Tagging of MADS domain proteins for chromatin immunoprecipitation

<p>Abstract</p> <p>Background</p> <p>Most transcription factors fulfill their role in complexes and regulate their target genes upon binding to DNA motifs located in upstream regions or introns. To date, knowledge about transcription factor target genes and their corresponding transcription factor binding sites are still very limited. Two related methods that allow <it>in vivo </it>identification of transcription factor binding sites are chromatin immunoprecipitation (ChIP) and chromatin affinity purification (ChAP). For ChAP, the protein of interest is tagged with a peptide or protein, which can be used for affinity purification of the protein-DNA complex and hence, the identification of the target gene.</p> <p>Results</p> <p>Here, we present the results of experiments aiming at the development of a generic tagging approach for the Arabidopsis MADS domain proteins AGAMOUS, SEPALLATA3, and FRUITFULL. For this, Arabidopsis wild type plants were transformed with constructs containing a MADS-box gene fused to either a double <it>Strep</it>-tag^®II-FLAG-tag, a triple HA-tag, or an eGFP-tag, all under the control of the constitutive double 35S Cauliflower Mosaic Virus (CaMV) promoter. Strikingly, in all cases, the number of transformants with loss-of-function phenotypes was much larger than those with an overexpression phenotype. Using endogenous promoters in stead of the 35S CaMV resulted in a dramatic reduction in the frequency of loss-of-function phenotypes. Furthermore, pleiotropic defects occasionally caused by an overexpression strategy can be overcome by using the native promoter of the gene. Finally, a ChAP result is presented using GFP antibody on plants carrying a genomic fragment of a MADS-box gene fused to GFP.</p> <p>Conclusion</p> <p>This study revealed that MADS-box proteins are very sensitive to fusions with small peptide tags and GFP tags. Furthermore, for the expression of chimeric versions of MADS-box genes it is favorable to use the entire genomic region in frame to the tag of choice. Interestingly, though unexpected, it appears that the use of chimeric versions of MADS-box genes under the control of the strong 35S CaMV promoter is a very efficient method to obtain dominant-negative mutants, either caused by cosuppression or by alteration of the activity of the recombinant protein. Finally, we were able to demonstrate AGAMOUS binding to one of its targets by ChAP.</p

Conservation of the role of INNER NO OUTER in development of unitegmic ovules of the Solanaceae despite a divergence in protein function

The P-SlINO::SlINO-GFP transgene continues to be expressed after fertilization during the onset of fruit development. A-C: Ovules from P-SlINO::SlINO-GFP plants. D, E: Ovules from control plants. Images A (confocal) and B (DIC overlaid with GFP channel) show expression in the outer cell layer in an ovule post-anthesis. C-E are images of the surface cells of the integument of ovules taken from 3–4 mm fruits. C and D are images taken on an epifluorescence microscope (Axioplan) using a Chroma GFP filter set 41017 (Chroma, Bellows Falls, VT). E is a dark-field image of the same ovule in D. These images show expression is present in developing fruit. Scale bar in B represents 20 μm, scale bar in E represents 20 μm in C-E. (TIF 4435 kb

AG and SEP3 expression analysis and chromatin immunoprecipitation (ChIP)

<p><b>Copyright information:</b></p><p>Taken from "Tagging of MADS domain proteins for chromatin immunoprecipitation"</p><p>http://www.biomedcentral.com/1471-2229/7/47</p><p>BMC Plant Biology 2007;7():47-47.</p><p>Published online 14 Sep 2007</p><p>PMCID:PMC2071916.</p><p></p> Confocal Scanning Laser Microscopical (CSLM) imaging of (A) (pARC422) and (B) (pARC423) in the inflorescence. Top view (A, B) of an inflorescence with different floral bud stages (indicated by numbers). The GFP expression (green signal) is predominantly localized in the nuclei of floral meristem cells of flower buds from stage 3 onwards (comprising whorl 3 and 4 for AG, and whorl 2, 3, and 4 for SEP3, respectively). Autofluorescence is visible as red signal. (C) Anti-GFP Western blot with material from Arabidopsis WT and (pARC422) plants. Protein product is detectable in transgenic plants only. Bottom panel shows the Coomassie stained gel serving as loading control. (D) Enrichment of AG target DNA after ChAP with GFP antibody and compared with pre-immune. Quantification of target DNA was done by Real-time PCR using primers corresponding to sequences in the second intron of . FM, floral meristem, S, sepal, IM, inflorescence meristem, WT, wild-type

Phenotypes of transgenic Arabidopsis plants with different tagging constructs

<p><b>Copyright information:</b></p><p>Taken from "Tagging of MADS domain proteins for chromatin immunoprecipitation"</p><p>http://www.biomedcentral.com/1471-2229/7/47</p><p>BMC Plant Biology 2007;7():47-47.</p><p>Published online 14 Sep 2007</p><p>PMCID:PMC2071916.</p><p></p> (A) Wild-type Arabidopsis at the rosette stage, (D) at the inflorescence stage, and (G) a close-up of a flower. (B) Line with fusion construct showing an overexpression phenotype (pARC276). (C) Line with fusion construct showing a overexpression phenotype (pARC277). Rosette stage images (A-C) were taken from plants grown under the same conditions and were of the same age (bar indicates relative size). (E, H) Line with fusion construct showing an mutant phenotype (pARC308). (F, I) Line with fusion construct showing a partial -like mutant phenotype (pARC309). (J) Siliques of lines with fusion construct with either a overexpression (), mutant () phenotype, or wild-type phenotype (WT) (pARC310). (K) Arabidopsis root tip and (L) open silique with an ovule of a line expressing fusion construct (pARC310) observed by fluorescence microscopy. dz, dehiscence zone; v, valve; ov, ovule; n, nuclues; ca, carpel wall

Northern blot analysis of leaf tissue of different Arabidopsis lines containing various tagging constructs

<p><b>Copyright information:</b></p><p>Taken from "Tagging of MADS domain proteins for chromatin immunoprecipitation"</p><p>http://www.biomedcentral.com/1471-2229/7/47</p><p>BMC Plant Biology 2007;7():47-47.</p><p>Published online 14 Sep 2007</p><p>PMCID:PMC2071916.</p><p></p> (A) Expression analysis of (pARC276) lines. (B) Expression analysis of (pARC277) lines. (C) Expression analysis of --tagII-FLAG-tag (pARC117) lines, -like plants are indicated with 'm' and WT-like plants with 'n'. WT, wild-type; +, line with an overexpression phenotype