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. Author manuscript; available in PMC: 2016 Jul 12.
Published in final edited form as: Methods Enzymol. 2016 Feb 16;573:387–401. doi: 10.1016/bs.mie.2016.01.019

Characterization of How DNA Modifications Affect DNA Binding by C2H2 Zinc Finger Proteins

A Patel 1, H Hashimoto 1, X Zhang 1,1, X Cheng 1,1
PMCID: PMC4941819  NIHMSID: NIHMS799957  PMID: 27372763

Abstract

Much is known about vertebrate DNA methylation and oxidation; however, much less is known about how modified cytosine residues within particular sequences are recognized. Among the known methylated DNA-binding domains, the Cys2-His2 zinc finger (ZnF) protein superfamily is the largest with hundreds of members, each containing tandem ZnFs ranging from 3 to >30 fingers. We have begun to biochemically and structurally characterize these ZnFs not only on their sequence specificity but also on their sensitivity to various DNA modifications. Rather than following published methods of refolding insoluble ZnF arrays, we have expressed and purified soluble forms of ZnFs, ranging in size from a tandem array of two to six ZnFs, from seven different proteins. We also describe a fluorescence polarization assay to measure ZnFs affinity with oligonucleotides containing various modifications and our approaches for cocrystallization of ZnFs with oligonucleotides.

1. INTRODUCTION

The control of gene expression in mammals relies significantly on the modification status of DNA cytosine residues. DNA cytosine modification is a dynamic process catalyzed by specific DNA methyltransferases (DNMTs) that convert cytosine (C) to 5-methylcytosine (abbreviated 5mC or M; Bestor, Laudano, Mattaliano, & Ingram, 1988; Okano, Xie, & Li, 1998), usually within the sequence context of CpG (Bestor et al., 1988; Okano, Bell, Haber, & Li, 1999; Okano et al., 1998) or CpA (Gowher & Jeltsch, 2001; Kubo et al., 2015; Lister et al., 2013, 2009; Ramsahoye et al., 2000; Vlachogiannis et al., 2015). A subset of 5mC may then be oxidized to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) by the ten-eleven translocation (Tet) dioxygenases in three consecutive Fe(II) and α-ketoglutarate-dependent oxidation reactions (He et al., 2011; Ito et al., 2010, 2011; Tahiliani et al., 2009).

The best-known modified DNA-recognition domains are two that recognize methylated cytosine: methyl-binding domains (MBDs) recognize fully methylated CpG dinucleotides (Dhasarathy & Wade, 2008; Guy, Cheval, Selfridge, & Bird, 2011), and “SET and RING finger-associated” (SRA) domains that bind hemimethylated CpG sites generated transiently by DNA replication (Hashimoto, Horton, Zhang, & Cheng, 2009; Sharif & Koseki, 2011; reviewed in Hashimoto, Zhang, Vertino, & Cheng, 2015; Liu, Zhang, Blumenthal, & Cheng, 2013). Both MBD and SRA domains have been structurally characterized in complexes with 5mC (Arita, Ariyoshi, Tochio, Nakamura, & Shirakawa, 2008; Avvakumov et al., 2008; Hashimoto et al., 2008; Ho et al., 2008; Ohki et al., 2001; Scarsdale, Webb, Ginder, & Williams, 2011).

A third class of mammalian proteins that can recognize methylated DNA is the Cys2-His2 (C2H2) zinc finger (ZnF) proteins, which can preferentially bind to methylated CpG within a longer specific sequence (Sasai, Nakao, & Defossez, 2010). Kaiso is the first known methyl-binding ZnF protein that belongs to the BTB/POZ family (Prokhortchouk et al., 2001), which also includes ZBTB24, whose mutations are associated with immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome (Cerbone et al., 2012; Chouery et al., 2012; de Greef et al., 2011; Nitta et al., 2013), a disease also caused by mutations in a DNA methyl-transferase gene, DNMT3B (Hansen et al., 1999; Okano et al., 1999; Shirohzu et al., 2002; Xu et al., 1999). Recently, ZnF DNA-binding domains from five proteins, Kaiso, Zfp57, Klf4, Egr1, and WT1, have been structurally analyzed in complex with their respective methylated DNA elements (Buck-Koehntop et al., 2012; Hashimoto et al., 2014; Liu et al., 2014; Liu, Toh, Sasaki, Zhang, & Cheng, 2012; Zandarashvili, White, Esadze, & Iwahara, 2015), allowing comparison to other 5mC-binding proteins. In addition, WT1 binds 5caC DNA, as does a mutant Zfp57 (Hashimoto et al., 2014; Liu, Olanrewaju, Zhang, & Cheng, 2013).

Among the C2H2 ZnF proteins, KRAB-ZnF transcription factors (KRAB-ZnFs) act mostly as chromatin-modulating transcription repressors (Meylan et al., 2011). Of the >300 human or mouse KRAB-ZnF proteins examined, the number of tandem ZnFs ranges from 3 to 35, with a mode of around 11–13 fingers (Liu, Zhang, et al., 2013; Fig. 1A and B). The domain structures of a few examples of mammalian KRAB-ZnF proteins with known biological roles are shown (Fig. 1C). ZFP57 mutations have been found in patients with transient neonatal diabetes (Mackay et al., 2008). Zfp809 restricts retroviral transposition in embryonic stem cells (Wolf & Goff, 2009), and retroviral silencing has been suggested to be the ancestral role of KRAB-ZnFs (Thomas & Schneider, 2011). Regulator of sex limitation (Rsl1) regulates sex- and tissue-specific promoter methylation (Krebs, Schultz, & Robins, 2012). Zfp568 regulates extraembryonic tissue morphogenesis (Garcia-Garcia, Shibata, & Anderson, 2008). Like Zfp57 (Quenneville et al., 2011; Zuo et al., 2012), ZNF274 recruits the histone H3 lysine 9 methyltransferase SETDB1 (SET domain, bifurcated 1; Frietze, O’Geen, Blahnik, Jin, & Farnham, 2010) via the corepressor TRIM28 (tripartite motif-containing 28; also known as KAP1 for Krüppel-associated protein), an essential regulator of genomic imprinting (Messerschmidt et al., 2012). PRDM9 (PR domain zinc finger protein 9), a major determinant of meiotic recombination hotspots, contains a SET domain that methylates histone H3 lysine 4 (Mihola, Trachtulec, Vlcek, Schimenti, & Forejt, 2009). These examples further illustrate the coordinated chromatin controls between DNA methylation and the lysine methylation status of histone H3 (at residues 4 and 9; Cheng & Blumenthal, 2010).

Fig. 1.

Fig. 1

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KRAB-ZnF proteins. The SysZNF database was examined for Krüppel-associated box (KRAB)-ZnF proteins in human (A) or mouse (B). In each case, the distribution of KRAB-ZnF proteins containing a given number of ZnF repeats is shown. (C) Examples of mammalian KRAB-ZnF proteins with known biologic roles. The size of each protein (in amino acids) is shown on the right. The classic C2H2 ZnF motifs are shown in blue (gray in the print version) boxes and the gray or open boxes indicate degenerate ZnFs that contain mutations affecting zinc coordination. The red (gray in the print version) boxes of mouse Zfp57 and human PRDM9 indicate the structurally characterized ZnFs in complex with their recognition sequences shown below. The amino acids within the red (gray in the print version) box indicate the three or four residues of each ZnF involved in base specific interactions. The SCAN box, a leucine-rich region, was named after SRE-ZBP, CTfin51, AW-1 (ZNF174), Number 18 cDNA (ZnF20) (Williams, Khachigian, Shows, & Collins, 1995). The SET domain was named after Su(var)3–9, Enhancer of zeste, Trithorax (Jenuwein, Laible, Dorn, & Reuter, 1998). Modified from Liu, Y., Zhang, X., Blumenthal, R. M., & Cheng, X. (2013). A common mode of recognition for methylated CpG. Trends in Biochemical Sciences, 38, 177–183.

In the last few years, we have biochemically and structurally characterized mouse Zfp57 (2 ZnFs) (Liu, Olanrewaju, et al., 2013; Liu et al., 2012), mouse Krüppel-like factor 4 (Klf4) (3 ZnFs) (Liu et al., 2014), human early growth response protein (Egr1, also known as Zif268) (3 ZnFs) (Hashimoto et al., 2014), human Wilms tumor protein (WT1) (3 ZnFs) (Hashimoto et al., 2014), human PRDM9 allele-A (5 ZnFs) (Patel, Horton, Wilson, Zhang, & Cheng, 2016), human PRDM9 allele-C (6 ZnFs) (Patel et al., unpublished), and human CTCF (4 ZnFs) (Hashimoto et al., unpublished). Rather than following published methods of refolding insoluble proteins, such as WT1 (Laity, Chung, Dyson, & Wright, 2000) and Egr1/Zif268 (Pavletich & Pabo, 1991), we expressed and purified the tandem array of ZnF DNA-binding domains in soluble form as fusion proteins with glutathione S-transferase (GST). One key to our success in obtaining quality ZnF protein suitable for biochemistry analysis and crystallization is the use of polyethylenimine (PEI), a polymer with repeating unit composed of the amine and two carbon aliphatic spacer, and anion exchange column to completely remove bacterial nucleic acids associated with the ZnF proteins.

2. SOLUBLE EXPRESSION AND PURIFICATION OF ZnF PROTEINS

ZnF cDNA fragments were cloned into the BamHI site of pGEX6p-1 vector (GE Healthcare), leaving five extra residues at the N-terminal, Gly-Pro-Leu-Gly-Ser, after PreScission protease cleavage.

2.1 Expression

  1. Day 1: Inoculate 15 mL of noninducing MDAG media (Studier, 2005) supplemented with 100 µg/mL sterile ampicillin, with a single colony or glycerol freezer stock of Escherichia coli BL21 (DE3) Codon-plus RIL cells containing the expression plasmid. Incubate overnight with shaking at 37°C.

  2. Day 2: Inoculate 1 L LB medium supplemented with 100 µg/mL ampicillin with 2 mL of the starter culture. Grow at 37°C with shaking at 200 rpm until the A600 reaches ~0.5 when the shaker temperature is reduced to 16°C.
    Note: Monitor the liquid temperature with a thermometer until it has reached 16°C, which can take 0.5–1.5 h depending on shaker. Add 200 µL of 0.5 M ZnCl2 (to a final concentration of 100 µM) and induce the expression of protein by adding isopropyl β-d-1-thiogalactopyranoside (IPTG) to 0.2 mM final concentration. Incubate overnight with shaking at 16°C.

  3. Day 3: Harvest the cells by centrifugation at 3500 rpm for 30 min at 4°C and freeze the pellets at −20°C until purification.

2.2 Purification

The general scheme involves four-column chromatography (Fig. 2A).

Fig. 2.

Fig. 2

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One example of ZnF protein purification. (A) The general scheme of four-column chromatography. Inserted is a picture of tandem Q-SP columns. (B) A 12% SDS-PAGE showing total lysate (lane 2), supernatant after PEI treatment (lane 3), flowthrough (FT; lane 4), and washing through the GST column (lanes 5 and 6), five elutions by GSH (lanes 7–11), and before and after PreScission protease cleavage (lanes 12 and 13). Note that more soluble protein appears after PEI treatment (comparing lanes 2 and 3). (C) Elution profile of the HiTrap Q column. (D) Elution profile of the HiTrap SP column and an accompanying SDS-PAGE showing the fractions. (E) Elution profile of a Superdex-200 (16/60 GL) column and an accompanying SDS-PAGE showing the fractions.

Day 1

  1. Resuspend cells from 6 L culture into 120 mL of lysis buffer [20 mM Tris (pH 7.5), 5% (v/v) glycerol, 25 µM ZnCl2, 0.5mM tris(2-carboxyethyl) phosphine (TCEP), and 0.1 mM phenylmethylsulfonyl fluoride (PMSF)] containing 250–700 mM NaCl.
    Note: The salt concentration needs to be individually determined to assure maximum solubility.

  2. Lyse cells by sonication with 1 s on and 2 s off cycles for 8 min in total.

  3. Treat the lysate with PEI (Sigma—408727) neutralized by HCl to pH 7.
    Note: Slowly add 6 mL of 2% (w/v) PEI solution drop by drop into the lysate to a final concentration of 0.1% while stirring on an ice bath.

  4. Clear the lysate by centrifugation at 16,500 rpm for 45 min at 4°C.

  5. Load the supernatant onto a Glutathione Sepharose 4B column (GE Healthcare) with 5 mL bed volume equilibrated with 250–700 mM NaCl lysis buffer (see Step 1) at ~0.7 mL/min flow rate. Wash the column with 40 mL of lysis buffer followed by 25 mL of washing buffer containing 100 mM Tris (pH 8.0), 500 mM NaCl, 5% glycerol, 25 µM ZnCl2, and 0.5 mM TCEP. Elute the GST-tagged protein with 50 mL of elution buffer (washing buffer + 20 mM reduced glutathione) into fractions of 10 mL (Fig. 2B).

  6. Remove the GST tag by treating the eluted protein with ~100 µg of PreScission protease (GE Healthcare 27-0843-01 or purified in-house) at 4°C overnight.

Day 2

  1. Load the protein onto 5 mL HiTrap Q-SP columns connected in tandem (GE Healthcare; Fig. 2A), equilibrated with column buffer (lysis buffer minus PMSF) with 500 mM NaCl. After washing 20 mL with the same buffer, disconnect the Q and SP columns and elute separately with a 50 mL (or 75) linear gradient of 0.5–1 M NaCl (Fig. 2C and D).
    Note: DNA-free protein flows through the Q column and binds the SP column, while the DNA-containing protein binds to the Q column along with free DNA (Fig. 2C).

  2. Concentrate the protein eluted from the SP column to ~2 mL using a centrifugal concentrator such as Vivaspin. Load onto a Superdex-200 (16/60) column equilibrated with column buffer with 500 mM NaCl. Collect the protein eluted as a single peak (Fig. 2E). Concentrate to about 5 mg/mL and flash freeze with liquid nitrogen and store at −80°C in aliquots. Final yields of the protein range from 10 to 15 mg/6 L culture.

3. FLUORESCENCE POLARIZATION ASSAY FOR ANALYSIS OF DNA BINDING

  1. Synthesize 5′-FAM (6-carboxyfluorescein)-labeled oligonucleotides containing various cytosine modifications. Only one strand is labeled.
    Note: Generally, blunt-ended DNA duplex is preferred, although sometimes labeling at one (or two) base overhang at the 5′-end yields better signal (Hashimoto et al., 2014; Liu et al., 2014, 2012). The FAM label may not be compatible with 5caC modification within the same strand due to currently available synthesis chemistry.

  2. Mix twofold serially diluted protein solutions (1–10 µMstarting concentration, 10–15 points) with 1–5 nMfinal concentration of DNA probe in a Corning 3575 plate, using binding buffer of 20 mM Tris–HCl, pH 7.5, 5% glycerol, and 0.5 mM TCEP with varying NaCl concentration (150–300 mM). Incubate the mixture for 10 min at room temperature. Perform at least two duplicate experiments.

  3. Measure fluorescence polarization at 25°C on a Synergy 4 Microplate Reader (BioTek) using 485/20 nm and 528/20 nm filters for emission and excitation, respectively.
    Note: The presence of protein should cause no change in fluorescence intensity.
  4. Calculate the dissociation constants (KD) by fitting the experimental data to the following equation using GraphPad Prism software (version 6.0): [mP]=[maximum mP] × [C] / (KD + [C]) + [baseline mP], then replot the curve using % of saturation calculated as ([mP] − [baseline mP])/([maximum mP] − [baseline mP]), where mP is millipolarization and [C] is protein concentration.
    Note: The maximum increase of mP observed is protein and/or oligonucleotide dependent, and is most commonly between 50 and 100 mP. Very large mP change (>200) at high protein concentration (>1 µM) often indicates nonspecific binding.
  5. Effect of NaCl concentration: The KD values are extremely sensitive to the ionic strength of the binding buffer. Fig. 3 illustrated that the WT1 + KTS isoform binds most strongly to 5caC-containing DNA. Affinity is uniformly low in 300 mM NaCl (Fig. 3A) but considerably higher (>10-fold) in 200 mM NaCl (Fig. 3B; Hashimoto et al., 2014). The effect is even more pronounced for human PRDM9 allele-A: increasing NaCl concentration by 20 mM can result in as much as 3-fold increase in KD value (Fig. 3C), and a 50 mM increase of NaCl resulted in an 18-fold reduction in affinity. As documented in previous studies (Jantz & Berg, 2010), the double-logarithmic plot of KD as a function of NaCl concentration is linear (Fig. 3D).
    Note: Due to extreme salt sensitivity of the assay, different batches of the same buffer formulation can give noticeably different KD values. When possible, use the same batch of buffer for all assays in one study.

Fig. 3.

Fig. 3

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The effect of NaCl on binding affinity. The DNA-binding affinity of WT1 + KTS isoform is uniformly low in 300 mM NaCl (A) but increases markedly in 200 mM NaCl (B). (C) The DNA-binding affinity (KD) of human PRDM9 allele-A under NaCl concentrations from 260 to 310 mM with 10–20 mM increments. (D) The linear correlation of double-logarithmic plot of KD values and NaCl concentrations. Panel (B): Adopted from Hashimoto, H., Olanrewaju, Y. O., Zheng, Y., Wilson, G. G., Zhang, X., & Cheng, X. (2014). Wilms tumor protein recognizes 5-carboxylcytosine within a specific DNA sequence. Genes & Development, 28, 2304–2313.

4. CRYSTALLIZATION OF ZnF PROTEINS IN COMPLEX WITH DNA

  1. Design of oligonucleotide suitable for cocrystallization: both the length and the ends must be considered. Fig. 4 illustrates the process of obtaining diffraction quality crystals for human PRDM9 allele-A ZnF 8–12 by varying oligonucleotides. We started with a 15 + 1 base pairs (bp) double-stranded oligonucleotide (oligo)—the minimum length required for recognition by five ZnFs—plus a 5′-overhanging thymine or adenine on either strand (Fig. 4). This design was then lengthened 1 or 2 bp at a time to become 16 + 1 (1 bp increase on one end), 17 + 1 (1 bp increase on both ends), 18-, 19-, and 20-bp blunt ends, and 18 + 1, 19 + 1, and 20 + 1 bp with 5′-overhangs. In the end, only the 20 + 1 oligo yielded high-diffraction quality crystals. Alternatively, 3′-overhangings or asymmetric overhanging only on one strand could also be used.

  2. Purification of crude oligonucleotides: Due to cost consideration, we use crude unmodified oligos to screen the length and ends. Resuspend each single-stranded DNA into annealing buffer containing 10 mM Tris (pH 8.0), 50 mM NaCl, and 1 mM EDTA (ethylenediaminetetraacetic acid) to final concentration of ~1 mM. Mix complimentary stands of DNA in equimolar ratio, heat in a boiling water bath that is slowly cooled overnight to room temperature. Load the annealed double-stranded (ds) DNA to a 5 mL HiTrap Q column with 20 mM Tris (pH 8.0) as buffer A and 20 mM Tris (pH 8.0) and 1 M NaCl as buffer B. Collect DNA eluted as a single large peak between 0.55 and 0.65 MNaCl using a linear gradient of NaCl from 0.1 to 1 M. Pool peak fractions and measure DNA concentration by absorbance at 260 nm.

  3. Protein–DNA complex formation by dialysis: Mix ZnF protein with purified dsDNA in 1:1 molar ratio to a final concentration of 25 µM each in buffer containing 20 mM Tris (7.5), 500 mM NaCl, 5% glycerol, 25 µM ZnCl2, and 0.5 mM TCEP. Dialyze the mixture against low salt buffer (150–250 mM NaCl) at 4°C with two 500 mL changes of buffer every 6–8 h. The slow exchange to low salt buffer can potentially reduce nonspecific binding and promote specific complex formation between DNA and protein. [Alternatively, modified oligos are often HPLC or PAGE purified after synthesis, thus can be directly mixed with protein at high concentration (~1 mM).] After dialysis, centrifuge the protein–DNA complex at 4000 rpm for 15 min to remove any precipitate. Concentrate the supernatant to desired concentration and centrifuge at 13,000 rpm for 10 min before crystallization trial.

Fig. 4.

Fig. 4

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Examples of DNA oligonucleotide sequences used for cocrystallization with human PRDM9 allele-A, crystals observed, and quality of X-ray diffractions. Five examples of crystals and corresponding conditions were shown (A–E).

Acknowledgments

We sincerely thank Yusuf Olatunde Olanrewaju and Yiwei Liu for their early efforts in purifying soluble ZnF proteins. This work was supported by grant from the National Institutes of Health GM049245-22 to X.C. (who is a Georgia Research Alliance Eminent Scholar).

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