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. 2014;8(1):43-51.
doi: 10.4161/fly.26805. Epub 2013 Dec 13.

Bi-functional cross-linking reagents efficiently capture protein-DNA complexes in Drosophila embryos

Tsutomu Aoki  1 , Daniel Wolle  1 , Ella Preger-Ben Noon  1 , Qi Dai  2 , Eric C Lai  2 , Paul Schedl  3
Affiliations

Bi-functional cross-linking reagents efficiently capture protein-DNA complexes in Drosophila embryos

Tsutomu Aoki et al. Fly (Austin). 2014.

Abstract

Chromatin immunoprecipitation (ChIP) is widely used for mapping DNA-protein interactions across eukaryotic genomes in cells, tissues or even whole organisms. Critical to this procedure is the efficient cross-linking of chromatin-associated proteins to DNA sequences that are in close proximity. Since the mid-nineties formaldehyde fixation has been the method of choice. However, some protein-DNA complexes cannot be successfully captured for ChIP using formaldehyde. One such formaldehyde refractory complex is the developmentally regulated insulator factor, Elba. Here we describe a new embryo fixation procedure using the bi-functional cross-linking reagents DSG (disuccinimidyl glutarate) and DSP (dithiobis[succinimidyl propionate). We show that unlike standard formaldehyde fixation protocols, it is possible to capture Elba association with insulator elements in 2-5 h embryos using this new cross-linking procedure. We show that this new cross-linking procedure can also be applied to localize nuclear proteins that are amenable to ChIP using standard formaldehyde cross-linking protocols, and that in the cases tested the enrichment was generally superior to that achieved using formaldehyde cross-linking.

Keywords: ChIP; DNA binding; DSG DSP; Elba; Insensitive; bi-functional cross-linkers; chromatin immunoprecipitation; formadelhyde; insulators.

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Figures

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Figure 1. DSG or DSP cross-linking of embryo improves the chromatin IP of Elba1. (A) The Elba1 ChIP from formaldehyde (FA) cross-linked embryo did not give significant enrichment of the Fab-7 Elba recognition sequence. 2–5 h old Oregon R embryos were treated as indicated with formaldehyde, and the isolated nuclei were subjected to the ChIP processes with anti-Elba1 #2 antiserum of ref. . The relative amounts of the DNA fragments were measured by SYBR-green qPCR and the ratio of 'Immune serum IP/Pre-immune serum IP' (fold-enrichment) was calculated using the ∆∆Ct method. Each experiment was repeated more than 3 times to obtain the standard deviation shown as an error bar. (B) The cross-linking of embryo with NHS-esters improved the enrichment of Elba1 ChIP at the target site. The 2–5 h old Oregon R embryos were first cross-linked with indicated concentration of DSG or DSP in the PBS/heptane for one hour followed by 4% FA treatment for 15 min. The ChIP experiments and data analyses were applied as described above.
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Figure 2. Elba complex binds to a semi-consensus sequence in Fab-8 in vitro. The 28 base pair DNA fragment from Fab-8 that contains the 'CGAATAAG' sequence was end-labeled with 32P and subjected to the EMSA (Electrophoresis Mobility Shift Assay) with either A) increasing amounts of nuclear extract (NE) from 0–6 h embryos or B) with the products of a rabbit reticulocyte in vitro-translation primed with a control mRNA ('Control tsl', lane 2) or with a mixture of mRNAs encoding the 3 Elba protein ('Elba tsl', lanes 3–9) as described in ref. . In panel B, lanes 4–9, 50-fold (lanes 4, 6, and 8) or 100-fold (lanes 5, 7, and 9) excess amounts of unlabeled DNA fragments as indicated were added as cold competitors. Competitors. Fab-8 Elba wt: 28 bp wild-type DNA from Fab-8. Fab-8 Elba mut: 28 bp Fab-8 DNA fragment that has 8 bp alteration of 'ATCCGCCT ' instead of the semi-consensus sequence. Fab-7 Elba wt: 27 bp Fab-7 fragment that spans the original Elba site (See ref. and 10).
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Figure 3. It is possible to capture Elba complexes in Fab-8 and in the apterous boundary using the DSG-formaldehyde combination procedure. 2–5 h Oregon R embryos were cross-linked with either 1.8% formaldehyde (FA) or 2.5 mM DSG + 4% formaldehyde (FA) and then ChIP’d with Elba1 antibody (antiserum #1 in the ref. 9). The DNA recovered with the pre-immune and immune sera was analyzed by qPCR with primer pairs spanning the 28 fragment containing the Fab-8 Elba semi-consensus site (A) or 2 consensus (CCAATAAG) sequences at the far 5′ end of the apterous locus close to the lethal (2) 09851 gene (B). The ChIP enrichment was calculated as described in Figure 1. We also tested the enrichment of sequences 250 bp and 500 bp proximal (in iab-7) to the Fab-8 Elba site and sequences 250 bp and 500 bp from the first apterous Elba consensus sequence (closest to the apterous gene). For comparison, we have included the extent of enrichment at each site obtained using the 1.8% formaldehyde (FA) procedure.
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Figure 4. ChIP enrichment of Insensitive (Insv) protein is improved by using DSG-formaldehyde combination procedure. 2–5 h old Oregon R embryos were cross-linked with either 1.8% formaldehyde or 2.5 mM DSG + 4% formaldehyde and subjected to the ChIP with rabbit pre-immune serum and Insv serum as described in Figure 1 and 3. We used primers spanning 7 published Insv in vivo binding sites as well as 2 control loci Sex lethal (Sxl) and twine (twe) for qPCR. In vivo Insv sites showing < 15-fold enrichment with the DSG-formaldehyde combination procedure are presented in panel A, while those showing > 15-fold enrichment are presented in panel B. For comparison, we have included the extent of enrichment at each site obtained using the 1.8% formaldehyde (FA) procedure. The previously identified in vivo Insv binding sites are as follows: vg: vestigial. mir-263a: microRNA-263a or bereft. repo: reversed polarity. fng: fringe. ham: hamlet. fne: found in neurons. Dlic2: Dynein light intermediate chain 2.
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Figure 5. ChIP enrichment of RNA polymerase II (pol II) is improved at some promoters by the DSG-treatment of embryo. Ten - 12 h embryos were cross-linked with either 1.8% formaldehyde only or with 2.5 mM DSG followed by 1.8% formaldehyde. The embryos were disrupted by sonication and resulting chromatin was subjected to IP with either anti-pol II CTD (C-terminal domain) or control IgG. The relative amounts of specific genome sites were measured by qPCR, and the ChIP enrichments were calculated from the ratio of Pol II IP/control IgG IP. The results of promoter of RpL32 (Ribosomal protein L32) is shown here as the representative of the Pol II-bound promoters in this developmental stage. A region upstream of apterous transcription unit was used as a negative control (ap 5′ control).
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