RESUMEN
Previous studies have identified topologically associating domains (TADs) as basic units of genome organization. We present evidence of a previously unreported level of genome folding, where distant TAD pairs, megabases apart, interact to form meta-domains. Within meta-domains, gene promoters and structural intergenic elements present in distant TADs are specifically paired. The associated genes encode neuronal determinants, including those engaged in axonal guidance and adhesion. These long-range associations occur in a large fraction of neurons but support transcription in only a subset of neurons. Meta-domains are formed by diverse transcription factors that are able to pair over long and flexible distances. We present evidence that two such factors, GAF and CTCF, play direct roles in this process. The relative simplicity of higher-order meta-domain interactions in Drosophila, compared with those previously described in mammals, allowed the demonstration that genomes can fold into highly specialized cell-type-specific scaffolds that enable megabase-scale regulatory associations.
Asunto(s)
Cromosomas de Insectos , Drosophila , Animales , Cromatina/genética , Empaquetamiento del ADN , Drosophila/genética , Mamíferos/genética , Neurogénesis , Neuronas , Factores de Transcripción , Proteínas de Drosophila , Genoma de los Insectos , Regulación de la Expresión GénicaRESUMEN
Polycomb group (PcG) protein complexes repress developmental regulator genes by modifying their chromatin. How different PcG proteins assemble into complexes and are recruited to their target genes is poorly understood. Here, we report the crystal structure of the core of the Drosophila PcG protein complex Pleiohomeotic (Pho)-repressive complex (PhoRC), which contains the Polycomb response element (PRE)-binding protein Pho and Sfmbt. The spacer region of Pho, separated from the DNA-binding domain by a long flexible linker, forms a tight complex with the four malignant brain tumor (4MBT) domain of Sfmbt. The highly conserved spacer region of the human Pho ortholog YY1 binds three of the four human 4MBT domain proteins in an analogous manner but with lower affinity. Comparison of the Drosophila Pho:Sfmbt and human YY1:MBTD1 complex structures provides a molecular explanation for the lower affinity of YY1 for human 4MBT domain proteins. Structure-guided mutations that disrupt the interaction between Pho and Sfmbt abolish formation of a ternary Sfmbt:Pho:DNA complex in vitro and repression of developmental regulator genes in Drosophila. PRE tethering of Sfmbt by Pho is therefore essential for Polycomb repression in Drosophila. Our results support a model where DNA tethering of Sfmbt by Pho and multivalent interactions of Sfmbt with histone modifications and other PcG proteins create a hub for PcG protein complex assembly at PREs.
Asunto(s)
Proteínas de Drosophila/química , Drosophila melanogaster , Regulación del Desarrollo de la Expresión Génica , Modelos Moleculares , Proteínas del Grupo Polycomb/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/química , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Humanos , Datos de Secuencia Molecular , Mutación/genética , Proteínas del Grupo Polycomb/química , Proteínas del Grupo Polycomb/genética , Unión Proteica , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Factor de Transcripción YY1/química , Factor de Transcripción YY1/metabolismoRESUMEN
O-linked ß-N-Acetylglucosamine (O-GlcNAc) is a posttranslational modification that is catalyzed by O-GlcNAc transferase (Ogt) and found on a plethora of nuclear and cytosolic proteins in animals and plants. Studies in different model organisms revealed that while O-GlcNAc is required for selected processes in Caenorhabditis elegans and Drosophila, it has evolved to become required for cell viability in mice, and this has challenged investigations to identify cellular functions that critically require this modification in mammals. Nevertheless, a principal cellular process that engages O-GlcNAcylation in all of these species is the regulation of gene transcription. Here, we revisit several of the primary experimental observations that led to current models of how O-GlcNAcylation affects gene expression. In particular, we discuss the role of the stable association of Ogt with the transcription factors Hcf1 and Tet, the two main Ogt-interacting proteins in nuclei of mammalian cells. We also critically evaluate the evidence that specific residues on core histones, including serine 112 of histone 2B (H2B-S112), are O-GlcNAcylated in vivo and discuss possible physiological effects of these modifications. Finally, we review our understanding of the role of O-GlcNAcylation in Drosophila, where recent studies suggest that the developmental defects in Ogt mutants are all caused by lack of O-GlcNAcylation of a single transcriptional regulator, the Polycomb repressor protein Polyhomeotic (Ph). Collectively, this reexamination of the experimental evidence suggests that a number of recently propagated models about the role of O-GlcNAcylation in transcriptional control should be treated cautiously.
Asunto(s)
Cromatina/metabolismo , Regulación de la Expresión Génica , Histonas/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Animales , Proteínas de Unión al ADN , Proteínas de Drosophila , Humanos , N-Acetilglucosaminiltransferasas/fisiología , Complejo Represivo Polycomb 1 , Procesamiento Proteico-PostraduccionalRESUMEN
Polycomb group (PcG) proteins exist in multiprotein complexes that modify chromatin to repress transcription. Drosophila PcG proteins Sex combs extra (Sce; dRing) and Posterior sex combs (Psc) are core subunits of PRC1-type complexes. The Sce:Psc module acts as an E3 ligase for monoubiquitylation of histone H2A, an activity thought to be crucial for repression by PRC1-type complexes. Here, we created an Sce knockout allele and show that depletion of Sce results in loss of H2A monoubiquitylation in developing Drosophila. Genome-wide profiling identified a set of target genes co-bound by Sce and all other PRC1 subunits. Analyses in mutants lacking individual PRC1 subunits reveals that these target genes comprise two distinct classes. Class I genes are misexpressed in mutants lacking any of the PRC1 subunits. Class II genes are only misexpressed in animals lacking the Psc-Su(z)2 and Polyhomeotic (Ph) subunits but remain stably repressed in the absence of the Sce and Polycomb (Pc) subunits. Repression of class II target genes therefore does not require Sce and H2A monoubiquitylation but might rely on the ability of Psc-Su(z)2 and Ph to inhibit nucleosome remodeling or to compact chromatin. Similarly, Sce does not provide tumor suppressor activity in larval tissues under conditions in which Psc-Su(z)2, Ph and Pc show such activity. Sce and H2A monoubiquitylation are therefore only crucial for repression of a subset of genes and processes regulated by PRC1-type complexes. Sce synergizes with the Polycomb repressive deubiquitinase (PR-DUB) complex to repress transcription at class I genes, suggesting that H2A monoubiquitylation must be appropriately balanced for their transcriptional repression.
Asunto(s)
Proteína con Homeodominio Antennapedia/metabolismo , Cromatina/fisiología , Proteínas de Drosophila/metabolismo , Drosophila/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/fisiología , Complejos Multiproteicos/metabolismo , Proteínas Represoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Proteína con Homeodominio Antennapedia/genética , Cromatina/metabolismo , Cartilla de ADN/genética , Proteínas de Unión al ADN/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Técnicas de Inactivación de Genes , Nucleosomas/fisiología , Complejo Represivo Polycomb 1 , Proteínas del Grupo Polycomb , Interferencia de ARN , UbiquitinaciónRESUMEN
To regulate expression, enhancers must come in proximity to their target gene. However, the relationship between the timing of enhancer-promoter (E-P) proximity and activity remains unclear, with examples of uncoupled, anticorrelated and correlated interactions. To assess this, we selected 600 characterized enhancers or promoters with tissue-specific activity in Drosophila embryos and performed Capture-C in FACS-purified myogenic or neurogenic cells during specification and tissue differentiation. This enabled direct comparison between E-P proximity and activity transitioning from OFF-to-ON and ON-to-OFF states across developmental conditions. This showed remarkably similar E-P topologies between specified muscle and neuronal cells, which are uncoupled from activity. During tissue differentiation, many new distal interactions emerge where changes in E-P proximity reflect changes in activity. The mode of E-P regulation therefore appears to change as embryogenesis proceeds, from largely permissive topologies during cell-fate specification to more instructive regulation during terminal tissue differentiation, when E-P proximity is coupled to activation.
Asunto(s)
Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Animales , Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica/genética , Regiones Promotoras Genéticas/genética , Drosophila/genética , Diferenciación Celular/genéticaRESUMEN
Boundaries in animal genomes delimit contact domains with enhanced internal contact frequencies and have debated functions in limiting regulatory cross-talk between domains and guiding enhancers to target promoters. Most mammalian boundaries form by stalling of chromosomal loop-extruding cohesin by CTCF, but most Drosophila boundaries form CTCF independently. However, how CTCF-independent boundaries form and function remains largely unexplored. Here, we assess genome folding and developmental gene expression in fly embryos lacking the ubiquitous boundary-associated factor Cp190. We find that sequence-specific DNA binding proteins such as CTCF and Su(Hw) directly interact with and recruit Cp190 to form most promoter-distal boundaries. Cp190 is essential for early development and prevents regulatory cross-talk between specific gene loci that pattern the embryo. Cp190 was, in contrast, dispensable for long-range enhancer-promoter communication at tested loci. Cp190 is thus currently the major player in fly boundary formation and function, revealing that diverse mechanisms evolved to partition genomes into independent regulatory domains.
RESUMEN
Vertebrate genomes are partitioned into contact domains defined by enhanced internal contact frequency and formed by two principal mechanisms: compartmentalization of transcriptionally active and inactive domains, and stalling of chromosomal loop-extruding cohesin by CTCF bound at domain boundaries. While Drosophila has widespread contact domains and CTCF, it is currently unclear whether CTCF-dependent domains exist in flies. We genetically ablate CTCF in Drosophila and examine impacts on genome folding and transcriptional regulation in the central nervous system. We find that CTCF is required to form a small fraction of all domain boundaries, while critically controlling expression patterns of certain genes and supporting nervous system function. We also find that CTCF recruits the pervasive boundary-associated factor Cp190 to CTCF-occupied boundaries and co-regulates a subset of genes near boundaries together with Cp190. These results highlight a profound difference in CTCF-requirement for genome folding in flies and vertebrates, in which a large fraction of boundaries are CTCF-dependent and suggest that CTCF has played mutable roles in genome architecture and direct gene expression control during metazoan evolution.
Asunto(s)
Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Drosophila/genética , Genoma , Animales , Cromatina , Cromosomas/metabolismo , Biología Evolutiva , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Técnicas de Inactivación de Genes , Masculino , Proteínas Asociadas a Microtúbulos/metabolismoRESUMEN
We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes.
Asunto(s)
Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/fisiología , Evolución Biológica , Cromosomas/ultraestructura , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Eucariontes/genética , Genoma , Complejos Multiproteicos/genética , Complejos Multiproteicos/fisiología , Adenosina Trifosfatasas/química , Algoritmos , Animales , Nucléolo Celular/ultraestructura , Núcleo Celular/ultraestructura , Centrómero/ultraestructura , Cromosomas/química , Cromosomas Humanos/química , Cromosomas Humanos/ultraestructura , Proteínas de Unión al ADN/química , Genoma Humano , Genómica , Heterocromatina/ultraestructura , Humanos , Interfase , Mitosis , Modelos Biológicos , Complejos Multiproteicos/química , Telómero/ultraestructuraRESUMEN
Development is orchestrated by regulatory elements that turn genes ON or OFF in precise spatial and temporal patterns. Many safety mechanisms prevent inappropriate action of a regulatory element on the wrong gene promoter. In flies and mammals, dedicated DNA elements (insulators) recruit protein factors (insulator binding proteins, or IBPs) to shield promoters from regulatory elements. In mammals, a single IBP called CCCTC-binding factor (CTCF) is known, whereas genetic and biochemical analyses in Drosophila have identified a larger repertoire of IBPs. How insulators function at the molecular level is not fully understood, but it is currently thought that they fold chromosomes into conformations that affect regulatory element-promoter communication. Here, we review the discovery of insulators and describe their properties. We discuss recent genetic studies in flies and mice to address the question: Is gene insulation important for animal development? Comparing and contrasting observations in these two species reveal that they have different requirements for insulation, but that insulation is a conserved and critical gene regulation strategy.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Elementos Aisladores , Animales , Drosophila melanogaster , Humanos , Transcripción GenéticaRESUMEN
Insulator binding proteins (IBPs) play an important role in regulating gene expression by binding to specific DNA sites to facilitate appropriate gene regulation. There are several IBPs in Drosophila, each defined by their ability to insulate target gene promoters in transgenic assays from the activating or silencing effects of neighboring regulatory elements. Of these, only CCCTC-binding factor (CTCF) has an obvious ortholog in mammals. CTCF is essential for mammalian cell viability and is an important regulator of genome architecture. In flies, CTCF is both maternally deposited and zygotically expressed. Flies lacking zygotic CTCF die as young adults with homeotic defects, suggesting that specific Hox genes are misexpressed in inappropriate body segments. The lack of any major embryonic defects was assumed to be due to the maternal supply of CTCF protein, as maternally contributed factors are often sufficient to progress through much of embryogenesis. Here, we definitively determined the requirement of CTCF for developmental progression in Drosophila We generated animals that completely lack both maternal and zygotic CTCF and found that, contrary to expectation, these mutants progress through embryogenesis and larval life. They develop to pharate adults, which fail to eclose from their pupal case. These mutants show exacerbated homeotic defects compared to zygotic mutants, misexpressing the Hox gene Abdominal-B outside of its normal expression domain early in development. Our results indicate that loss of Drosophila CTCF is not accompanied by widespread effects on gene expression, which may be due to redundant functions with other IBPs. Rather, CTCF is required for correct Hox gene expression patterns and for the viability of adult Drosophila.
Asunto(s)
Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Animales , Cromatina/genética , ADN/metabolismo , Drosophila melanogaster , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox , Genoma , Elementos Aisladores , Regiones Promotoras Genéticas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Represoras/genéticaRESUMEN
The glycosyltransferase Ogt adds O-linked N-Acetylglucosamine (O-GlcNAc) moieties to nuclear and cytosolic proteins. Drosophila embryos lacking Ogt protein arrest development with a remarkably specific Polycomb phenotype, arising from the failure to repress Polycomb target genes. The Polycomb protein Polyhomeotic (Ph), an Ogt substrate, forms large aggregates in the absence of O-GlcNAcylation both in vivo and in vitro. O-GlcNAcylation of a serine/threonine (S/T) stretch in Ph is critical to prevent nonproductive aggregation of both Drosophila and human Ph via their C-terminal sterile alpha motif (SAM) domains in vitro. Full Ph repressor activity in vivo requires both the SAM domain and O-GlcNAcylation of the S/T stretch. We demonstrate that Ph mutants lacking the S/T stretch reproduce the phenotype of ogt mutants, suggesting that the S/T stretch in Ph is the key Ogt substrate in Drosophila. We propose that O-GlcNAcylation is needed for Ph to form functional, ordered assemblies via its SAM domain.
Asunto(s)
Acetilglucosamina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Proteínas Represoras/metabolismo , Animales , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Técnicas de Inactivación de Genes/métodos , Humanos , N-Acetilglucosaminiltransferasas/metabolismo , Complejo Represivo Polycomb 1/genéticaRESUMEN
Polycomb group proteins are conserved transcriptional repressors that control animal and plant development. Here, we found that the Drosophila Polycomb group gene super sex combs (sxc) encodes Ogt, the highly conserved glycosyltransferase that catalyzes the addition of N-acetylglucosamine (GlcNAc) to proteins in animals and plants. Genome-wide profiling in Drosophila revealed that GlcNAc-modified proteins are highly enriched at Polycomb response elements. Among different Polycomb group proteins, Polyhomeotic is glycosylated by Sxc/Ogt in vivo. sxc/Ogt-null mutants lacked O-linked GlcNAcylation and failed to maintain Polycomb transcriptional repression even though Polycomb group protein complexes were bound at their target sites. Polycomb repression appears to be a critical function of Sxc/Ogt in Drosophila and may be mediated by the glycosylation of Polyhomeotic.