Optimization of a Method for Chromatin Immunoprecipitation Assays in the Marine Invertebrate Chordate Ciona

Abstract

Chromatin immunoprecipitation (ChIP) assays allow the efficient characterization of the in vivo occupancy of genomic regions by DNA-binding proteins, and thus facilitate the prediction of cis-regulatory sequences in silico and guide their validation in vivo. For these reasons, these assays and their permutations (e.g., ChIP-on-chip, ChIP-Sequencing) are currently being extended to several non-mainstream model organisms, as the availability of specific antibodies increases. Here we describe the development of a polyclonal antibody against the Brachyury protein of the marine invertebrate chordate Ciona intestinalis and provide a detailed ChIP protocol that should be easily adaptable to other marine organisms.

Functional Brachyury Binding Sites Establish a Temporal Read-out of Gene Expression in the Ciona Notochord

“…We had tested in previous studies some of the putative Ci-Bra binding sites identified in notochord CRMs for their ability to be bound in vitro by Ci-Bra via electrophoretic mobility shift assays (EMSA) (Table 1); here we assessed the occupancy of these sites in vivo through ChIP assays on mid-tailbud stage embryos (Figure 8A) using a polyclonal Ci-Bra antibody (Figure 8B) [46]. To test the specificity of the binding by Ci-Bra, we carried out ChIP over a 10-kb stretch encompassing part of the Ci-FCol1 locus and its neighboring gene (Figure 8A).…”

“…Colored boxes below the graph symbolize exons (green, gene KH.C7.121; grey, 5′-UTR; pink, Ci-FCol1 ); the lines connecting them represent introns. (B) Fluorescence microphotograph of a late tailbud Ciona embryo carrying the Ci-Bra>GFP transgene [27], immunostained with the Ci-Bra-specific antibody [46]. The nuclei of the 40 notochord cells are stained by the antibody in red; GFP expression is detected in a subset of the 40 notochord cells, owing to mosaic incorporation of the transgene.…”

“…Immunohistochemistry was performed on either wild-type or transgenic C. intestinalis embryos carrying the Ci-Bra>GFP construct [27], essentially as previously described [66], using the published anti-Ci-Bra polyclonal antibody [46]. After an overnight incubation at 4°C with a goat anti-rabbit Alexa Fluor 546 fluorescent antibody (Invitrogen) in PBS, the embryos were washed six times for 10 min in PBS, mounted with Vectashield mounting medium containing DAPI (Vector Laboratories) and imaged using a Leica DMR microscope.…”

“…ChIP assays were carried out as previously published [46]. qPCR was performed in triplicate on each of the biological replicates, using SYBR green (USB) in an Applied Biosystems (ABI) Prism 7700 Real-Time qPCR thermocycler.…”

Cis-regulatory interfaces reveal the molecular mechanisms underlying the notochord gene regulatory network of Ciona

“…As in the case of Lmx1-r , we employed an in vivo trans -activation assay to verify the results obtained through the cis -regulatory analysis, taking advantage of the notochord-specific expression of Ci-Bra 28 and of a Ci-Bra-specific antibody developed in our lab 57 , 80 . We used the 2.6-kb Foxa.a promoter region to drive ectopic expression of Ci-Bra in endoderm and CNS precursors ( Foxa.a>Bra plasmid 81 ) and verified the expression of the Ci-Bra protein and the incorporation of the CRMs fused to the LacZ reporter through double immunostaining with the Ci-Bra antibody (Fig.…”