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1.
Cell ; 186(19): 4100-4116.e15, 2023 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-37643610

RESUMO

Nucleosomes block access to DNA methyltransferase, unless they are remodeled by DECREASE in DNA METHYLATION 1 (DDM1LSH/HELLS), a Snf2-like master regulator of epigenetic inheritance. We show that DDM1 promotes replacement of histone variant H3.3 by H3.1. In ddm1 mutants, DNA methylation is partly restored by loss of the H3.3 chaperone HIRA, while the H3.1 chaperone CAF-1 becomes essential. The single-particle cryo-EM structure at 3.2 Å of DDM1 with a variant nucleosome reveals engagement with histone H3.3 near residues required for assembly and with the unmodified H4 tail. An N-terminal autoinhibitory domain inhibits activity, while a disulfide bond in the helicase domain supports activity. DDM1 co-localizes with H3.1 and H3.3 during the cell cycle, and with the DNA methyltransferase MET1Dnmt1, but is blocked by H4K16 acetylation. The male germline H3.3 variant MGH3/HTR10 is resistant to remodeling by DDM1 and acts as a placeholder nucleosome in sperm cells for epigenetic inheritance.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Metilação de DNA , Histonas , Nucleossomos , Montagem e Desmontagem da Cromatina , DNA , Metilases de Modificação do DNA , Epigênese Genética , Histonas/genética , Nucleossomos/genética , Sêmen , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo
2.
Genome Res ; 30(4): 576-588, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32303559

RESUMO

In Arabidopsis, LTR retrotransposons are activated by mutations in the chromatin gene DECREASE in DNA METHYLATION 1 (DDM1), giving rise to 21- to 22-nt epigenetically activated siRNA (easiRNA) that depend on RNA DEPENDENT RNA POLYMERASE 6 (RDR6). We purified virus-like particles (VLPs) from ddm1 and ddm1rdr6 mutants in which genomic RNA is reverse transcribed into complementary DNA. High-throughput short-read and long-read sequencing of VLP DNA (VLP DNA-seq) revealed a comprehensive catalog of active LTR retrotransposons without the need for mapping transposition, as well as independent of genomic copy number. Linear replication intermediates of the functionally intact COPIA element EVADE revealed multiple central polypurine tracts (cPPTs), a feature shared with HIV in which cPPTs promote nuclear localization. For one member of the ATCOPIA52 subfamily (SISYPHUS), cPPT intermediates were not observed, but abundant circular DNA indicated transposon "suicide" by auto-integration within the VLP. easiRNA targeted EVADE genomic RNA, polysome association of GYPSY (ATHILA) subgenomic RNA, and transcription via histone H3 lysine-9 dimethylation. VLP DNA-seq provides a comprehensive landscape of LTR retrotransposons and their control at transcriptional, post-transcriptional, and reverse transcriptional levels.


Assuntos
Arabidopsis/genética , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Retroelementos , Biologia Computacional/métodos , Bases de Dados Genéticas , Interferência de RNA , Processamento Pós-Transcricional do RNA , RNA Interferente Pequeno/genética , Sequências Repetidas Terminais , Navegador
3.
Biochem Soc Trans ; 49(5): 2241-2251, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34495315

RESUMO

Plant genomes are largely comprised of retrotransposons which can replicate through 'copy and paste' mechanisms. Long terminal repeat (LTR) retrotransposons are the major class of retrotransposons in plant species, and importantly they broadly affect the expression of nearby genes. Although most LTR retrotransposons are non-functional, active retrotranspositions have been reported in plant species or mutants under normal growth condition and environmental stresses. With the well-defined reference genome and numerous mutant alleles, Arabidopsis studies have significantly expanded our understanding of retrotransposon regulation. Active LTR retrotransposon loci produce virus-like particles to perform reverse transcription, and their complementary DNA can be inserted into new genomic loci. Due to the detrimental consequences of retrotransposition, plants like animals, have developed transcriptional and post-transcriptional silencing mechanisms. Recently several different genome-wide techniques have been developed to understand LTR retrotransposition in Arabidopsis and different plant species. Transposome, methylome, transcriptome, translatome and small RNA sequencing data have revealed how host silencing mechanisms can affect multiple steps of retrotransposition. These recent advances shed light on future mechanistic studies of retrotransposition as well as retrotransposon diversity.


Assuntos
Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Retroelementos , Replicação do DNA , Epigênese Genética , Inativação Gênica , Genes de Plantas , Ativação Transcricional
4.
PLoS Biol ; 12(9): e1001950, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25226037

RESUMO

Transcriptional activation in response to hypoxia in plants is orchestrated by ethylene-responsive factor group VII (ERF-VII) transcription factors, which are stable during hypoxia but destabilized during normoxia through their targeting to the N-end rule pathway of selective proteolysis. Whereas the conditionally expressed ERF-VII genes enable effective flooding survival strategies in rice, the constitutive accumulation of N-end-rule-insensitive versions of the Arabidopsis thaliana ERF-VII factor RAP2.12 is maladaptive. This suggests that transcriptional activation under hypoxia that leads to anaerobic metabolism may need to be fine-tuned. However, it is presently unknown whether a counterbalance of RAP2.12 exists. Genome-wide transcriptome analyses identified an uncharacterized trihelix transcription factor gene, which we named HYPOXIA RESPONSE ATTENUATOR1 (HRA1), as highly up-regulated by hypoxia. HRA1 counteracts the induction of core low oxygen-responsive genes and transcriptional activation of hypoxia-responsive promoters by RAP2.12. By yeast-two-hybrid assays and chromatin immunoprecipitation we demonstrated that HRA1 interacts with the RAP2.12 protein but with only a few genomic DNA regions from hypoxia-regulated genes, indicating that HRA1 modulates RAP2.12 through protein-protein interaction. Comparison of the low oxygen response of tissues characterized by different levels of metabolic hypoxia (i.e., the shoot apical zone versus mature rosette leaves) revealed that the antagonistic interplay between RAP2.12 and HRA1 enables a flexible response to fluctuating hypoxia and is of importance to stress survival. In Arabidopsis, an effective low oxygen-sensing response requires RAP2.12 stabilization followed by HRA1 induction to modulate the extent of the anaerobic response by negative feedback regulation of RAP2.12. This mechanism is crucial for plant survival under suboptimal oxygenation conditions. The discovery of the feedback loop regulating the oxygen-sensing mechanism in plants opens new perspectives for breeding flood-resistant crops.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Oxigênio/farmacologia , Fatores de Transcrição/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Hipóxia Celular/genética , Imunoprecipitação da Cromatina , DNA de Plantas/genética , DNA de Plantas/metabolismo , Proteínas de Ligação a DNA , Retroalimentação Fisiológica , Oxigênio/metabolismo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/genética , Brotos de Planta/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ativação Transcricional , Técnicas do Sistema de Duplo-Híbrido
5.
Nature ; 479(7373): 415-8, 2011 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-22020279

RESUMO

Plants and animals are obligate aerobes, requiring oxygen for mitochondrial respiration and energy production. In plants, an unanticipated decline in oxygen availability (hypoxia), as caused by roots becoming waterlogged or foliage submergence, triggers changes in gene transcription and messenger RNA translation that promote anaerobic metabolism and thus sustain substrate-level ATP production. In contrast to animals, oxygen sensing has not been ascribed to a mechanism of gene regulation in response to oxygen deprivation in plants. Here we show that the N-end rule pathway of targeted proteolysis acts as a homeostatic sensor of severe low oxygen levels in Arabidopsis, through its regulation of key hypoxia-response transcription factors. We found that plants lacking components of the N-end rule pathway constitutively express core hypoxia-response genes and are more tolerant of hypoxic stress. We identify the hypoxia-associated ethylene response factor group VII transcription factors of Arabidopsis as substrates of this pathway. Regulation of these proteins by the N-end rule pathway occurs through a characteristic conserved motif at the amino terminus initiating with Met-Cys. Enhanced stability of one of these proteins, HRE2, under low oxygen conditions improves hypoxia survival and reveals a molecular mechanism for oxygen sensing in plants via the evolutionarily conserved N-end rule pathway. SUB1A-1, a major determinant of submergence tolerance in rice, was shown not to be a substrate for the N-end rule pathway despite containing the N-terminal motif, indicating that it is uncoupled from N-end rule pathway regulation, and that enhanced stability may relate to the superior tolerance of Sub1 rice varieties to multiple abiotic stresses.


Assuntos
Arabidopsis/metabolismo , Hipóxia Celular , Homeostase , Aclimatação , Anaerobiose/efeitos dos fármacos , Anaerobiose/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Etilenos/farmacologia , Inundações , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Imersão , Oryza/efeitos dos fármacos , Oryza/metabolismo , Oxigênio/metabolismo , Oxigênio/farmacologia , Proteólise/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Fatores de Transcrição/metabolismo
6.
Physiol Plant ; 148(3): 322-33, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23517122

RESUMO

Over 13% of all genes in the Arabidopsis thaliana genome encode for proteins classified as having a completely unknown function, with the function of >30% of the Arabidopsis proteome poorly characterized. Although empirical data in the form of mRNA and proteome profiling experiments suggest that many of these proteins play an important role in different biological processes, their functional characterization remains one of the major challenges in modern biology. To expand the annotation of genes with unknown function involved in the response of Arabidopsis to different environmental stress conditions, we selected 1007 such genes and tested the response of their corresponding homozygous T-DNA insertional mutants to salinity, oxidative, osmotic, heat, cold and hypoxia stresses. Depending on the specific abiotic stresses tested, 12-31% of mutants had an altered stress-response phenotype. Interestingly, 832 out of 1007 mutants showed tolerance or sensitivity to more than one abiotic stress treatment, suggesting that genes of unknown function could play an important role in abiotic stress-response signaling, or general acclimation mechanisms. Further analysis of multiple stress-response phenotypes within different populations of mutants revealed interesting links between acclimation to heat, cold and oxidative stresses, as well as between sensitivity to ABA, osmotic, salinity, oxidative and hypoxia stresses. Our findings provide a significant contribution to the biological characterization of genes with unknown function in Arabidopsis and demonstrate that many of these genes play a key role in the response of plants to abiotic stresses.


Assuntos
Arabidopsis/genética , Arabidopsis/fisiologia , Genes de Plantas/genética , Ensaios de Triagem em Larga Escala , Estresse Fisiológico/genética , Ácido Abscísico/farmacologia , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/genética , DNA Bacteriano/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Mutagênese Insercional/efeitos dos fármacos , Mutagênese Insercional/genética , Mutação/genética , Fenótipo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Plântula/efeitos dos fármacos , Plântula/genética , Estresse Fisiológico/efeitos dos fármacos
7.
Prostate Int ; 11(2): 113-121, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37409096

RESUMO

Background: DNA methylation markers are considered robust diagnostic features in various cancer types, as epigenetic marks are commonly altered during cancer progression. Differentiation between benign prostatic hyperplasia (BPH) and early-stage prostate cancer (PCa) is clinically difficult, relying on the information of the patient's symptoms or levels of prostate-specific antigen. Methods: A total of 42 PCa patients and 11 BPH patients were recruited. Genomic DNA was purified from tissues and used for the library preparation of the target-enriched methylome with enzymatic conversion and a Twist 85 Mbp EM-seq panel. Paired-end sequencing (150 bp) was performed using NovaSeq 6000 or NextSeq 550. After quality control, including adapter trimming and de-duplication of raw sequencing data, differential methylation patterns were analyzed between the BPH and PCa groups. Results: We report DNA methylation patterns existing between BPH and PCa. The major finding is that broad hypermethylation occurred at genic loci in PCa tissues as compared to the BPH. Gene ontology analysis suggested that hypermethylation of genic loci involved in chromatin and transcriptional regulation is involved in cancer progression. We also compared PCa tissues with high Gleason scores to tissues with low Gleason scores. The high-Gleason PCa tissues showed hundreds of focal differentially methylated CpG sites corresponding to genes functioning in cancer cell proliferation or metastasis. This suggests that dissecting early-to-advanced-grade cancer stages requires an in-depth analysis of differential methylation at the single CpG site level. Conclusions: Our study reports that enzymatic methylome sequencing data can be used to distinguish PCa from BPH and advanced PCa from early-stage PCa. The stage-specific methylation patterns in this study will be valuable resources for diagnostic purposes as well as further development of liquid biopsy approaches for the early detection of PCa.

8.
bioRxiv ; 2023 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-37503143

RESUMO

Epigenetic inheritance refers to the faithful replication of DNA methylation and histone modification independent of DNA sequence. Nucleosomes block access to DNA methyltransferases, unless they are remodeled by DECREASE IN DNA METHYLATION1 (DDM1 Lsh/HELLS ), a Snf2-like master regulator of epigenetic inheritance. We show that DDM1 activity results in replacement of the transcriptional histone variant H3.3 for the replicative variant H3.1 during the cell cycle. In ddm1 mutants, DNA methylation can be restored by loss of the H3.3 chaperone HIRA, while the H3.1 chaperone CAF-1 becomes essential. The single-particle cryo-EM structure at 3.2 Å of DDM1 with a variant nucleosome reveals direct engagement at SHL2 with histone H3.3 at or near variant residues required for assembly, as well as with the deacetylated H4 tail. An N-terminal autoinhibitory domain binds H2A variants to allow remodeling, while a disulfide bond in the helicase domain is essential for activity in vivo and in vitro . We show that differential remodeling of H3 and H2A variants in vitro reflects preferential deposition in vivo . DDM1 co-localizes with H3.1 and H3.3 during the cell cycle, and with the DNA methyltransferase MET1 Dnmt1 . DDM1 localization to the chromosome is blocked by H4K16 acetylation, which accumulates at DDM1 targets in ddm1 mutants, as does the sperm cell specific H3.3 variant MGH3 in pollen, which acts as a placeholder nucleosome in the germline and contributes to epigenetic inheritance.

9.
New Phytol ; 190(2): 457-71, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21231933

RESUMO

• A detailed description of the molecular response of Arabidopsis thaliana to submergence can aid the identification of genes that are critical to flooding survival. • Rosette-stage plants were fully submerged in complete darkness and shoot and root tissue was harvested separately after the O(2) partial pressure of the petiole and root had stabilized at c. 6 and 0.1 kPa, respectively. As controls, plants were untreated or exposed to darkness. Following quantitative profiling of cellular mRNAs with the Affymetrix ATH1 platform, changes in the transcriptome in response to submergence, early darkness, and O(2)-deprivation were evaluated by fuzzy k-means clustering. This identified genes co-regulated at the conditional, developmental or organ-specific level. Mutants for 10 differentially expressed HYPOXIA-RESPONSIVE UNKNOWN PROTEIN (HUP) genes were screened for altered submergence tolerance. • The analysis identified 34 genes that were ubiquitously co-regulated by submergence and O(2) deprivation. The biological functions of these include signaling, transcription, and anaerobic energy metabolism. HUPs comprised 40% of the co-regulated transcripts and mutants of seven of these genes were significantly altered in submergence tolerance. • The results define transcriptomic adjustments in response to submergence in the dark and demonstrate that the manipulation of HUPs can alter submergence tolerance.


Assuntos
Arabidopsis/genética , Arabidopsis/fisiologia , Água/farmacologia , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Carbono/metabolismo , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Proliferação de Células/efeitos dos fármacos , Análise por Conglomerados , Escuridão , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas/genética , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Oxigênio/metabolismo , Pressão Parcial , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
Plant Physiol ; 152(3): 1484-500, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20097791

RESUMO

High-throughput technology has facilitated genome-scale analyses of transcriptomic adjustments in response to environmental perturbations with an oxygen deprivation component, such as transient hypoxia or anoxia, root waterlogging, or complete submergence. We showed previously that Arabidopsis (Arabidopsis thaliana) seedlings elevate the levels of hundreds of transcripts, including a core group of 49 genes that are prioritized for translation across cell types of both shoots and roots. To recognize low-oxygen responses that are evolutionarily conserved versus species specific, we compared the transcriptomic reconfiguration in 21 organisms from four kingdoms (Plantae, Animalia, Fungi, and Bacteria). Sorting of organism proteomes into clusters of putative orthologs identified broadly conserved responses associated with glycolysis, fermentation, alternative respiration, metabolite transport, reactive oxygen species amelioration, chaperone activity, and ribosome biogenesis. Differentially regulated genes involved in signaling and transcriptional regulation were poorly conserved across kingdoms. Strikingly, nearly half of the induced mRNAs of Arabidopsis seedlings encode proteins of unknown function, of which over 40% had up-regulated orthologs in poplar (Populus trichocarpa), rice (Oryza sativa), or Chlamydomonas reinhardtii. Sixteen HYPOXIA-RESPONSIVE UNKNOWN PROTEIN (HUP) genes, including four that are Arabidopsis specific, were ectopically overexpressed and evaluated for their effect on seedling tolerance to oxygen deprivation. This allowed the identification of HUPs coregulated with genes associated with anaerobic metabolism and other processes that significantly enhance or reduce stress survival when ectopically overexpressed. These findings illuminate both broadly conserved and plant-specific low-oxygen stress responses and confirm that plant-specific HUPs with limited phylogenetic distribution influence low-oxygen stress endurance.


Assuntos
Arabidopsis/genética , Perfilação da Expressão Gênica , Oxigênio/metabolismo , Arabidopsis/metabolismo , Chlamydomonas/genética , Chlamydomonas/metabolismo , Análise por Conglomerados , Hibridização Genômica Comparativa , Biologia Computacional , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Hipóxia , Oryza/genética , Oryza/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Populus/genética , Populus/metabolismo , Especificidade da Espécie , Estresse Fisiológico
13.
Trends Plant Sci ; 17(3): 129-38, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22280796

RESUMO

Plant-specific group VII Ethylene Response Factor (ERF) transcription factors have emerged as pivotal regulators of flooding and low oxygen responses. In rice (Oryza sativa), these proteins regulate contrasting strategies of flooding survival. Recent studies on Arabidopsis thaliana group VII ERFs show they are stabilized under hypoxia but destabilized under oxygen-replete conditions via the N-end rule pathway of targeted proteolysis. Oxygen-dependent sequestration at the plasma membrane maintains at least one of these proteins, RAP2.12, under normoxia. Remarkably, SUB1A, the rice group VII ERF that enables prolonged submergence tolerance, appears to evade oxygen-regulated N-end rule degradation. We propose that the turnover of group VII ERFs is of ecological relevance in wetland species and might be manipulated to improve flood tolerance of crops.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Oxigênio/metabolismo , Estresse Fisiológico , Animais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Inundações , Humanos , Áreas Alagadas
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