Comment[ArrayExpressAccession] E-GEOD-14600 Public Release Date 2009-04-20 Investigation Title Target genes of the MADS transcription factor SEPALLATA3, ChIP-seq Comment[Submitted Name] Target genes of the MADS transcription factor SEPALLATA3, ChIP-seq Experiment 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. Date of Experiment Term Source Name EFO Term Source Version Term Source File http://www.ebi.ac.uk/efo/efo.owl Person Last Name Angenent Kaufmann Muino Jauregui Airoldi Smaczniak Krajewski Angenent Person First Name Gerco Kerstin Jose Ruy Chiara Cezary Pawel Gerco Person Mid Initials M Person Email gerco.angenent@wur.nl Person Affiliation Plant Research International Person Phone Person Fax Person Address Plant Research International, Bornsesteeg 65, Wageningen, Netherlands Person Roles submitter Person Roles Term Source REF Person Roles Term Accession Number Normalization Type Normalization Term Accession Number Normalization Term Source REF Replicate Type Replicate Term Accession Number Replicate Term Source REF Experimental Design Experimental Design Term Accession Number Experimental Design Term Source REF Quality Control Type Quality Control Term Accession Number Quality Control Term Source REF Protocol Name P-GSE14600-1 P-GSE14600-3 P-GSE14600-2 P-GSE14600-5 P-GSE14600-4 Protocol Description Inflorescences were harvested and fixed using FAA following a protocol published in Gomez-Mena et al. 2005 (PMID: 15634696) with modifications in de Folter et al. 2007 (PMID: 17868439) ChIP experiments were performed as described in Gomez-Mena et al. 2005 (PMID: 15634696) with modifications in de Folter et al. 2007 (PMID: 17868439) Arabidopsis thaliana, wild-type (Col-0), sep3-1 (Col-0) and ag-1 (Ler) mutant plants were grown under standard greenhouse conditions (20°C, long day light regime: 16 h light, 8 hours dark cycle). Flower material was harvested from primary and secondary inflorescences of 5-7 weeks old plants Reads were mapped to the unmasked Arabidopsis reference genome (ATH1.1con.01222004; ftp://ftp.arabidopsis.org/) using the SOAP software.Only uniquely mapped reads were retained. Reads were extended directionally to 300 nt. For each nucleotide position enrichment was tested using a Poisson model. Reads were mapped to the unmasked Arabidopsis reference genome (ATH1.1con.01222004; ftp://ftp.arabidopsis.org/) using the SOAP software.Only uniquely mapped reads were retained. Reads were extended directionally to 300 nt. For each nucleotide position enrichment was tested using a Poisson model. We have processed SEP3_ChIPSeq_wt_1 and SEP3_ChIPSeq_wt_2 sample records together on the file wkk1b_aligned.txt Protocol Software Protocol Hardware Protocol Contact Protocol Type specified_biomaterial_action nucleic_acid_extraction grow feature_extraction feature_extraction Protocol Term Source REF Protocol Term Accession Number Experimental Factor Name VARIABLE Experimental Factor Type variable Experimental Factor Term Source REF Experimental Factor Term Accession Number Publication Title Target genes of the MADS transcription factor SEPALLATA3: integration of developmental and hormonal pathways in the Arabidopsis flower. Publication Author List Kaufmann K, Muiño JM, Jauregui R, Airoldi CA, Smaczniak C, Krajewski P, Angenent GC PubMed ID 19385720 Publication DOI 10.1371/journal.pbio.1000090 Publication Status Publication Status Term Source REF Publication Status Term Accession Number Comment[SecondaryAccession] GSE14600 SRP000783 Comment[GEOLastUpdateDate] 2012-05-08 Comment[AEExperimentType] ChIP-seq Comment[GEOReleaseDate] 2009-04-19 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR016810-SRR016814 Comment[ArrayExpressSubmissionDate] 2009-01-27 SDRF File E-GEOD-14600.sdrf.txt