RESUMO
The histone variant H2A.Z plays key functions in transcription and genome stability in all eukaryotes ranging from yeast to human, but the molecular mechanisms by which H2A.Z is incorporated into chromatin remain largely obscure. Here, we characterized the two homologs of yeast Chaperone for H2A.Z-H2B (Chz1) in Arabidopsis thaliana, AtChz1A and AtChz1B. AtChz1A/AtChz1B were verified to bind to H2A.Z-H2B and facilitate nucleosome assembly in vitro. Simultaneous knockdown of AtChz1A and AtChz1B, which exhibit redundant functions, led to a genome-wide reduction in H2A.Z and phenotypes similar to those of the H2A.Z-deficient mutant hta9-1hta11-2, including early flowering and abnormal flower morphologies. Interestingly, AtChz1A was found to physically interact with ACTIN-RELATED PROTEIN 6 (ARP6), an evolutionarily conserved subunit of the SWR1 chromatin-remodeling complex. Genetic interaction analyses showed that atchz1a-1atchz1b-1 was hypostatic to arp6-1. Consistently, genome-wide profiling analyses revealed partially overlapping genes and fewer misregulated genes and H2A.Z-reduced chromatin regions in atchz1a-1atchz1b-1 compared with arp6-1. Together, our results demonstrate that AtChz1A and AtChz1B act as histone chaperones to assist the deposition of H2A.Z into chromatin via interacting with SWR1, thereby playing critical roles in the transcription of genes involved in flowering and many other processes.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Montagem e Desmontagem da Cromatina , Chaperonas de Histonas , Adenosina Trifosfatases/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cromatina/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Photomorphogenesis is a critical developmental process bridging light-regulated transcriptional reprogramming with morphological changes in organisms. Strikingly, the chromatin-based transcriptional control of photomorphogenesis remains poorly understood. Here, we show that the Arabidopsis (Arabidopsis thaliana) ortholog of ATP-dependent chromatin-remodeling factor AtINO80 represses plant photomorphogenesis. Loss of AtINO80 inhibited hypocotyl cell elongation and caused anthocyanin accumulation. Both light-induced genes and dark-induced genes were affected in the atino80 mutant. Genome-wide occupancy of the H2A.Z histone variant and levels of histone H3 were reduced in atino80 In particular, AtINO80 bound the gene body of ELONGATED HYPOCOTYL 5 (HY5), resulting in lower chromatin incorporations of H2A.Z and H3 at HY5 in atino80 Genetic analysis revealed that AtINO80 acts in a phytochrome B- and HY5-dependent manner in the regulation of photomorphogenesis. Together, our study elucidates a mechanism wherein AtINO80 modulates nucleosome density and H2A.Z incorporation and represses the transcription of light-related genes, such as HY5, to fine tune plant photomorphogenesis.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Histonas/metabolismo , Luz , Morfogênese/efeitos da radiação , Nucleossomos/metabolismo , Adenosina Trifosfatases/genética , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Escuridão , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Histonas/genética , Mutação/genética , Transcriptoma/genéticaRESUMO
As the largest group of MYB family transcription factors, R2R3-MYB proteins play essential roles during plant growth and development. However, the structural basis underlying how R2R3-MYBs recognize the target DNA remains elusive. Here, we report the crystal structure of Arabidopsis WEREWOLF (WER), an R2R3-MYB protein, in complex with its target DNA. Structural analysis showed that the third α-helices in both the R2 and R3 repeats of WER fit in the major groove of the DNA, specifically recognizing the DNA motif 5'-AACNGC-3'. In combination with mutagenesis, in vitro binding and in vivo luciferase assays, we showed that K55, N106, K109 and N110 are critical for the function of WER. Although L59 of WER is not involved in DNA binding in the structure, ITC analysis suggested that L59 plays an important role in sensing DNA methylation at the fifth position of cytosine (5mC). Like 5mC, methylation at the sixth position of adenine (6mA) in the AAC element also inhibits the interaction between WER and its target DNA. Our study not only unravels the molecular basis of how WER recognizes its target DNA, but also suggests that 5mC and 6mA modifications may block the interaction between R2R3-MYB transcription factors and their target genes.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/genética , DNA de Plantas/química , Proteínas de Ligação a DNA/química , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/genética , Sequência de Aminoácidos , Animais , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Galinhas/genética , Galinhas/metabolismo , Sequência Conservada , Cristalografia por Raios X , Metilação de DNA , DNA de Plantas/genética , DNA de Plantas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mamíferos , Modelos Moleculares , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Histones are highly basic proteins involved in packaging DNA into chromatin, and histone modifications are fundamental in epigenetic regulation in eukaryotes. Among the numerous chromatin modifiers identified in Arabidopsis (Arabidopsis thaliana), MORF-RELATED GENE (MRG)1 and MRG2 have redundant functions in reading histone H3 lysine 36 trimethylation (H3K36me3). Here, we show that MRG2 binds histone chaperones belonging to the NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1) family, including NAP1-RELATED PROTEIN (NRP)1 and NRP2. Characterization of the loss-of-function mutants mrg1 mrg2, nrp1 nrp2 and mrg1 mrg2 nrp1 nrp2 revealed that MRG1/MRG2 and NRP1/NRP2 regulate flowering time through fine-tuning transcription of floral genes by distinct molecular mechanisms. In particular, the physical interaction between NRP1/NRP2 and MRG1/MRG2 inhibited the binding of MRG1/MRG2 to the transcription factor CONSTANS (CO), leading to a transcriptional repression of FLOWERING LOCUS T (FT) through impeded H4K5 acetylation (H4K5ac) within the FT chromatin. By contrast, NRP1/NRP2 and MRG1/MRG2 act together, likely in a multiprotein complex manner, in promoting the transcription of FLOWERING LOCUS C (FLC) via an increase of both H4K5ac and H3K9ac in the FLC chromatin. Because the expression pattern of FLC represents the major category of differentially expressed genes identified by genome-wide RNA-sequencing analysis in the mrg1 mrg2, nrp1 nrp2 and mrg1 mrg2 nrp1 nrp2 mutants, it is reasonable to speculate that the NRP1/NRP2-MRG1/MRG2 complex may be involved in transcriptional activation of genes beyond FLC and flowering time control.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Proteínas Cromossômicas não Histona/fisiologia , Flores/crescimento & desenvolvimento , Chaperonas de Histonas/fisiologia , Chaperonas Moleculares/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Flores/metabolismo , Flores/fisiologia , Genes de Plantas/genética , Genes de Plantas/fisiologia , Estudo de Associação Genômica Ampla , Chaperonas de Histonas/metabolismo , Código das Histonas , Chaperonas Moleculares/metabolismoRESUMO
Chromatin modifications play important roles in plant adaptation to abiotic stresses, but the precise function of histone H3 lysine 36 (H3K36) methylation in drought tolerance remains poorly evaluated. Here, we report that SDG708, a specific H3K36 methyltransferase, functions as a positive regulator of drought tolerance in rice. SDG708 promoted abscisic acid (ABA) biosynthesis by directly targeting and activating the crucial ABA biosynthesis genes NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (OsNCED3) and NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 5 (OsNCED5). Additionally, SDG708 induced hydrogen peroxide accumulation in the guard cells and promoted stomatal closure to reduce water loss. Overexpression of SDG708 concomitantly enhanced rice drought tolerance and increased grain yield under normal and drought stress conditions. Thus, SDG708 is potentially useful as an epigenetic regulator in breeding for grain yield improvement.
Assuntos
Oryza , Ácido Abscísico , Secas , Regulação da Expressão Gênica de Plantas , Histona Metiltransferases , Histonas , Metiltransferases/genética , Oryza/genética , Oryza/metabolismo , Melhoramento Vegetal , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Estresse Fisiológico/genéticaRESUMO
ß0/ß0 thalassemia is the most severe type of transfusion-dependent ß-thalassemia (TDT) and is still a challenge facing lentiviral gene therapy. Here, we report the interim analysis of a single-center, single-arm pilot trial (NCT05015920) evaluating the safety and efficacy of a ß-globin expression-optimized and insulator-engineered lentivirus-modified cell product (BD211) in ß0/ß0 TDT. Two female children were enrolled, infused with BD211, and followed up for an average of 25.5 months. Engraftment of genetically modified hematopoietic stem and progenitor cells was successful and sustained in both patients. No unexpected safety issues occurred during conditioning or after infusion. Both patients achieved transfusion independence for over 22 months. The treatment extended the lifespan of red blood cells by over 42 days. Single-cell DNA/RNA-sequencing analysis of the dynamic changes of gene-modified cells, transgene expression, and oncogene activation showed no notable adverse effects. Optimized lentiviral gene therapy may safely and effectively treat all ß-thalassemia.
Assuntos
Terapia Genética , Lentivirus , Globinas beta , Talassemia beta , Humanos , Talassemia beta/terapia , Talassemia beta/genética , Projetos Piloto , Feminino , Lentivirus/genética , Globinas beta/genética , Criança , Transfusão de Sangue , Pré-EscolarRESUMO
While the yeast Chz1 acts as a specific histone-chaperone for H2A.Z, functions of CHZ-domain proteins in multicellular eukaryotes remain obscure. Here, we report on the functional characterization of OsChz1, a sole CHZ-domain protein identified in rice. OsChz1 interacts with both the canonical H2A-H2B dimer and the variant H2A.Z-H2B dimer. Within crystal structure the C-terminal region of OsChz1 binds H2A-H2B via an acidic region, pointing to a previously unknown recognition mechanism. Knockout of OsChz1 leads to multiple plant developmental defects. At genome-wide level, loss of OsChz1 causes mis-regulations of thousands of genes and broad alterations of nucleosome occupancy as well as reductions of H2A.Z-enrichment. While OsChz1 associates with chromatin regions enriched of repressive histone marks (H3K27me3 and H3K4me2), its loss does not affect the genome landscape of DNA methylation. Taken together, it is emerging that OsChz1 functions as an important H2A/H2A.Z-H2B chaperone in dynamic regulation of chromatin for higher eukaryote development.
Assuntos
Cromatina/metabolismo , Histonas/metabolismo , Oryza/genética , Proteínas de Plantas/metabolismo , Sistemas CRISPR-Cas , Cromatina/genética , Metilação de DNA , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Histonas/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Nucleossomos/genética , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Multimerização ProteicaRESUMO
BACKGROUND: In animals, H3K4me2 and H3K4me3 are enriched at the transcription start site (TSS) and function as epigenetic marks that regulate gene transcription, but their functions in plants have not been fully characterized. RESULTS: We used chromatin immunoprecipitation sequencing to analyze the rice genome-wide changes to H3K4me1/H3K4me2/H3K4me3 following the loss of an H3K4-specific methyltransferase, SDG701. The knockdown of SDG701 resulted in a global decrease in H3K4me2/H3K4me3 levels throughout the rice genome. An RNA-sequencing analysis revealed that many genes related to diverse developmental processes were misregulated in the SDG701 knockdown mutant. In rice, H3K4me3 and H3K36me3 are positively correlated with gene transcription; however, surprisingly, the H3K4me2 level was negatively associated with gene transcription levels. Furthermore, the H3K4me3 level at the TSS region decreased significantly in the genes that exhibited down-regulated expression in the SDG701 knockdown mutant. In contrast, the genes with up-regulated expression in the mutant were associated with a considerable decrease in H3K4me2 levels over the gene body region. CONCLUSION: A comparison of the genome-wide distributions of H3K4me2 in eukaryotes indicated that the H3K4me2 level is not correlated with the gene transcription level in yeast, but is positively and negatively correlated with gene expression in animals and plants, respectively. Our results uncovered H3K4me2 as a novel repressive mark in plants.