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

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.     

Immunoprecipitation of DNA-protein complexes cross-linked by cis-diamminedichloroplatinum

For the study of in vitro and in vivo DNA-protein interactions, cross-linking reactions driven by UV or formaldehyde have been frequently used, followed by standard protocols of immunoprecipitation and analysis of the DNA isolated from the complexes. Here we present a basically modified method to analyze the DNA-protein cross-linked complexes obtained by an alternative cross-linking reagent. The innovations presented here include cross-linking by cis-diamminedichloroplatinum II, a fast method to isolate DNA-protein complexes using gel-filtration chromatography, and a modified procedure to obtain specific immunocomplexes that can be analyzed either for DNA or for protein content. The application of this method to two nuclear proteins from chicken liver nuclei is described.     

A single fixation protocol for proteome-wide immunofluorescence localization studies

Immunofluorescence microscopy is a valuable tool for analyzing protein expression and localization at a subcellular level thus providing information regarding protein function, interaction partners and its role in cellular processes. When performing sample fixation, parameters such as difference in accessibility of proteins present in various cellular compartments as well as the chemical composition of the protein to be studied, needs to be taken into account. However, in systematic and proteome-wide efforts, a need exists for standard fixation protocol(s) that works well for the majority of all proteins independent of subcellular localization. Here, we report on a study with the goal to find a standardized protocol based on the analysis of 18 human proteins localized in 11 different organelles and subcellular structures. Six fixation protocols were tested based on either dehydration by alcohols (methanol, ethanol or iso-propanol) or cross-linking by paraformaldehyde followed by detergent permeabilization (Triton X-100 or saponin) in three human cell lines. Our results show that cross-linking is essential for proteome-wide localization studies and that cross-linking using paraformaldehyde followed by Triton X-100 permeabilization successfully can be used as a single fixation protocol for systematic studies.     

Effects of formaldehyde fixation on protein secondary structure: a calorimetric and infrared spectroscopic investigation

We investigated the effects of formaldehyde fixation on the secondary structure of isolated proteins (bovine serum albumin, ribonuclease A, and hemoglobin) using high-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy. Whereas thermograms obtained by scanning calorimetry on unfixed purified proteins demonstrated denaturation transitions in the 70-90 degrees C temperature range, the thermograms showed no denaturation transitions in this temperature range when the proteins had been placed in formaldehyde solutions. Thus, fixation destroyed the denaturation transition of bovine serum albumin, ribonuclease A, and hemoglobin. Infrared spectra obtained on the unfixed and fixed proteins were essentially identical. This demonstrates that the "fixed" proteins retain the secondary structure present before fixation. We therefore conclude that the cross-linking of proteins that occurs in the process of formaldehyde fixation "locks in" the secondary structure of these protein molecules.     

Importance of fixation in immunohistochemistry: use of formaldehyde solutions at variable pH for the localization of tyrosine hydroxylase

Adequate fixative in immunohistochemistry requires not only a rapid and total immobilization of the antigen, but also a sufficient preservation of its immunoreactivity and maintenance of its accessibility to the immunochemical reagents for localization. Thus, the optimal fixation condition for a specific antigen necessitates a compromise between these opposing variables and can be determined by the preparation of a series of tissues with a progressively increasing degree of fixation. Unless the results of localization using such a series is available, one must be satisfied with adequate but less than optimal results. In the present study, this principle is demonstrated using the localization of tyrosine hydroxylase in the dopaminergic system with formaldehyde as the fixative. The rate and degree of fixation with formaldehyde was shown to be highly pH dependent. By perfusing the tissue with formaldehyde at pH 6.5 (where the rate of fixation is extremely slow) it is possible to rapidly distribute the fixative homogeneously into the tissue. By suddenly changing to a formaldehyde perfusate of higher pH, the cross-linking reaction is rapidly increased. This two-step fixation procedure provides a means of obtaining a rapid and uniform immobilization of the antigen, so that its translocation can be avoided. The final degree of fixation is controlled by the duration and pH of the second fixative solution. The results obtained by increasing the pH of the second solution demonstrated that complete fixation of tyrosine hydroxylase in the dopaminergic system with formaldehyde maybe obtained using a very basic formaldehyde solution (pH 11) while still retaining immunoreactivity of the enzyme. The localization that was achieved at lower pH appeared adequate until it was compared to the results obtained by perfusion at pH 11 in the second step.     

Fixated on fixation: using ChIP to interrogate the dynamics of chromatin interactions

A new study exploits the time-dependence of formaldehyde cross-linking in the commonly used chromatin immunoprecipitation (ChIP) assay to infer the on and off rates for site-specific chromatin interactions.     

Rumors of the death of consumer genomics are greatly exaggerated

A new study exploits the time-dependence of formaldehyde cross-linking in the commonly used chromatin immunoprecipitation (ChIP) assay to infer the on and off rates for site-specific chromatin interactions.     

A modified chromatin-immunoprecipitation protocol for silkmoth ovarian follicular cells reveals C/EBP and GATA binding modes on an early chorion gene promoter

Standard Chromatin immunoprecipitation protocols have been designed to suit studies performed on cell line cultures or yeast cells growing in liquid cultures. In these cases cross-linking/fixation takes place directly in the growing medium of the cells by the addition of a general fixation reagent. When applied on whole isolated silkmoth follicles, this procedure results in poor release of follicular cells from the basal membrane and lower yield of cross-linked chromatin. We present a modification to the standard protocol, where detachment of follicular cells from the basal membrane of the egg and nuclei isolation precedes formaldehyde-mediated cross-linking. We also discuss application of the modified method for the identification of distinct BmC/EBP and BmGATAβ binding modes on a chorion gene promoter from the Er1.A/B early gene pair.     

In vivo protein-protein and protein-DNA crosslinking for genomewide binding microarray

Chromatin immunoprecipitation (ChrIP or ChIP) has commonly been used to map protein-DNA interaction sites at specific genomic loci through use of formaldehyde-induced crosslinking. However, formaldehyde alone has proved inadequate for crosslinking of certain proteins such as the yeast histone deacetylase Rpd3. We report here a modified crosslinking procedure that includes a protein-protein crosslinking agent in addition to formaldehyde. Using this double crosslinking method, we have successfully mapped Rpd3 binding sites in vivo. We also describe the use of ChrIP in combination with DNA microarrays (ChrIP-array) to determine the pattern of Rpd3 binding genomewide. This approach couples the versatility of ChrIP with that of microarrays to identify binding patterns that would otherwise be hidden in a gene-by-gene survey.     

Ultrasound-facilitated formalin fixation of biological specimens

Abstract

In a previous study, we showed that ultrasound can dramatically reduce the time required for tissue fixation in formalin. It generally is believed that ultrasound increases the speed of tissue fixation in two possible ways: 1) increasing the speed of penetration of fixative molecules into tissue samples and 2) increasing the speed of cross-linking reactions. We addressed here the second possible way by using protein solutions and cultured cells, which minimized the effects of the penetration factor. Proteins or cultured cells in solution were fixed with formalin with or without ultrasound irradiation. Fixed proteins and cell lysates then were separated by SDS-poly acrylamide gel electrophoresis and subjected to Western blotting to examine cross-linking formation in certain proteins. Unexpectedly, irradiation with ultrasound did not produce an observable difference in the rate of cross-linking in protein solutions. In similar experiments using cultured cells, however, we observed a significant reduction in recovery of certain proteins from cells fixed by formalin under the influence of ultrasound, which indicated that the ultrasound fixation procedure accelerated cross-linking formation within cells. Studies on protein and cell fixation without ultrasound showed that cross-linking formation was closely related to incubation temperature, which indicates that the heating function, which is inherently associated with ultrasound is another major factor in the ability of ultrasound to accelerate cross-linking.     

Cross-linking of estrogen receptor to chromatin in intact MCF-7 human breast cancer cells: optimization and effect of ligand

To investigate the effect of ligand (be it hormone, antihormone, or no hormone) on the interaction between estrogen receptor (ER) and chromatin, we have used formaldehyde as a cross-linking agent in intact MCF-7 human breast cancer cells. After a 1- to 2-h hormone treatment, the cells are exposed for 8 min to formaldehyde, which is added directly to their culture medium to minimize environmental perturbation. Nuclei are prepared from formaldehyde-treated cells and their contents are fractionated on CsCl density gradients to separate DNA-protein complexes from free protein. Peak gradient fractions are assayed for the presence of specific proteins by immunoblot of sodium dodecyl sulfate-polyacrylamide gel patterns. Using this approach, we find that 0.15% formaldehyde is optimal for cross-linking ER to chromatin. We detect ER and the large subunit of RNA polymerase II with DNA from formaldehyde-treated, but not from untreated cells. On the other hand, actin (a cytoplasmic protein) and small nuclear ribonucleoprotein particle proteins (nuclear RNA binding proteins) are not cross-linked to DNA. Therefore, cross-linking appears to be selective and fractionation is efficient. Interestingly, we detect similar levels of ER (as well as RNA polymerase II) with DNA from formaldehyde-treated cells, regardless of whether the cells are preexposed to estrogen (17 beta-estradiol at 10(-8) M), antiestrogen (ICI 164,384 at 10(-7) or 10(-6) M), or no hormone. These results, using covalent cross-linking in intact cells, indicate that both ligand-occupied and unoccupied ER are associated with chromatin.     

DNA from formalin-fixed tissue: extraction or repair? That is the question

Establishing effective DNA-based protocols for use on archival material fixed in formaldehyde (formalin) is a particularly challenging task. Formalin fixation induces cross-linking with nucleic acids and proteins, thereby reducing the amount and quality of the extracted DNA. Previous attempts have primarily focused on optimizing DNA extraction protocols. Here we focus on the use of enzymes capable of in vitro repair of DNA extracts prior to amplification of the nucleic acids by the polymerase chain reaction (PCR). The amplification success of mitochondrial DNA was greater using the repair enzyme assay (56%) than with the regular PCR assay (20%), and even more convincing results were obtained with the amplified nuclear ribosomal region (91% versus 21%). These results indicate that in vitro repair of DNA damage (depurinated sites, strand nicks and base modifications) increases the number of samples that amplify, amplify to a greater extent and amplify fewer ancillary bands and that DNA repair has been overlooked as a way of improving the efficiency of molecular methods used on formalin-fixed samples. Fidelity has not been specifically investigated, but preliminary results indicate that misincorporation is not a major problem.     

CrossWork: software-assisted identification of cross-linked peptides

The increased interest in chemical cross-linking for probing protein structure and interaction has led to a large increase in literature describing new cross-linkers and search programs. However, this has not led to a corresponding increase in the analysis of large and complex proteins. A major obstacle is that the new cross-linkers are either not readily available and/or have a low reactivity. In combination with aging search programs that are slow and have low sensitivity, or new search programs that are described but not released, these efforts do little to advance the field of cross-linking. Here we present a method pipeline for chemical cross-linking, using two standard cross-linkers, BS3 and BS2G, combined with our freely available CrossWork search program. By this approach we generate cross-link data sufficient to derive structural information for large and complex proteins. CrossWork searches batches of tandem mass-spectrometric data, and identifies cross-linked and non-cross-linked peptides using a standard PC. We tested CrossWork by searching mass-spectrometric datasets of cross-linked complement factor C3 against small (1 protein) and large (1000 proteins) search spaces, and show that the resulting distance constraints agree with the established structures. We further investigated the structure of the multi-domain ERp72, and combined the individual domains of ERp72 into a single structure.     

A simplified formaldehyde fixation and immunoprecipitation technique for studying protein-DNA interactions

Using the single cell eukaryote Tetrahymena thermophila, a simple method was developed for studying protein-DNA associations by cross-linking proteins to DNA with formaldehyde and immunoprecipitating the solubilized chromatin fragments with a specific antiserum. The protocol uses crude antiserum and involves only three steps: cross-linking, shearing to solubilize the chromatin, and immunoprecipitation. Methods for optimizing certain critical parameters, such as fixation time and NaCl concentration, are described. The method is likely to be generally useful for a variety of nuclear antigens.     

Fixation requirements for the immunohistochemical reactivity of PCNA antibody PC10 on cryostat sections

Summary In contrast to that in paraffin-embedded tissue, the reactivity of monoclonal PCNA antibody PC10 on cryostat sections requires a special fixation procedure as the target epitope is seemingly not accessible to its antibody. A panel of 18 fixation protocols was investigated. Chilled methanol or acetone, or PLP (paraformaldehyde-lysine-periodate) was found to be unsuitable for skin preparations. A two-step fixation protocol was developed for normal skin and basal cell carcinomas. They were fixed first in 3.4% buffered formaldehyde, followed by fixation in 2:1 v/v ethanol-acetic acid. Following this fixation regime, cryostat sections displayed the same PCNA/PC10 labelling pattern as paraffin sections of formalin-fixed tissue.