RESUMO
Centromeres are epigenetically defined by the centromere-specific histone H3 variant CENP-A. Specialized loading machinery, including the histone chaperone HJURP/Scm3, participates in CENP-A nucleosome assembly. However, Scm3/HJURP is missing from multiple lineages, including nematodes, with CENP-A-dependent centromeres. Here, we show that the extended N-terminal tail of Caenorhabditis elegans CENP-A contains a predicted structured region that is essential for centromeric chromatin assembly; removal of this region prevents CENP-A loading, resulting in failure of kinetochore assembly and defective chromosome condensation. By contrast, the N-tail mutant CENP-A localizes normally in the presence of endogenous CENP-A. The portion of the N-tail containing the predicted structured region binds to KNL-2, a conserved SANTA domain and Myb domain-containing protein (referred to as M18BP1 in vertebrates) specifically involved in CENP-A chromatin assembly. This direct interaction is conserved in the related nematode Caenorhabditis briggsae, despite divergence of the N-tail and KNL-2 primary sequences. Thus, the extended N-tail of CENP-A is essential for CENP-A chromatin assembly in C. elegans and partially substitutes for the function of Scm3/HJURP, in that it mediates a direct interaction between CENP-A and KNL-2. These results highlight an evolutionary variation on centromeric chromatin assembly in the absence of a dedicated CENP-A-specific chaperone/targeting factor of the Scm3/HJURP family.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteína Centromérica A/metabolismo , Centrômero/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Caenorhabditis elegans/genética , Proteína Centromérica A/genética , Ligação Proteica , Conformação Proteica , Domínios ProteicosRESUMO
Centromeres are defined epigenetically in the majority of eukaryotes by the presence of chromatin containing the centromeric histone H3 variant CENP-A. Most species have a single gene encoding a centromeric histone variant whereas C. elegans has two: HCP-3 (also known as CeCENP-A) and CPAR-1. Prior RNAi replacement experiments showed that HCP-3 is the functionally dominant isoform, consistent with CPAR-1 not being detectable in embryos. GFP::CPAR-1 is loaded onto meiotic chromosomes in diakinesis and is enriched on bivalents until meiosis I. Here we show that GFP::CPAR-1 signal loss from chromosomes precisely coincides with homolog segregation during anaphase I. This loss of GFP::CPAR-1 signal reflects proteolytic cleavage between GFP and the histone fold of CPAR-1, as CPAR-1::GFP, in which GFP is fused to the C-terminus of CPAR-1, does not exhibit any loss of GFP signal. A focused candidate screen implicated separase, the protease that initiates anaphase by cleaving the kleisin subunit of cohesin, in this cleavage reaction. Examination of the N-terminal tail sequence of CPAR-1 revealed a putative separase cleavage site and mutation of the signature residues in this site eliminated the cleavage reaction, as visualized by retention of GFP::CPAR-1 signal on separating homologous chromosomes at the metaphase-anaphase transition of meiosis I. Neither cleaved nor uncleavable CPAR-1 were centromere-localized in mitosis and instead localized throughout chromatin, indicating that centromere activity has not been retained in CPAR-1. Although the functions of CPAR-1 and of its separase-dependent cleavage remain to be elucidated, this effort reveals a new substrate of separase and provides an in vivo biosensor to monitor separase activity at the onset of meiosis I anaphase.
Assuntos
Anáfase , Autoantígenos/metabolismo , Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/citologia , Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/metabolismo , Meiose , Metáfase , Separase/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Proteína Centromérica A , Segregação de Cromossomos , Cromossomos/metabolismo , Duplicação Gênica , Proteínas de Fluorescência Verde/metabolismo , Histonas/metabolismo , Dados de Sequência Molecular , Mutação/genética , Oócitos/citologia , Oócitos/metabolismo , Separase/antagonistas & inibidoresRESUMO
Centromeres are chromosomal loci that direct segregation of the genome during cell division. The histone H3 variant CENP-A (also known as CenH3) defines centromeres in monocentric organisms, which confine centromere activity to a discrete chromosomal region, and holocentric organisms, which distribute centromere activity along the chromosome length. Because the highly repetitive DNA found at most centromeres is neither necessary nor sufficient for centromere function, stable inheritance of CENP-A nucleosomal chromatin is postulated to propagate centromere identity epigenetically. Here, we show that in the holocentric nematode Caenorhabditis elegans pre-existing CENP-A nucleosomes are not necessary to guide recruitment of new CENP-A nucleosomes. This is indicated by lack of CENP-A transmission by sperm during fertilization and by removal and subsequent reloading of CENP-A during oogenic meiotic prophase. Genome-wide mapping of CENP-A location in embryos and quantification of CENP-A molecules in nuclei revealed that CENP-A is incorporated at low density in domains that cumulatively encompass half the genome. Embryonic CENP-A domains are established in a pattern inverse to regions that are transcribed in the germline and early embryo, and ectopic transcription of genes in a mutant germline altered the pattern of CENP-A incorporation in embryos. Furthermore, regions transcribed in the germline but not embryos fail to incorporate CENP-A throughout embryogenesis. We propose that germline transcription defines genomic regions that exclude CENP-A incorporation in progeny, and that zygotic transcription during early embryogenesis remodels and reinforces this basal pattern. These findings link centromere identity to transcription and shed light on the evolutionary plasticity of centromeres.
Assuntos
Caenorhabditis elegans/genética , Centrômero/genética , Cromatina/genética , Células Germinativas/metabolismo , Transcrição Gênica , Animais , Autoantígenos/metabolismo , Evolução Biológica , Caenorhabditis elegans/embriologia , Proteína Centromérica A , Proteínas Cromossômicas não Histona/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Fertilização , Regulação da Expressão Gênica no Desenvolvimento , Genoma Helmíntico , Gônadas/citologia , Gônadas/metabolismo , Organismos Hermafroditas , Masculino , MeioseRESUMO
The vertebrate hindbrain contains various sensory-motor networks controlling movements of the eyes, jaw, head, and body. Here we show that stripes of neurons with shared neurotransmitter phenotype that extend throughout the hindbrain of young zebrafish reflect a broad underlying structural and functional patterning. The neurotransmitter stripes contain cell types with shared gross morphologies and transcription factor markers. Neurons within a stripe are stacked systematically by extent and location of axonal projections, input resistance, and age, and are recruited along the axis of the stripe during behavior. The implication of this pattern is that the many networks in hindbrain are constructed from a series of neuronal components organized into stripes that are ordered from top to bottom according to a neuron's age, structural and functional properties, and behavioral roles. This simple organization probably forms a foundation for the construction of the networks underlying the many behaviors produced by the hindbrain.
Assuntos
Padronização Corporal/fisiologia , Interneurônios/metabolismo , Modelos Neurológicos , Rede Nervosa , Neurotransmissores/metabolismo , Rombencéfalo/anatomia & histologia , Peixe-Zebra/anatomia & histologia , Fatores Etários , Animais , Animais Geneticamente Modificados , Cálcio/metabolismo , Eletrofisiologia , Interneurônios/citologia , Microscopia Confocal , Rombencéfalo/fisiologia , Peixe-Zebra/fisiologiaRESUMO
The Caenorhabditis elegans gonad and early embryo have recently emerged as an attractive metazoan model system for studying cell and developmental biology. The success of this system is attributable to the stereotypical architecture and reproducible cell divisions of the gonad/early embryo, coupled with penetrant RNAi-mediated protein depletion. These features have facilitated the development of visual assays with high spatiotemporal resolution to monitor specific subcellular processes. Assay development has relied heavily on the emergence of methods to circumvent germline silencing to allow the expression of transgenes encoding fluorescent fusion proteins. In this chapter, we discuss methods for the expression and imaging of fluorescent proteins in the C. elegans germline, including the design of transgenes for optimal expression, the generation of transgenic worm lines by ballistic bombardment, the construction of multimarker lines by mating, and methods for live imaging of the gonad and early embryo.
Assuntos
Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Embrião não Mamífero , Técnicas Genéticas , Proteínas de Fluorescência Verde/genética , Proteínas Luminescentes/genética , Animais , Animais Geneticamente Modificados , Regulação da Expressão Gênica no Desenvolvimento , Vetores Genéticos , Gônadas/embriologia , Proteínas de Fluorescência Verde/análise , Íntrons , Proteínas Luminescentes/análise , Proteínas Recombinantes de Fusão , TransgenesRESUMO
Nucleosomes containing the centromere-specific histone H3 variant centromere protein A (CENP-A) create the chromatin foundation for kinetochore assembly. To understand the mechanisms that selectively target CENP-A to centromeres, we took a functional genomics approach in the nematode Caenorhabditis elegans, in which failure to load CENP-A results in a signature kinetochore-null (KNL) phenotype. We identified a single protein, KNL-2, that is specifically required for CENP-A incorporation into chromatin. KNL-2 and CENP-A localize to centromeres throughout the cell cycle in an interdependent manner and coordinately direct chromosome condensation, kinetochore assembly, and chromosome segregation. The isolation of KNL-2-associated chromatin coenriched CENP-A, indicating their close proximity on DNA. KNL-2 defines a new conserved family of Myb DNA-binding domain-containing proteins. The human homologue of KNL-2 is also specifically required for CENP-A loading and kinetochore assembly but is only transiently present at centromeres after mitotic exit. These results implicate a new protein class in the assembly of centromeric chromatin and suggest that holocentric and monocentric chromosomes share a common mechanism for CENP-A loading.
Assuntos
Autoantígenos/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Família Multigênica , Sequência de Aminoácidos , Animais , Autoantígenos/genética , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/química , Centrômero/metabolismo , Proteína Centromérica A , Proteínas Cromossômicas não Histona/genética , Genômica , Histonas/metabolismo , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/química , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-myb/química , Interferência de RNA , Alinhamento de SequênciaRESUMO
Two distinct chromosome architectures are prevalent among eukaryotes: monocentric, in which localized centromeres restrict kinetochore assembly to a single chromosomal site, and holocentric, in which diffuse kinetochores form along the entire chromosome length. During mitosis, both chromosome types use specialized chromatin, containing the histone H3 variant CENP-A, to direct kinetochore assembly. For the segregation of recombined homologous chromosomes during meiosis, monocentricity is thought to be crucial for limiting spindle-based forces to one side of a crossover and to prevent recombined chromatids from being simultaneously pulled towards both spindle poles. The mechanisms that allow holocentric chromosomes to avert this fate remain uncharacterized. Here, we show that markedly different mechanisms segregate holocentric chromosomes during meiosis and mitosis in the nematode Caenorhabditis elegans. Immediately prior to oocyte meiotic segregation, outer-kinetochore proteins were recruited to cup-like structures on the chromosome surface via a mechanism that is independent of CENP-A. In striking contrast to mitosis, both oocyte meiotic divisions proceeded normally following depletion of either CENP-A or the closely associated centromeric protein CENP-C. These findings highlight a pronounced difference between the segregation of holocentric chromosomes during meiosis and mitosis and demonstrate the potential to uncouple assembly of outer-kinetochore proteins from CENP-A chromatin.