RESUMEN
Networks of coordinated alternative splicing (AS) events play critical roles in development and disease. However, a comprehensive knowledge of the factors that regulate these networks is lacking. We describe a high-throughput system for systematically linking trans-acting factors to endogenous RNA regulatory events. Using this system, we identify hundreds of factors associated with diverse regulatory layers that positively or negatively control AS events linked to cell fate. Remarkably, more than one-third of the regulators are transcription factors. Further analyses of the zinc finger protein Zfp871 and BTB/POZ domain transcription factor Nacc1, which regulate neural and stem cell AS programs, respectively, reveal roles in controlling the expression of specific splicing regulators. Surprisingly, these proteins also appear to regulate target AS programs via binding RNA. Our results thus uncover a large "missing cache" of splicing regulators among annotated transcription factors, some of which dually regulate AS through direct and indirect mechanisms.
Asunto(s)
Empalme Alternativo , Redes Reguladoras de Genes , Análisis de Secuencia de ARN/métodos , Factores de Transcripción/metabolismo , Animales , Línea Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células HEK293 , Humanos , Ratones , Neuronas/citología , Neuronas/metabolismo , ARN Mensajero/genéticaRESUMEN
La-related protein 1 (LARP1) has been identified as a key translational inhibitor of terminal oligopyrimidine (TOP) mRNAs downstream of the nutrient sensing protein kinase complex, mTORC1. LARP1 exerts this inhibitory effect on TOP mRNA translation by binding to the mRNA cap and the adjacent 5'TOP motif, resulting in the displacement of the cap-binding protein eIF4E from TOP mRNAs. However, the involvement of additional signaling pathway in regulating LARP1-mediated inhibition of TOP mRNA translation is largely unexplored. In the present study, we identify a second nutrient sensing kinase GCN2 that converges on LARP1 to control TOP mRNA translation. Using chromatin-immunoprecipitation followed by massive parallel sequencing (ChIP-seq) analysis of activating transcription factor 4 (ATF4), an effector of GCN2 in nutrient stress conditions, in WT and GCN2 KO mouse embryonic fibroblasts, we determined that LARP1 is a GCN2-dependent transcriptional target of ATF4. Moreover, we identified GCN1, a GCN2 activator, participates in a complex with LARP1 on stalled ribosomes, suggesting a role for GCN1 in LARP1-mediated translation inhibition in response to ribosome stalling. Therefore, our data suggest that the GCN2 pathway controls LARP1 activity via two mechanisms: ATF4-dependent transcriptional induction of LARP1 mRNA and GCN1-mediated recruitment of LARP1 to stalled ribosomes.
Asunto(s)
Aminoácidos , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas , Secuencia de Oligopirimidina en la Región 5' Terminal del ARN , ARN Mensajero , Proteínas de Unión al ARN , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Aminoácidos/metabolismo , Animales , Técnicas de Cultivo de Célula , Inmunoprecipitación de Cromatina , Factor 4E Eucariótico de Iniciación/metabolismo , Fibroblastos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
C2H2 zinc finger proteins represent the largest and most enigmatic class of human transcription factors. Their C2H2-ZF arrays are highly variable, indicating that most will have unique DNA binding motifs. However, most of the binding motifs have not been directly determined. In addition, little is known about whether or how these proteins regulate transcription. Most of the â¼700 human C2H2-ZF proteins also contain at least one KRAB, SCAN, BTB, or SET domain, suggesting that they may have common interacting partners and/or effector functions. Here, we report a multifaceted functional analysis of 131 human C2H2-ZF proteins, encompassing DNA binding sites, interacting proteins, and transcriptional response to genetic perturbation. We confirm the expected diversity in DNA binding motifs and genomic binding sites, and provide motif models for 78 previously uncharacterized C2H2-ZF proteins, most of which are unique. Surprisingly, the diversity in protein-protein interactions is nearly as high as diversity in DNA binding motifs: Most C2H2-ZF proteins interact with a unique spectrum of co-activators and co-repressors. Thus, multiparameter diversification likely underlies the evolutionary success of this large class of human proteins.
Asunto(s)
ADN/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Sitios de Unión , Dedos de Zinc CYS2-HIS2 , Evolución Molecular , Regulación de la Expresión Génica , Células HEK293 , Humanos , Unión Proteica , Mapas de Interacción de Proteínas , Análisis de Secuencia de ADN , Análisis de Secuencia de ARNRESUMEN
Development of an accurate protein-DNA recognition code that can predict DNA specificity from protein sequence is a central problem in biology. C2H2 zinc fingers constitute by far the largest family of DNA binding domains and their binding specificity has been studied intensively. However, despite decades of research, accurate prediction of DNA specificity remains elusive. A major obstacle is thought to be the inability of current methods to account for the influence of neighbouring domains. Here we show that this problem can be addressed using a structural approach: we build structural models for all C2H2-ZF-DNA complexes with known binding motifs and find six distinct binding modes. Each mode changes the orientation of specificity residues with respect to the DNA, thereby modulating base preference. Most importantly, the structural analysis shows that residues at the domain interface strongly and predictably influence the binding mode, and hence specificity. Accounting for predicted binding mode significantly improves prediction accuracy of predicted motifs. This new insight into the fundamental behaviour of C2H2-ZFs has implications for both improving the prediction of natural zinc finger-binding sites, and for prioritizing further experiments to complete the code. It also provides a new design feature for zinc finger engineering.
Asunto(s)
Proteínas de Unión al ADN/química , ADN/química , Dedos de Zinc , Sitios de Unión , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Bases de Datos de Proteínas , Modelos Moleculares , Unión Proteica , Conformación Proteica , Análisis de Secuencia de ProteínaRESUMEN
Histones are the primary protein component of chromatin, the mixture of DNA and proteins that packages the genetic material in eukaryotes. Large amounts of histones are required during the S phase of the cell cycle when genome replication occurs. However, ectopic expression of histones during other cell cycle phases is toxic; thus, histone expression is restricted to the S phase and is tightly regulated at multiple levels, including transcriptional, post-transcriptional, translational, and post-translational. In this review, we discuss mechanisms of regulation of histone gene expression with emphasis on the transcriptional regulation of the replication-dependent histone genes in the model yeast Saccharomyces cerevisiae.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Histonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Activación TranscripcionalRESUMEN
Rtt109 is a fungal histone acetyltransferase (HAT) that catalyzes histone H3 acetylation functionally associated with chromatin assembly. Rtt109-mediated H3 acetylation involves two histone chaperones, Asf1 and Vps75. In vivo, Rtt109 requires both chaperones for histone H3 lysine 9 acetylation (H3K9ac) but only Asf1 for full H3K56ac. In vitro, Rtt109-Vps75 catalyzes both H3K9ac and H3K56ac, whereas Rtt109-Asf1 catalyzes only H3K56ac. In this study, we extend the in vitro chaperone-associated substrate specificity of Rtt109 by showing that it acetylates vertebrate linker histone in the presence of Vps75 but not Asf1. In addition, we demonstrate that in Saccharomyces cerevisiae a short basic sequence at the carboxyl terminus of Rtt109 (Rtt109C) is required for H3K9ac in vivo. Furthermore, through in vitro and in vivo studies, we demonstrate that Rtt109C is required for optimal H3K56ac by the HAT in the presence of full-length Asf1. When Rtt109C is absent, Vps75 becomes important for H3K56ac by Rtt109 in vivo. In addition, we show that lysine 290 (K290) in Rtt109 is required in vivo for Vps75 to enhance the activity of the HAT. This is the first in vivo evidence for a role for Vps75 in H3K56ac. Taken together, our results contribute to a better understanding of chaperone control of Rtt109-mediated H3 acetylation.
Asunto(s)
Histona Acetiltransferasas/fisiología , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/enzimología , Acetilación , Secuencia de Aminoácidos , Animales , Proteínas Aviares/química , Dominio Catalítico , Proteínas de Ciclo Celular/química , Pollos , Técnicas de Inactivación de Genes , Histona Acetiltransferasas/química , Histonas/química , Lisina/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Cys2-His2 zinc finger (C2H2-ZF) proteins represent the largest class of putative human transcription factors. However, for most C2H2-ZF proteins it is unknown whether they even bind DNA or, if they do, to which sequences. Here, by combining data from a modified bacterial one-hybrid system with protein-binding microarray and chromatin immunoprecipitation analyses, we show that natural C2H2-ZFs encoded in the human genome bind DNA both in vitro and in vivo, and we infer the DNA recognition code using DNA-binding data for thousands of natural C2H2-ZF domains. In vivo binding data are generally consistent with our recognition code and indicate that C2H2-ZF proteins recognize more motifs than all other human transcription factors combined. We provide direct evidence that most KRAB-containing C2H2-ZF proteins bind specific endogenous retroelements (EREs), ranging from currently active to ancient families. The majority of C2H2-ZF proteins, including KRAB proteins, also show widespread binding to regulatory regions, indicating that the human genome contains an extensive and largely unstudied adaptive C2H2-ZF regulatory network that targets a diverse range of genes and pathways.
Asunto(s)
Proteínas Portadoras/metabolismo , Genoma Humano , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Retroelementos/genética , Proteínas Portadoras/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Humanos , Proteínas Nucleares/genética , Unión Proteica , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Represoras/genéticaRESUMEN
Condensin is a central regulator of mitotic genome structure with mutants showing poorly condensed chromosomes and profound segregation defects. Here, we identify NCT, a complex comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02), casein kinase II (CKII), and several TAFs, as a regulator of condensin function. We show that NCT and condensin bind similar genomic regions but only briefly colocalize during the periods of chromosome condensation and decondensation. This pattern of NCT binding at the core centromere, the region of maximal condensin enrichment, tracks the abundance of acetylated histone H4, as regulated by the Hat1-Mis16 acetyltransferase complex and recognized by the first Nrc1 bromodomain. Strikingly, mutants in NCT or Hat1-Mis16 restore the formation of segregation-competent chromosomes in cells containing defective condensin. These results are consistent with a model where NCT targets CKII to chromatin in a cell-cycle-directed manner in order to modulate the activity of condensin during chromosome condensation and decondensation.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Quinasa de la Caseína II/metabolismo , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Acetilación , Centrómero/metabolismo , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Mitosis/fisiología , Levaduras/metabolismoRESUMEN
How the eukaryotic cell specifies distinct chromatin domains is a central problem in molecular biology. The ciliate protozoan Tetrahymena thermophila features a separation of structurally and functionally distinct germ-line and somatic chromatin into two distinct nuclei, the micronucleus (MIC) and macronucleus (MAC) respectively. To address questions about how distinct chromatin states are assembled in the MAC and MIC, we have initiated studies to define protein-protein interactions for T. thermophila chromatin-related proteins. Affinity purification followed by mass spectrometry analysis of the conserved Asf1 histone chaperone in T. thermophila revealed that it forms a complex with an importin ß, ImpB6. Furthermore, these proteins co-localized to both the MAC and MIC in growth and development. We suggest that newly synthesized histones H3 and H4 in T. thermophila are transported via Asf1-ImpB6 in an evolutionarily conserved pathway to both nuclei where they then enter nucleus-specific chromatin assembly pathways. These studies set the stage for further use of functional proteomics to elucidate details of the characterization and functional analysis of the unique chromatin domains in T. thermophila. BIOLOGICAL SIGNIFICANCE: Asf1 is an evolutionarily conserved chaperone of H3 and H4 histones that functions in replication dependent and independent chromatin assembly. Although Asf1 has been well studied in humans and yeast (members of the Opisthokonta lineage of eukaryotes), questions remain concerning its mechanism of function. To obtain additional insight into the Asf1 function we have initiated a proteomic analysis in the ciliate protozoan T. thermophila, a member of the Alveolata lineage of eukaryotes. Our results suggest that an evolutionarily conserved function of Asf1 is mediating the nuclear transport of newly synthesized histones H3 and H4.