Measuring the Formaldehyde Protein–DNA Cross-Link Reversal Rate (2024)

Protein–DNA binding interactionsplay critical roles inimportant cellular processes such as gene expression, cell division,and chromosomal organization. Techniques to identify and characterizethese interactions often utilize formaldehyde cross-linking for stabilizationof the complexes. Advantages of formaldehyde as a cross-linking reagentinclude cell permeability, relatively fast cross-linking kinetics,and short cross-linker length. In addition, formaldehyde cross-linksare reversible, which has the advantage of allowing complexes to bedissociated if desired but may also present a problem if undesireddissociation occurs in the course of an experiment. While the kineticsof formaldehyde cross-link formation have been well-established innumerous studies, there have been no reports of the rate of cross-linkdissociation, even though it is clearly a critical variable when developinga biochemical protocol involving formaldehyde cross-linking. We presenthere a method for measurement of the rate of formaldehyde cross-linkreversal based upon the Formaldehyde-Assisted Isolation of RegulatoryElements (FAIRE) procedure and use it to determine the rate of cross-linkreversal for cross-linked protein–DNA complexes from yeastcell lysate. The half-life of the protein–DNA cross-links variesfrom 179 h at 4 °C to 11.3 h at 47 °C, with a rate thatincreases exponentially with temperature and is independent of saltconcentration.

Materials

Saccharomyces cerevisiae strain Y1788 was obtained from Professor David Mitchell (Universityof Texas). Yeast extract peptone dextrose (Y1375, abbreviated YPD),37% formaldehyde (F38775), phenol–chloroform–isoamylsolution 25:24:1 (77617, abbreviated phenol–chloroform), andprotease inhibitors for fungal growth (P8215) were purchased fromSigma-Aldrich Co. (St. Louis, MO). The 20% sodium dodecyl sulfate(SDS) (161-0418) was purchased from Bio-Rad (Hercules, CA). The 10×phosphate buffered saline (PBS) (P0191), 5 M Tris pH = 8 (T5581),1 M Tris pH = 7 (T1070), 5 M sodium chloride (NaCl) (S0250), and 500mM tetraethylenediaminetetraacetic acid (EDTA) (E0307-06) were purchasedfrom Teknova (Hollister, CA). RNaseA (12091-039) was purchased fromLife Technologies (Carlsbad, CA). qPCR probes were ordered from IntegratedDNA Technologies (Coralville, IA). The 96-well plates (04729692001)and Master Mix solutions (0470749001) for qPCR were purchased fromRoche USA (Nutley, NJ). Proteinase K (P8107S) was purchased from NewEngland Biolabs (Ipswich, MA).

Cell Lysate Preparation

Yeast cells were grown to saturationin 5 mL of YPD overnight at 30 °C and shaken at 200 rpm in anAmerex 747 shaker/incubator. The cells were diluted into 1.5 L ofYPD and grown to an OD₆₀₀ ∼2.0 as measured usingan Agilent 8453 UV–vis spectrophotometer. Formaldehyde wasadded to a final concentration just under 3% (122 mL) and incubatedfor 30 min at room temperature, after which unreacted formaldehydewas quenched with 250 mL of 5 M Tris. The cells were collected usingan Avanti J-25I centrifuge at 5 000g for 20min. The cell pellet was washed once with 1× PBS and either usedright away or stored at −80 °C. Cells from 500 mL of culture(∼2–3 mL cell pellet) were resuspended in 50 mL of lysisbuffer (20 mM EDTA, 200 mM NaCl, 50 mM Tris pH7, and protease inhibitors(1/200 from stock)). The cells were lysed at 30 kpsi using a ConstantSystems TS Series Cell Disruptor. SDS was added to the lysate solutionto a final concentration of 1% and the lysate was incubated at 65°C for 5 min. The cross-linked chromatin was sonicated in 50mL volumes using a MisoniX Ultrasonic Processor S4000 at 20 V fora total of 3.5 min with alternating intervals of 4 s on and 4 s off.The sample was then centrifuged at 8 000g for12 min to separate the cellular debris from the soluble chromatin.The supernatant was removed from the pellet and diluted 5-fold intolysis buffer to decrease the total SDS concentration to 0.2%. RNaseAwas added to a final concentration of 60 μg/mL, and the solutionwas shaken at 150 rpm at 37 °C for 60 min.

Phenol–ChloroformExtraction to Purify Protein-Free DNA

Samples of cell lysate(10 mL) were used for each formaldehydecross-linking reversal measurement. Aliquots (700 μL) were removedfrom each sample prior to incubation to provide a reference samplefor determination of the total amount of DNA present (referred toas input samples). Ten units of proteinase K were added to each ofthese aliquots followed by overnight incubation at 37 °C in orderto degrade all proteins present and release the free DNA. The sampleswere incubated at 4, 23, 37, or 47 °C, and three additional 700 μLaliquots were removed at each time point (0, 2.5, 5, 10, and 20 h).Phenol–chloroform (700 μL) was added to each aliquotfollowed by vigorous vortexing for 30 s. The samples were centrifugedat maximum speed (14 000g) for 5 min in aEppendorf 5417R centrifuge, after which the organic and aqueous layersare completely separated. A volume of 500 μL of the aqueous(upper) layer was removed and placed in a new eppendorf tube, followedby addition of a second 500 μL portion of phenol–chloroform.Vortexing and centrifugation were repeated, 200 μL of the aqueousphase was removed, and 200 μL of the phenol–chloroformwas added and the vortexing/centrifugation steps were repeated fora final time. A volume of 80 μL of the aqueous phase was removedand diluted 10-fold into 1× TE (10 mM Tris pH = 7, 1 mM EDTA).These samples were stored at −20 °C prior to qPCR analysis.The input samples treated with proteinase K were subjected to thesame phenol–chloroform extraction procedure.

qPCR Analysis

DNA isolated from each sample was measuredusing Taqman assays for each of the three genes studied (see the Supporting Information). Dilutions of purifiedyeast genomic DNA were used for a standard curve. Each DNA samplewas analyzed in duplicate in a 96-well microtiter plate. Each wellcontained 5 μL of sample, 10 μL of LightCycler 480 probemaster mix, 4.5 μL of water, and 0.5 μL of 40× primerprobe mix. After pipetting, each plate was centrifuged for 2 min at2 000g. The samples were then analyzed usinga Roche 480 LightCycler. The qPCR runs included a 5 min preincubationstep at 95 °C, amplification cycles, and a 2 min cooling stepat 40 °C. Each amplification cycle was composed of a 10 s 95°C incubation with a temperature ramp of 4.4 °C/s, a 30s incubation at 60 °C with a temperature ramp of 2.2 °C/s,and a third 1 s incubation at 72 °C with a temperature ramp of4.4 °C/s. Detection of the FAM fluorophore was performed duringthe 72 °C incubation using a 483–533 filter set. Analysisof the resultant qPCR curves and calculation of Cp values were performedusing the Roche 480 LightCycler software and the second quant/2ndderivative function. Absolute DNA amounts were calculated from thegenomic DNA standard curve, and % protein-free DNA values are givenby the ratio (protein-free DNA in aqueous phase/input DNA) ×100%).

Measuring the FormaldehydeCross-Link Reversal Rate

Formaldehyde cross-link reversalinvolves dissociation of the methylenelinkage between the protein and DNA moieties (Figure ​(Figure1).1). The nature of these cross-links varies depending upon theamino acid and nucleotides involved and the chemical landscape withina cell subjected to formaldehyde is likely quite complex because ofthe various linkages present. Lu et al. recently reported the cross-linkedspecies that result from addition of formaldehyde to mixtures of eachamino acid and nucleotide.¹² They foundthat cross-linking occurs between the amino acids cysteine (Cys),histidine, tryptophan, and lysine (Lys) and the deoxynucleosides deoxyadenosine,deoxycytosine, and/or deoxyguanosine (dG), with the predominant reactionbeing that between Lys and dG to form an aminal linkage, and the secondmost prominent reaction being that between Cys and dG to form a hemiaminalthioether linkage.¹² A detailed reactionmechanism for the prototypic Lys–dG coupling reaction is shownin Figure ​Figure1.1. In the present work, we determinethe aggregate cross-link reversal rate of formaldehyde protein–DNAcomplexes isolated from cross-linked yeast cells, which are likelyto reflect the predominant Lys-dG and Cys-dG cross-links as well assmall contributions from the other amino acid-deoxynucleoside pairs.

In general, to measure the rateof cross-link reversal, eitherthe disappearance of reactants or the appearance of products mustbe measured. In the case of formaldehyde cross-links, this means thedisappearance of protein–DNA complexes or the appearance ofnative protein and DNA moieties without the methylene linkages. Manymethods exist to measure total DNA or protein levels including PicoGreenbinding¹³ for the former and BCA¹⁴ and A260¹⁵ assaysfor the latter. In the complex background of cell lysate, however,the disappearance of a single methylene bridge is not a substantialperturbation and these assays do not distinguish between cross-linkedand non-cross-linked species. Therefore, an additional purificationstep is useful to isolate one component of the reaction and allowfor specific analysis.

FAIRE was developed as a method to identifyprotein-free regionsof the genome.⁶ The assay uses phenol–chloroformextraction as a means to separate protein-free DNA fragments fromcross-linked protein–DNA complexes and free protein. When subjectedto phenol–chloroform extraction, proteins partition into theorganic phase, while DNA remains in the aqueous phase. Protein–DNAcomplexes reside at the interface between the two phases. Identificationof the DNA sequences found in the aqueous phase, the protein-freefraction, reveals those sequences in the genome that are not associatedwith protein, thereby providing a means of determining protein occupancyacross the genome. We utilized FAIRE to determine the kinetics ofcross-link reversal for formaldehyde-cross-linked protein–DNAcomplexes from yeast. Cross-linked cells were lysed, sonicated, andcentrifuged, to shear the DNA and clear the lysate sample of debris.Aliquots of the lysate sample were then periodically subjected tophenol–chloroform extraction to isolate the protein-free DNAfraction of the lysate. The aqueous material was then interrogatedusing qPCR assays specific for three genes (INO1,X-element, and 25S rDNA) to measure the relative amounts of DNA correspondingto each gene present as a function of time. The DNA levels presentin the aqueous phase of the phenol–chloroform extraction wereplotted as a function of time and the cross-link reversal rate correspondsto the slope of the line (Figure ​(Figure2).2). As verysimilar results were obtained for all three genes, the results forall three genes were averaged to provide an overall measure of cross-linkreversal rate.

Temperature Effects

It is well-knownthat heat reversesformaldehyde cross-links;¹⁶ however, therates have not been measured nor has the dependence of the rate ontemperature. We measured the cross-link reversal rate at four differenttemperatures: 4, 23, 37, and 47 °C. The first three temperatureswere selected as they correspond to commonly employed temperaturesin biochemical protocols, and the 47 °C temperature was chosenarbitrarily as a more extreme case (dissociation rates at other temperaturesmay be estimated if desired from the exponential fit to the data shownin Figure ​Figure2B).2B). The percent of protein-freeDNA relative to total input DNA is plotted versus time for each ofthe four temperatures examined (Figure ​(Figure2A).The2A).The slope of each line is given in Table 1 asthe reaction rate. As expected, the reaction rate increases with temperature,with the slowest reaction rate at the lowest temperature measured(0.3% protein-free DNA/h at 4 °C) and the fastest reaction rateat the highest temperature measured (4.4% protein-free DNA/h at 47°C). Half-lives were also calculated for each temperature assuminga zero-order rate law for a unimolecular dissociation. The longesthalf-life was 179 h, at 4 °C, while the shortest half-life was11.3 h, at 47 °C.

The temperaturedependence of the cross-link reversal rate fitswell to an exponential function, as shown in Figure ​Figure2B.2B. On the basis of this dependence, we were able to fit thedata to both the linear Arrhenius eq 1 and Eyringeq 2.

Fromthe Arrhenius plot, the activation energy(E_a) was determined to be 47 kJ mol^–1, while from the Eyring plot, the enthalpy of activation(ΔH^⧧) and entropy of activation(ΔS^⧧) were determined tobe 44 kJ mol^–1 and −0.1 kJ mol^–1 K^–1, respectively.

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