Project description:Nuclear Factor Y (NF-Y) is a heterotrimeric transcription factor that binds CCAAT elements. The NF-Y trimer is composed of a Histone Fold Domain (HFD) dimer (NF-YB/NF-YC) and NF-YA, which confers DNA sequence specificity. NF-YA shares a conserved domain with the CONSTANS, CONSTANS-LIKE, TOC1 (CCT) proteins. We show that CONSTANS (CO/B-BOX PROTEIN1 BBX1), a master flowering regulator, forms a trimer with Arabidopsis thaliana NF-YB2/NF-YC3 to efficiently bind the CORE element of the FLOWERING LOCUS T promoter. Using saturation mutagenesis, electrophoretic mobility shift assays, and RNA-sequencing profiling of co, nf-yb, and nf-yc mutants, we identify CCACA elements as the core NF-CO binding site. CO physically interacts with the same HFD surface required for NF-YA association, as determined by mutations in NF-YB2 and NF-YC9, and tested in vitro and in vivo. The co-7 mutation in the CCT domain, corresponding to an NF-YA arginine directly involved in CCAAT recognition, abolishes NF-CO binding to DNA
Project description:Five members of the Arabidopsis thaliana NF-YA gene family are strongly induced by several stress conditions via transcriptional and miR169-related posttranscriptional mechanisms. These transcription factors participate in gene regulation via two different mechanisms, one depending on binding to the CCAAT-box in the promoter of regulated genes and the other, independent of the CCAAT-box, in which NF-YA prevents the interaction of the NF-YB/YC heterodimer with transcription factors.
Project description:Cell type-specific master transcription factors (MTFs) play vital roles in defining cell identity and function. However, the roles ubiquitous factors play in the specification of cell identity remain underappreciated. Here we show that all three subunits of the ubiquitous heterotrimeric CCAAT-binding NF-Y complex are required for the maintenance of embryonic stem cell (ESC) identity, and establish NF-Y as a novel component of the core pluripotency network. Genome-wide occupancy and transcriptomic analyses in ESCs and neurons reveal that not only does NF-Y regulate genes with housekeeping functions through cell type-invariant promoter-proximal binding, but also genes required for cell identity by binding to cell type-specific enhancers with MTFs. Mechanistically, NF-Y's distinctive DNA-binding mode promotes MTF binding at enhancers by facilitating a permissive chromatin conformation. Our studies unearth a novel function for NF-Y in promoting chromatin accessibility, and suggest that other proteins with analogous structural and DNA-binding properties may function in similar ways. Genome-wide mapping of NF-YA, NF-YB, and NF-YC subunits of the NF-Y complex in mouse ESCs, and microarray gene expression profiling of control knockdown (KD), NF-YA KD, NF-YB KD, NF-YC KD, and NF-YA/NF-YB/NF-YC triple KD ESCs.
Project description:Cell type-specific master transcription factors (MTFs) play vital roles in defining cell identity and function. However, the roles ubiquitous factors play in the specification of cell identity remain underappreciated. Here we show that all three subunits of the ubiquitous heterotrimeric CCAAT-binding NF-Y complex are required for the maintenance of embryonic stem cell (ESC) identity, and establish NF-Y as a novel component of the core pluripotency network. Genome-wide occupancy and transcriptomic analyses in ESCs and neurons reveal that not only does NF-Y regulate genes with housekeeping functions through cell type-invariant promoter-proximal binding, but also genes required for cell identity by binding to cell type-specific enhancers with MTFs. Mechanistically, NF-Y's distinctive DNA-binding mode promotes MTF binding at enhancers by facilitating a permissive chromatin conformation. Our studies unearth a novel function for NF-Y in promoting chromatin accessibility, and suggest that other proteins with analogous structural and DNA-binding properties may function in similar ways. Genome-wide mapping of NF-YA, NF-YB, and NF-YC subunits of the NF-Y complex in mouse ESCs, and microarray gene expression profiling of control knockdown (KD), NF-YA KD, NF-YB KD, NF-YC KD, and NF-YA/NF-YB/NF-YC triple KD ESCs.
Project description:Investigation of the binding behaviour of Sp1, Sp2, Sp3 and NF-ya, NF-yb and NF-yc in mouse embryonic fibroblasts and of Sp1, Sp2 and Sp3 in HEK-293 cells reveals distinct binding of the seemingly similar transcription factors Sp1/3 and Sp2.
Project description:Five members of the Arabidopsis thaliana NF-YA gene family are strongly induced by several stress conditions via transcriptional and miR169-related posttranscriptional mechanisms. These transcription factors participate in gene regulation via two different mechanisms, one depending on binding to the CCAAT-box in the promoter of regulated genes and the other, independent of the CCAAT-box, in which NF-YA prevents the interaction of the NF-YB/YC heterodimer with transcription factors. Three biological and two technical (in swap) replicates for each genotype were obtained for each treatment (DMSO (mock) and estradiol 24h after induction). Mock samples were pooled and used as a reference.
Project description:NF-Y, a trimeric transcription factor (TF) composed of two histone-like subunits (NF-YB (NFYB) and NF-YC (NFYC)) and a sequence-specific subunit (NF-YA), binds to the CCAAT motif, a common promoter element. Genome-wide mapping reveals 5,000-15,000 NF-Y binding sites depending on the cell type, with the NF-YA and NF-YB subunits binding asymmetrically with respect to the CCAAT motif. Despite being characterized as a proximal promoter TF, only 25% of NF-Y sites map to promoters. A comparable number of NF-Y sites are located at enhancers, many of which are tissue specific, and nearly half of NF-Y sites are in select subclasses of HERV LTR repeats. Unlike most TFs, NF-Y can access its target DNA motif in inactive (non-modified) or polycomb-repressed chromatin domains. Unexpectedly, NF-Y extensively co-localizes with FOS in all genomic contexts, and at promoters and enhancers this often occurs in the absence of JUN and the AP-1 motif. NF-Y also co-associates with a select cluster of growth-controlling and oncogenic TFs, consistent with the abundance of CCAAT motifs in the promoters of genes overexpressed in cancer. Interestingly, NF-Y and several growth-controlling TFs bind in a stereo-specific manner, suggesting a mechanism for cooperative action at promoters and enhancers. Our results indicate that NF-Y is not merely a commonly-used, proximal promoter TF, but rather performs a more diverse set of biological functions, many of which are likely to involve co-association with FOS. Scrambled control (shSCM) and NF-YA pLKO.1-shRNAs were designed by Sigma-Aldrich. The puromycin resistance cassette was replaced with an EGFP cassette. Viral production and transduction were carried out as previously described (Benatti et al. 2011). HeLaS3 cells were transduced with shSCM or shNF-YA viral supernatants, in triplicate, and cells collected after 48 hr of incubation. Total RNA was prepared by Trizol extraction and Qiagen RNeasy kit purification, converted to biotinylated aRNA and hybridized to U133 Plus 2.0 GeneChip expression arrays using the 3’ IVT Express Kit (Affymetrix, USA) following the manufacturer’s protocol. Arrays were RMA normalized (Irizarry et al. 2003), gene expression levels calculated, differential expression determined using the following R packages from the Bioconductor project: affy (Gautier et al. 2004), limma (Smyth 2004). HelaS3 cells were transduced with shSCM or shNF-YA in triplicate for 48hrs.
Project description:We generated 18 Gb of high-quality sequencing data (~3 Gb per sample) and catalogued the expression profiles of 27,416 annotated Arabidopsis thaliana genes in each sample. The analysis showed differences of transcriptomes between wild-type Col and Nuclear Factor Y (NF-Y) C1,3,4,9 loss of function mutant nf-yc quadruple (nf-ycQ) seedlings treated with 2 days red light. We identified numerous differentially expressed genes that exhibited distinct expression patterns. These genes have known or potential roles in growth and development of Arabidopsis thaliana. Therefore, they are appealing candidates for further investigation of the gene expression and associated regulatory mechanisms related to NF-YCs in light signaling pathway.
Project description:To understand how the NF-YC-RGL2 complex functions in repressing seed germination, a genome-wide transcriptomic analysis was carried out using germinating seeds of rgl2, nf-ycT and the wild-type (Col) grown on the medium containing 5 µM PAC and Col grown on mock treatment. Basing on the criteria of 1.5-fold cutoff for the genes with 5% false discovery rate, we first identified the differentially expressed genes in Col_PAC vs Col_mock, nf-ycT_PAC vs Col_PAC, and rgl2_PAC vs Col_PAC subsets, which are referred to as PAC-, NF-YC-, and RGL2-regulated genes.These data reveal that NF-YCs and RGL2 co-target a set of common genes in response to phytohormone signals, strongly supporting the role of NF-YC-RGL2 in seed germination regulation.