts across the cell cycle. Using a combined experimental and computational approach to deconvolve measured expression values into "single-cell" expression profiles, we were able to overcome the limitations inherent in synchronizing nontransformed mammalian cells. This allowed us to identify 480 periodically expressed genes in primary human foreskin fibroblasts. Analysis of the reconstructed primary cell profiles and comparison with published expression datasets from synchronized transformed cells reveals a large number of genes that cycle exclusively in primary cells. This conclusion was supported by both bioinformatic analysis and experiments performed on other cell types. We suggest that this approach will help pinpoint genetic elements contributing to normal cell growth and cellular transformation.