RESUMO
Vitis vinifera is an economically important crop and a useful model in which to study chromatin dynamics. In contrast to the small and relatively simple genome of Arabidopsis thaliana, grapevine contains a complex genome of 487 Mb that exhibits extensive colonization by transposable elements. We used Hi-C, ChIP-seq and ATAC-seq to measure how chromatin features correlate to the expression of 31 845 grapevine genes. ATAC-seq revealed the presence of more than 16 000 open chromatin regions, of which we characterize nearly 5000 as possible distal enhancer candidates that occur in intergenic space >â 2 kb from the nearest transcription start site (TSS). A motif search identified more than 480 transcription factor (TF) binding sites in these regions, with those for TCP family proteins in greatest abundance. These open chromatin regions are typically within 15 kb from their nearest promoter, and a gene ontology analysis indicated that their nearest genes are significantly enriched for TF activity. The presence of a candidate cis-regulatory element (cCRE) >â 2 kb upstream of the TSS, location in the active nuclear compartment as determined by Hi-C, and the enrichment of H3K4me3, H3K4me1 and H3K27ac at the gene are correlated with gene expression. Taken together, these results suggest that regions of intergenic open chromatin identified by ATAC-seq can be considered potential candidates for cis-regulatory regions in V. vinifera. Our findings enhance the characterization of a valuable agricultural crop, and help to clarify the understanding of unique plant biology.
Assuntos
Cromatina/genética , Histonas/genética , Sequências Reguladoras de Ácido Nucleico , Vitis/genética , Sítios de Ligação , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Metilação de DNA , DNA Intergênico , Regulação da Expressão Gênica de Plantas , Ontologia Genética , Histonas/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Sítio de Iniciação de TranscriçãoRESUMO
Pdd1, a specialized HP1-like protein, is required for genome-wide DNA rearrangements that restructure a previously silent germ line genome into an active somatic genome during macronuclear differentiation of Tetrahymena thermophila. We deleted or otherwise mutated conserved regions of the protein to investigate how its different domains promote the excision of thousands of internal eliminated sequences (IESs). Previous studies revealed that Pdd1 contributes to recognition of IES loci after they are targeted by small-RNA-guided methylation of histone H3 on lysine 27 (H3K27), subsequently aids the establishment of H3K9 methylation, and recruits proteins that lead to excision. The phenotypes we observed for different Pdd1 alleles showed that each of the two chromodomains and the chromoshadow domain (CSD) have distinct contributions during somatic genome differentiation. Chromodomain 1 (CD1) is essential for conjugation as either its deletion or the substitution of two key aromatic amino acid residues (the W97A W100A mutant) is lethal. These mutations caused mislocalization of a cyan fluorescent protein (CFP)-tagged protein, prevented the establishment of histone H3 dimethylated on K9 (H3K9me2), and abolished IES excision. Nevertheless, the requirement for CD1 could be bypassed by recruiting Pdd1 directly to an IES by addition of a specific DNA binding domain. Chromodomain 2 (CD2) was necessary for producing viable progeny, but low levels of H3K9me2 and IES excision still occurred. A mutation in the chromoshadow domain (CSD) prevented Pdd1 focus formation but still permitted â¼17% of conjugants to produce viable progeny. However, this mutant was unable to stimulate excision when recruited to an ectopic IES, indicating that this domain is important for recruitment of excision factors.
Assuntos
Heterocromatina/metabolismo , Histonas/metabolismo , Mutação , Proteínas Nucleares/genética , Fosfoproteínas/genética , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/genética , Tetrahymena thermophila/genética , Sequência de Aminoácidos , DNA de Protozoário/metabolismo , Metilação , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Tetrahymena thermophila/metabolismoRESUMO
Over the past two centuries, introgression through repeated backcrossing has introduced disease resistance from wild grape species into the domesticated lineage Vitis vinifera subsp. sativa. Introgression lines are being cultivated over increasing vineyard surface areas, as their wines now rival in quality those obtained from preexisting varieties. There is, however, a lot of debate about whether and how wine laws defining commercial product categories, which are based on the classification of V. vinifera and interspecific hybrid grapes, should be revised to accommodate novel varieties that do not fit either category. Here, we developed a method of multilocus genotype analysis using short-read resequencing to identify haplotypic blocks of wild ancestry in introgression lines and quantify the physical length of chromosome segments free-of-introgression or with monoallelic and biallelic introgression. We used this genomic data to characterize species, hybrids and introgression lines and show that newly released resistant varieties contain 76.5-94.8% of V. vinifera DNA. We found that varietal wine ratings are not always commensurate with the percentage of V. vinifera ancestry and linkage drag of wild alleles around known resistance genes persists over at least 7.1-11.5 Mb, slowing down the recovery of the recurrent parental genome. This method also allowed us to identify the donor species of known resistance haplotypes, define the ancestry of wild genetic background in introgression lines with complex pedigrees, validate the ancestry of the historic varieties Concord and Norton, and unravel sample curation errors in public databases.
RESUMO
In order to elucidate the still controversial processes that originated European wine grapes from its wild progenitor, here we analyse 204 genomes of Vitis vinifera and show that all analyses support a single domestication event that occurred in Western Asia and was followed by numerous and pervasive introgressions from European wild populations. This admixture generated the so-called international wine grapes that have diffused from Alpine countries worldwide. Across Europe, marked differences in genomic diversity are observed in local varieties that are traditionally cultivated in different wine producing countries, with Italy and France showing the largest diversity. Three genomic regions of reduced genetic diversity are observed, presumably as a consequence of artificial selection. In the lowest diversity region, two candidate genes that gained berry-specific expression in domesticated varieties may contribute to the change in berry size and morphology that makes the fruit attractive for human consumption and adapted for winemaking.
Assuntos
Genoma de Planta , Vitis/genética , Europa (Continente) , Frutas/classificação , Frutas/genética , Variação Genética , Fenótipo , Vitis/classificação , Vinho/análiseRESUMO
Nucleophosmin (NPM) (B23) is an essential protein in mouse development and cell growth; however, it has been assigned numerous roles in very diverse cellular processes. Here, we present a unified mechanism for NPM's role in cell growth; NPM directs the nuclear export of both 40S and 60S ribosomal subunits. NPM interacts with rRNA and large and small ribosomal subunit proteins and also colocalizes with large and small ribosomal subunit proteins in the nucleolus, nucleus, and cytoplasm. The transduction of NPM shuttling-defective mutants or the loss of Npm1 inhibited the nuclear export of both the 40S and 60S ribosomal subunits, reduced the available pool of cytoplasmic polysomes, and diminished overall protein synthesis without affecting rRNA processing or ribosome assembly. While the inhibition of NPM shuttling can block cellular proliferation, the dramatic effects on ribosome export occur prior to cell cycle inhibition. Modest increases in NPM expression amplified the export of newly synthesized rRNAs, resulting in increased rates of protein synthesis and indicating that NPM is rate limiting in this pathway. These results support the idea that NPM-regulated ribosome export is a fundamental process in cell growth.