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1.
Nature ; 512(7515): 449-52, 2014 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-25164756

RESUMO

Genome function is dynamically regulated in part by chromatin, which consists of the histones, non-histone proteins and RNA molecules that package DNA. Studies in Caenorhabditis elegans and Drosophila melanogaster have contributed substantially to our understanding of molecular mechanisms of genome function in humans, and have revealed conservation of chromatin components and mechanisms. Nevertheless, the three organisms have markedly different genome sizes, chromosome architecture and gene organization. On human and fly chromosomes, for example, pericentric heterochromatin flanks single centromeres, whereas worm chromosomes have dispersed heterochromatin-like regions enriched in the distal chromosomal 'arms', and centromeres distributed along their lengths. To systematically investigate chromatin organization and associated gene regulation across species, we generated and analysed a large collection of genome-wide chromatin data sets from cell lines and developmental stages in worm, fly and human. Here we present over 800 new data sets from our ENCODE and modENCODE consortia, bringing the total to over 1,400. Comparison of combinatorial patterns of histone modifications, nuclear lamina-associated domains, organization of large-scale topological domains, chromatin environment at promoters and enhancers, nucleosome positioning, and DNA replication patterns reveals many conserved features of chromatin organization among the three organisms. We also find notable differences in the composition and locations of repressive chromatin. These data sets and analyses provide a rich resource for comparative and species-specific investigations of chromatin composition, organization and function.


Assuntos
Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Cromatina/genética , Cromatina/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Animais , Linhagem Celular , Centrômero/genética , Centrômero/metabolismo , Cromatina/química , Montagem e Desmontagem da Cromatina/genética , Replicação do DNA/genética , Elementos Facilitadores Genéticos/genética , Epigênese Genética , Heterocromatina/química , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Anotação de Sequência Molecular , Lâmina Nuclear/metabolismo , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Regiões Promotoras Genéticas/genética , Especificidade da Espécie
2.
Genome Res ; 23(8): 1339-47, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23550086

RESUMO

RNA polymerase transcription initiation sites are largely unknown in Caenorhabditis elegans. The initial 5' end of most protein-coding transcripts is removed by trans-splicing, and noncoding initiation sites have not been investigated. We characterized the landscape of RNA Pol II transcription initiation, identifying 73,500 distinct clusters of initiation. Bidirectional transcription is frequent, with a peak of transcriptional pairing at 120 bp. We assign transcription initiation sites to 7691 protein-coding genes and find that they display features typical of eukaryotic promoters. Strikingly, the majority of initiation events occur in regions with enhancer-like chromatin signatures. Based on the overlap of transcription initiation clusters with mapped transcription factor binding sites, we define 2361 transcribed intergenic enhancers. Remarkably, productive transcription elongation across these enhancers is predominantly in the same orientation as that of the nearest downstream gene. Directed elongation from an upstream enhancer toward a downstream gene could potentially deliver RNA polymerase II to a proximal promoter, or alternatively might function directly as a distal promoter. Our results provide a new resource to investigate transcription regulation in metazoans.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Cromatina/genética , Anotação de Sequência Molecular , Análise de Sequência de DNA , Sítio de Iniciação de Transcrição , Ativação Transcricional
3.
PLoS Genet ; 8(9): e1002933, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23028348

RESUMO

The Caenorhabditis elegans dosage compensation complex (DCC) equalizes X-chromosome gene dosage between XO males and XX hermaphrodites by two-fold repression of X-linked gene expression in hermaphrodites. The DCC localizes to the X chromosomes in hermaphrodites but not in males, and some subunits form a complex homologous to condensin. The mechanism by which the DCC downregulates gene expression remains unclear. Here we show that the DCC controls the methylation state of lysine 20 of histone H4, leading to higher H4K20me1 and lower H4K20me3 levels on the X chromosomes of XX hermaphrodites relative to autosomes. We identify the PR-SET7 ortholog SET-1 and the Suv4-20 ortholog SET-4 as the major histone methyltransferases for monomethylation and di/trimethylation of H4K20, respectively, and provide evidence that X-chromosome enrichment of H4K20me1 involves inhibition of SET-4 activity on the X. RNAi knockdown of set-1 results in synthetic lethality with dosage compensation mutants and upregulation of X-linked gene expression, supporting a model whereby H4K20me1 functions with the condensin-like C. elegans DCC to repress transcription of X-linked genes. H4K20me1 is important for mitotic chromosome condensation in mammals, suggesting that increased H4K20me1 on the X may restrict access of the transcription machinery to X-linked genes via chromatin compaction.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Mecanismo Genético de Compensação de Dose , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Metiltransferases/genética , Animais , Cromatina/genética , Transtornos do Desenvolvimento Sexual/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Ligados ao Cromossomo X , Histona-Lisina N-Metiltransferase/metabolismo , Masculino , Metilação , Interferência de RNA , Cromossomo X/genética
4.
NPJ Breast Cancer ; 4: 39, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30534596

RESUMO

Serial monitoring of plasma DNA mutations in estrogen receptor positive metastatic breast cancer (ER + MBC) holds promise as an early predictor of therapeutic response. Here, we developed dPCR-SEQ, a customized assay that utilizes digital PCR-based target enrichment followed by next-generation sequencing to analyze plasma DNA mutations in ESR1, PIK3CA, and TP53. We validated dPCR-SEQ in a prospective cohort of 58 patients with ER + MBC and demonstrate excellent concordance with hotspot ESR1 mutation abundance measured by conventional digital PCR. The dPCR-SEQ assay revealed ESR1, PIK3CA, and TP53 plasma ctDNA mutations in 55%, 32%, and 32% of the study patients, respectively. We also observed dynamic changes in ESR1, PIK3CA, and TP53 ctDNA mutant allele fraction (MAF) that were frequently discordant between the different genes. Thus, monitoring plasma DNA mutation dynamics using a dPCR-SEQ assay is feasible, accurate, and may be investigated as a biomarker of therapeutic response in ER + MBC.

5.
Mech Dev ; 136: 8-29, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25748605

RESUMO

We recently reported that the T-box transcription factor midline (mid) functions within the Notch-Delta signaling pathway to specify sensory organ precursor (SOP) cell fates in early-staged pupal eye imaginal discs and to suppress apoptosis (Das et al.). From genetic and allelic modifier screens, we now report that mid interacts with genes downstream of the insulin receptor(InR)/Akt, c-Jun-N-terminal kinase (JNK) and Notch signaling pathways to regulate interommatidial bristle (IOB) formation and cell survival. One of the most significant mid-interacting genes identified from the modifier screen is dFOXO, a transcription factor exhibiting a nucleocytoplasmic subcellular distribution pattern. In common with dFOXO, we show that Mid exhibits a nucleocytoplasmic distribution pattern within WT third-instar larval (3(o)L) tissue homogenates. Because dFOXO is a stress-responsive factor, we assayed the effects of either oxidative or metabolic stress responses on modifying the mid mutant phenotype which is characterized by a 50% loss of IOBs within the adult compound eye. While metabolic starvation stress does not affect the mid mutant phenotype, either 1 mM paraquat or 20% coconut oil, oxidative stress inducers, partially suppresses the mid mutant phenotype resulting in a significant recovery of IOBs. Another significant mid-interacting gene we identified is groucho (gro). Mid and Gro are predicted to act as corepressors of the enhancer-of-split gene complex downstream of Notch. Immunolabeling WT and dFOXO null 3(o)L eye-antennal imaginal discs with anti-Mid and anti-Engrailed (En) antibodies indicate that dFOXO is required to activate Mid and En expression within photoreceptor neurons of the eye disc. Taken together, these studies show that Mid and dFOXO serve as critical effectors of cell fate specification and survival within integrated Notch, InR/dAkt, and JNK signaling pathways during 3(o)L and pupal eye imaginal disc development.


Assuntos
Sobrevivência Celular/fisiologia , Proteínas de Drosophila/metabolismo , Insulina/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Estresse Fisiológico/fisiologia , Proteínas com Domínio T/metabolismo , Animais , Drosophila , Proteínas de Drosophila/genética , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Discos Imaginais/crescimento & desenvolvimento , Discos Imaginais/metabolismo , Receptores Notch/metabolismo , Inanição/genética , Inanição/metabolismo , Proteínas com Domínio T/genética
6.
Mech Dev ; 130(11-12): 577-601, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23962751

RESUMO

We report that the T-box transcription factor Midline (Mid), an evolutionary conserved homolog of the vertebrate Tbx20 protein, functions within the Notch-Delta signaling pathway essential for specifying the fates of sensory organ precursor (SOP) cells. These findings complement an established history of research showing that Mid regulates the cell-fate specification of diverse cell types within the developing heart, epidermis and central nervous system. Tbx20 has been detected in unique neuronal and epithelial cells of embryonic eye tissues in both mice and humans. However, the mechanisms by which either Mid or Tbx20 function to regulate cell-fate specification or other critical aspects of eye development including cell survival have not yet been elucidated. We have also gathered preliminary evidence suggesting that Mid may play an indirect, but vital role in selecting SOP cells within the third-instar larval eye disc by regulating the expression of the proneural gene atonal. During subsequent pupal stages, Mid specifies SOP cell fates as a member of the Notch-Delta signaling hierarchy and is essential for maintaining cell viability by inhibiting apoptotic pathways. We present several new hypotheses that seek to understand the role of Mid in regulating developmental processes downstream of the Notch receptor that are critical for specifying unique cell fates, patterning the adult eye and maintaining cellular homeostasis during eye disc morphogenesis.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Olho/metabolismo , Discos Imaginais/metabolismo , Larva/genética , Pupa/genética , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais , Proteínas com Domínio T/genética , Animais , Apoptose/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Padronização Corporal/genética , Sobrevivência Celular/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Olho/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Discos Imaginais/crescimento & desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Larva/crescimento & desenvolvimento , Larva/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Células Receptoras Sensoriais/citologia , Proteínas com Domínio T/metabolismo
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