Project description:Characterization of the activities of the transcription factor that AG encodes throughout flower development using perturbation assays and ChIP-Seq in combination with a floral induction system (FIS) that allows a stage-specific analysis of flower development. Examination of genomic regions bound by fully functional AG-GFP protein at approx floral stage 4-5 as compared to a negative control sample.
Project description:Characterization of the activities of the transcription factors that AP3 and PI encode throughout flower development using perturbation and ChIPSeq assays in combination with a floral induction system (FIS) that allows a stage-specific analysis of flower development. Examination of genomic regions bound by fully functional AP3-GFP and PI-GFP proteins at approx floral stage 4-5 as compared to a negative control sample
Project description:The emerging picture of transcriptional regulation is one of unexpected complexity. It is now clear that single transcription factors control hundreds, if not thousands, of direct targets by binding their genomic loci, but it is not understood how many of these are major players and how many are supporting cast. To address this, we leverage a well-characterized developmental network in Arabidopsis and map genome-wide binding of related proteins in multiple tissues. The transcription factor APETALA2 (AP2) has numerous functions, including roles in floral organ identity, seed development and stem cell maintenance. We focus on the role of AP2 in the floral transition and map direct targets on a genome-wide scale. We show that ap2 mutants flower early in long and short days, and that AP2 binds to many loci, most prominently floral pathway integrators, microRNAs and floral organ identity genes, many of which exhibit AP2-dependent transcription. Opposing, logical effects are evident in AP2 binding to two developmental microRNA genes that control AP2 expression, with AP2 positively regulating miR156 and negatively regulating miR172, forming a complex direct feedback loop, which also included all but one of the AP2-like miR172 target clade members. We also seek conserved targets by comparing the genome-wide direct target repertoire of AP2 with that of SCHLAFMÜTZE (SMZ), another member of the AP2-like miR172 target clade that shares partial redundancy, as evidenced by a hexuple mutant for the entire clade that flowered extremely early. Clear similarities and divergence are exposed in the AP2 and SMZ direct target repertoires. Finally, using an inducible expression system, we demonstrate that AP2 has dual molecular roles. It functions both as a transcriptional activator and repressor, directly inducing the expression of the floral repressor AGAMOUS-LIKE 15 (AGL15), and directly repressing the transcription of floral activators like SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1). ChIP-Seq of two biological replicates for ATH-AP2 and respective control samples
Project description:This SuperSeries is composed of the following subset Series: GSE38358: Molecular basis for the specification of floral organs by APETALA3 and PISTILLATA (ChIP-Seq) GSE38362: Molecular basis for the specification of floral organs by APETALA3 and PISTILLATA (mRNA) Refer to individual Series
Project description:Despite great advances in sequencing capacity, generating functional information for non-model organisms remains a challenge. One solution lies in an improved ability to predict genetic circuits based on primary DNA sequence combined with the characterization of regulatory molecules from model species. Here, we focus on the LEAFY (LFY) transcription factor, a conserved master regulator of floral development. Starting with biochemical and structural information, we built a biophysical model describing LFY DNA binding specificity in vitro that accurately predicts in vivo LFY binding sites in the Arabidopsis thaliana genome. Extending the model to other species, we show that it can correctly identify functional homologs of known LFY targets from Arabidopsis thaliana in other angiosperms, even if a functional shift between orthologs and paralogs has occurred. Moreover, this model demonstrates the evolutionary fluidity of the link between LFY and one of its target genes, underlining how this regulatory interaction can be conserved despite changes in position, sequence and affinity of the LFY binding sites. Our study shows that the cis-element fluidity recently illustrated in animals also exists in plants, and that it can be detected without any experimental work in each individual species, using a biophysical transcription factor model. A. thaliana LEAFY ChIP-seq w control, 2 replicates
Project description:Transcription profiling was performed of second branchial arches of E11.5 embryos from Hoxa2+/- intercrosses. After genotyping the embryos, wild type and Hoxa2-/- were profiled by microarray.
Project description:The molecular mechanisms by which floral homeotic genes act as major developmental switches to specify the identity of floral organs, are still largely unknown. Floral homeotic genes encode transcription factors of the MADS-box family, which are supposed to assemble in a combinatorial fashion into organ-specific multimeric protein complexes. Major mediators of protein interactions are MADS-domain proteins of the SEPALLATA subfamily, which play a crucial role in the development of all types of floral organs. In order to characterize the roles of the SEPALLATA3 transcription factor complexes at the molecular level, we analyzed genome-wide the direct targets of SEPALLATA3. We used chromatin immunoprecipitation followed by ultrahigh-throughput sequencing or hybridization to whole-genome tiling arrays to obtain genome-wide DNA-binding patterns of SEPALLATA3. The results demonstrate that SEPALLATA3 binds to thousands of sites in the genome. Most potential target sites that were strongly bound in wild-type inflorescences, are also bound in the floral homeotic agamous mutant, which displays only the perianth organs, sepals and petals. Characterization of the target genes shows that SEPALLATA3 integrates and modulates different growth-related and hormonal pathways in a combinatorial fashion with other MADS-box proteins and possibly with non-MADS transcription factors. In particular, the results suggest multiple links between SEPALLATA3 and auxin signaling pathways. Our gene expression analyses link the genomic binding site data with the phenotype of plants expressing a dominant repressor version of SEPALLATA3, suggesting that it modulates auxin response to facilitate floral organ outgrowth and morphogenesis. Furthermore, the binding of the SEPALLATA3 protein to cis-regulatory elements of other MADS-box genes and expression analyses reveal that this protein is a key component in the regulatory transcriptional network underlying the formation of floral organs. ChIP experiments were performed on Arabidopsis wildtype and agamous mutant inflorescences using an antibody raised against a C-terminal peptide of SEP3. As control, ChIP experiments were performed on the sep3 mutant.
Project description:Initiation of mineralisation during endochondral ossification is a multistep process and was assumed to correlate with specific interactions of annexins and collagens. Annexins A5 and A6 are postulated to represent the essential annexins promoting cartilage mineralisation. However, skeletal development appears to be normal in annexin A5 or A6 deficient mice. The highly conserved structures of annexins led to the assumption that annexins A5 and A6 may fulfill redundant functions. We now generated mice deficient for both proteins, annexins A5 and A6. Mice were viable, fertile and showed no obvious abnormalities. Assessment of skeletal elements using histological, ultrastructural and peripheral quantitative computed tomography methods revealed that mineralisation and development of the skeleton was not significantly affected in mutant mice. In respect of the lack of an obvious phenotype we now applied microarray analysis to the growth plate to define changes in the transcriptome of juvenile murine growth plates from mutant mice. Global gene expression analysis revealed subtle phenotypes at the transcriptome level of genes involved in cell growth and intermediate metabolism in mutant mice. These data demonstrate that both annexins are dispensable for proper cartilage mineralisation but may affect cell proliferation processes at the transcriptomic level.