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ChIP-chip analysis of GBF1 in the presence and absence of its heterodimerizing partners, HY5 and HYH

ABSTRACT: Transcription factors are key components of light signalling as they amplify the signal, which results in massive changes in genome-wide expression during photomorphogenesis. Three bZIP transcription factors (TFs), namely GBF1, HY5, and HYH, form heterodimers with each other and regulate photomorphogenesis in an interdependent manner. GBF1 acts as both a positive and negative regulator of photomorphogenesis, whereas HY5 and HYH mainly act as positive regulators of photomorphogenesis. In this study, the impact of heterodimerization of GBF1 with HY5 and HYH was analyzed for genome-wide binding of GBF1 through ChIP-chip in GBF1. We identified more than 2000 direct targets of GBF1 in the presence of HY5 and HYH. However, in the absence of HY5, very few binding sites were found, and in the absence of functional HYH protein, the number of GBF1 direct targets reduced to only half compared to when functional HYH was present.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE36965 | GEO | 2014/03/24

SECONDARY ACCESSION(S): PRJNA157299

REPOSITORIES: GEO

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ChIP-chip analysis of GBF1 in the presence and absence of its heterodimerizing partners, HY5 and HYH

Project description:Transcription factors are key components of light signalling as they amplify the signal, which results in massive changes in genome-wide expression during photomorphogenesis. Three bZIP transcription factors (TFs), namely GBF1, HY5, and HYH, form heterodimers with each other and regulate photomorphogenesis in an interdependent manner. GBF1 acts as both a positive and negative regulator of photomorphogenesis, whereas HY5 and HYH mainly act as positive regulators of photomorphogenesis. In this study, the impact of heterodimerization of GBF1 with HY5 and HYH was analyzed for genome-wide binding of GBF1 through ChIP-chip in GBF1. We identified more than 2000 direct targets of GBF1 in the presence of HY5 and HYH. However, in the absence of HY5, very few binding sites were found, and in the absence of functional HYH protein, the number of GBF1 direct targets reduced to only half compared to when functional HYH was present. ChIPed DNA with GBF1 antibody from GBF1OE, hy5 GBF1OE, and hyh GBF1OE lines vs. ChIPed DNA with GBF1 antibody from wild-type.

2014-03-24 | E-GEOD-36965 | biostudies-arrayexpress

Expression data from gbf1, gbf1 hy5, and gbf1 hyh mutants

2016-04-01 | GSE36964 | GEO

Expression data from gbf1, gbf1 hy5, and gbf1 hyh mutants

Project description:Three bZIP transcription factors (TFs), namely GBF1, HY5, and HYH, form heterodimers with each other and regulate photomorphogenesis in an interdependent manner. GBF1 acts as both a positive and negative regulator of photomorphogenesis, whereas HY5 and HYH mainly act as positive regulators of photomorphogenesis. To study the effect of HY5 and HYH on GBF1-mediated genome-wide expression, transcript profiling was performed in the gbf1 single mutant and gbf1hy5 and gbf1hyh double mutants. Our study revealed that GBF1 mainly acts antagonistic to HY5 and HYH for genome-wide expression. Four-day-old constant white-light grown Arabidipsis seedlings of wild-type (Col-0), gbf1 mutant, gbf1 hy5 double mutant and gbf1 hyh double mutant were harvested for total RNA extraction and Affymetrix microarray hybridisation. Two biological replicates of each sample were used for hybridisation and data analysis. Data were analysed using GeneSpring software by utilising the RMA algorithm. To draw the conclusions from this study, probe level intensity of gbf1, gbf1 hy5, and gbf1 hyh was normalised against the probe level intensity of wild-type, and also the probe level intensity of gbf1 hy5 and gbf1 hyh double mutants was normalised against the probe level intensity of the gbf1 single mutant.

2016-04-01 | E-GEOD-36964 | biostudies-arrayexpress

ELONGATED HYPOCOTYL (HY5) AND HY5 HOMOLOG (HYH) maintain shade avoidance suppression in sunlight

Project description:We show that longer-term inhibition of shade avoidance in Arabidopsis is sustained by ELONGATED HYPOCOTYL 5 (HY5) and HY5 HOMOLOG (HYH) which, together, regulate transcriptional reprogramming of genes involved in hormone signalling and cell wall modification.

2023-06-07 | GSE192469 | GEO

Analysis of transcription factor HY5 genomic binding sites in Arabidopsis

Project description:The transcription factor HY5 acts downstream of multiple families of the photoreceptors and promotes photomorphogenesis. Although it is well accepted that HY5 acts to regulate target gene expression, in vivo binding of HY5 to any of its target gene promoters has yet to be demonstrated. Here we used a chromatin immunoprecipitation procedure to verify suspected in vivo HY5 binding sites. We demonstrated that in vivo association of HY5 with promoter targets is not altered under distinct light qualities or during light-to-dark transition. Coupled with DNA chip hybridization using high density 60-nucleotide oligomer microarray that contains one probe for every 500 nucleotides over the entire Arabidopsis genome, we mapped genome wide in vivo HY5 binding sites. This analysis showed that HY5 binds preferentially to promoter regions in vivo and revealed over 3 thousand chromosomal sites as putative HY5 binding targets. HY5 binding targets tend to be enriched in the early light responsive genes and transcription factor genes. Our data thus supports a model in which HY5 is a high hierarchical regulator of the transcriptional cascades for photomorphogenesis. Keywords: ChIP-chip

2007-03-01 | GSE6510 | GEO

Transcription profiling of hy5 single mutant, hyh single mutant, hy5 hyh double mutant and wild type Arabidopsis seedlings of two different ecotypes and with or without auxin treatment

Project description:Arabidopsis thaliana seedlings comprising hy5 single mutant , hyh single mutant, hy5 hyh double mutant and wild-type plants were germinated in the dark on agar medium on usual Murashig and Skoog medium (pH 5.8) without sucrose. After 3 days of germination, etiolated seedlings were transfered to light conditions in liquid media similar to previous one but without agar . After 5 hours, auxin (10 micromolar IAA) was added to all treated samples except to controls (Mock treatment). all samples were harvested after one-hour treatment and frozen for further RNA extraction. Aim of the project was to detect impact of exogenous auxin treatment on each genotype

2006-11-08 | E-MEXP-715 | biostudies-arrayexpress

ELONGATED HYPOCOTYL (HY5) AND HY5 HOMOLOG (HYH) maintain shade avoidance suppression in sunlight

Project description:ELONGATED HYPOCOTYL (HY5) AND HY5 HOMOLOG (HYH) maintain shade avoidance suppression in sunlight

| PRJNA791625 | ENA

Co-condensation with photoexcited Cryptochromes facilitates MOS4-associated complex subunits 3A/3B to antagonize HY5 activities during photomorphogenesis in Arabidopsis

Project description:Cryptochromes (CRYs) is known as the key blue light receptors that promote photomorphogenesis in Arabidopsis, but to date, the underlying mechanisms are still not fully understood. Through interrogating the CRY2 interactome, we identified MOS4-ASSOCIATED COMPLEX subunits 3A and 3B (MAC3A and MAC3B) as blue light-independent CRY2 interacting partners. MAC3A/B proteins could be assembled into liquid nuclear condensates of CRYs in a blue light-dependent manner. Hypocotyl elongation is markedly repressed in mac3ab double knock-out mutants under various light conditions, which uncovers a previously unknown role of MAC3A/B as negative regulators in plant photomorphogenesis. Our results also uncover the noncanonical activities of MAC3A as the DNA-binding proteins that regulate transcription. Genome-wide mapping of MAC3A-binding sites reveals that blue light facilitates the binding of MAC3A to its targets, which is weakened in cry1cry2 mutants, suggesting that CRYs may enhance MAC3A activities in blue light to negatively influence photomorphogenesis. Interestingly, we observe that the genomic binding sites of MAC3A and HY5 are largely overlapped, and physical interactions between MAC3A and HY5 are detected as well. In addition, the in vitro DNA-binding assay shows that both proteins compete for the same DNA probe. These results indicate that MAC3A may antagonize the function of HY5 by competing for the common binding sites across the genome. Taken together, we propose that cryptochromes may fine-tune Arabidopsis photomorphogenesis by balancing the positive and negative effects on HY5 activities.

2023-08-21 | GSE220945 | GEO

Photoregulatory Protein Kinases Fine-tune Plant Photomorphogenesis by Directing a Bifunctional Phospho-code on HY5

Project description:The core regulatory mechanisms affecting the function of ELONGATED HYPOCOTYL 5 (HY5) during plant photomorphogenesis include dynamics in the transcriptional activity and protein stability of HY5 in response to changes in ambient light; however, little is known about the mediators of these processes. Here, we identified PHOTOREGULATORY PROTEIN KINASE 1 (PPK1), which interacts directly with and phosphorylates HY5, as one such mediator. PPK1 and its homologs play redundant roles in the negative regulation of photomorphogenesis. PPK1 phosphorylates HY5 at Thr55, Ser56, and Ser60. The phosphorylation of these residues is essential to establish high-affinity binding with B-BOX PROTEIN 24 and CONSTITUTIVE PHOTOMORPHOGENIC 1, which inhibit the transcriptional activity and promote the ubiquitination and degradation of HY5, respectively. As such, PPKs regulate not only the binding of HY5 to its target genes under light conditions,but also HY5 degradation when plants are transferred from light to dark conditions. Our data reveal a PPK-mediated phospho-code on HY5 that integrates the molecular mechanisms underlying the regulation of HY5 transcriptional activity and protein stability to precisely control plant photomorphogenesis.

2024-02-07 | PXD049266 |

DET1-mediated COP1 regulation avoids HY5 activity over second-site targets to tune plant photomorphogenesis

Project description:DE-ETIOLATED1 (DET1) is a negative regulator of plant photomorphogenesis acting as a component of the C3D complex, which can further associate to CULLIN4 to form a CRL4C3D E3 ubiquitin ligase. CRL4C3D is thought to act together with CRL4COP1SPA ubiquitin ligase, to promote the ubiquitin-mediated degradation of the master regulatory transcription factor ELONGATED HYPOCOTYL5 (HY5), thereby controlling photomorphogenic gene regulatory networks. Yet, functional links between COP1 and DET1 have long remained elusive. Here, upon mass spectrometry identification of DET1 and COP1-associated proteins, we provide in vivo evidence that DET1 associates with COP1 to promote its destabilization, a process necessary to dampen HY5 protein abundance. By regulating HY5 over-accumulation, DET1 is critical to avoid its association to second-site loci, including many PIF3 target genes. Accordingly, excessive HY5 levels result in an increased HY5 repressive activity and are sufficient to trigger fusca-like phenotypes otherwise observed typically in COP1 and COP9 signalosome mutant seedlings. This study therefore identifies that DET1-mediated regulation of COP1 stability tunes down HY5 cistrome and avoids hyper-photomorphogenic responses that might compromise plant viability.

2021-09-09 | GSE155147 | GEO

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