Structural Insights into How 5-hydroxymethylation Influences Transcription Factor Binding

Transcription factor binding and high resolution crystallographic studies (1.3 Å) of Dickerson-Drew duplexes with cytosine, methylcytosine and hydroxymethylcytosine bases provide evidence that C-5 cytosine modifications could regulate transcription by context dependent effects on DNA transcription factor interactions.


Additional background information
Crystallographic investigations of B-DNA reveal high similarity between C and 5mC duplexes, with methylation causing slight minor groove compaction 11 .Structures of 5mC in Z- 12,13 , A- 14,15 and E- 16 DNA forms (in CpG contexts) and of 5mC-binding proteins (MBD 17 , SRA 18 , zinc finger 19 ) complexed with 5mC DNA are also reported.A MBD4 structure in complex with 5hmC containing DNA is reported 20 , but the relatively low resolution (2.4 Å) and presence of protein, does not enable analysis of the effect of 5hmC on isolated dsDNA structure.

Oligonucleotide synthesis, purification and analysis
Standard DNA phosphoramidites, solid supports and additional reagents were purchased from Link Technologies and Applied Biosystems Ltd.Oligonucleotides were synthesized using an Applied Biosystems 394 automated DNA/ RNA synthesizer using a standard 1.0 mole phosphoramidite cycle of acid-catalyzed detritylation, coupling, capping, and iodine oxidation.Stepwise coupling efficiencies and overall yields were determined by an automated trityl cation conductivity monitoring facility and in all cases were >98.0%.-Cyanoethyl phosphoramidite monomers were dissolved in anhydrous acetonitrile to a concentration of 0.1 M immediately prior to use.The coupling time for normal A, G, C, and T monomers was 35 s, and the coupling time for the 5-methyl-2ˊ-deoxycytidine monomer was 60 s and for 5-hydroxmethyl-2ˊ-deoxycytidine monomer (5hmC) it was extended to 360 s.Cleavage of the oligonucleotides from the solid support and deprotection was achieved by exposure to concentrated aqueous ammonia solution (60 min.room temp.)followed by heating in a sealed tube (72 h, 65 °C).The fully deprotected oligonucleotides were purified by reversed-phase HPLC on a Gilson system using a Luna 10 µL C8 100Å pore Phenomenex 10x250 mm column with a gradient of acetonitrile in ammonium acetate (0% to 50% buffer B over 20 min, flow rate 4 mL/min), (buffer A: 0.1 M ammonium acetate, pH 7.0, buffer B: 0.1 M ammonium acetate, pH 7.0, with 50% acetonitrile).Elution was monitored by UV absorption at 295 nm.After HPLC purification, oligonucleotides were desalted using NAP-10 columns (GE Healthcare).The purified oligonucleotides were characterized by mass spectra on a Bruker micrOTOF TM II focus ESI -TOF MS instrument in ES -mode.

Crystallography
Crystals were grown at 20˚C by the hanging drop vapour diffusion method in 24 well Linbro plates using 22mm round cover slips and sealed using vacuum grease using the conditions in Supplementary Table S1.Crystals appeared within a few days and were harvested after 1 week.The MPD in the equilibrated drops was sufficient to serve as cryo-protectant and crystals were harvested directly from the crystal growth drop using a nylon loop and plunged into liquid nitrogen to cyro-cool.Crystals were stored under liquid nitrogen until they were mounted on a goniometer at the synchrotron beamline under a nitrogen gas stream at 100K.Complete data sets were collected for single crystals of each modification type and independently indexed, integrated and scaled using Xia2 21 or XDS/SCALA 22 as indicated.The structures were solved by molecular replacement using PHASER 23 and PDB entry 1BNA 24 and refined using PHENIX 25 .

Protein methods
MAX and USF were cloned from isolated cDNA into pGEM-T Easy vectors (Promega) using the following primers MAX_FW: ATGAGCGATAACGATGACATCGAGGTGG, MAX_RW: TTAGCTGGCCTCCATCCGGAGCTTC, USF_FW: ATGAAGGGGCAGCAGAAAACAGCTG, USF_RW: GGCCCAAAGCCCCTGAATCCCCA. MAX was subsequently subcloned into pET15b vector.MAX was expressed in BL21(DE3) Rosetta 2 and purified by IMAC and gel filtration.Plasmids encoding for hypoxic inducible factor (HIF)1-α and HIF1-β were a kind gift from Prof. Christoph W. Pugh.USF and Hif1-α/β were produced by IVTT using the TnT® Coupled Reticulocyte Lysate System (T7 for HIF, SP6 for USF; Promega; L4601 and L4611) using the manufacturer recommended conditions.For the control lanes, the manufacturer supplied luciferase plasmid was added to the IVTT mixture instead of the HIF1-α and -β plasmids. 26
The EPO template corresponds to the sequence responsible for the hypoxic upregulation of the erythropoietin / epo gene that contains a HRE (+97-156bp from the Electrophoretic mobility shift assays (EMSAs) 6 For assays with hypoxic inducible factor (HIF), the binding assays contained 2-5 µL of the IVTT mixture, 2 µL 10x binding buffer (final: 10 mM Tris pH 7.4, 50 mM NaCl, 50 mM KCl, 1 mM Ethylenediaminetetraacetic acid (EDTA), 5 mM DTT, 5% glycerol), 75ng dI/dC (1 µg/µL stock) diluted to 19 µL with water and incubated on ice for 10 min.Then 1 µL of radio-labeled DNA probe (0.08 ng/µL diluted from stock; see above for sequence) was added and the mixture was incubated on ice for an additional 30 min.The complete 20 µL EMSA mixture was then loaded onto a 5% PAGE gel (0,5x TBE (45 mM Tris-borate, 1 mM EDTA, pH 8.3), 0.7 mm, cast 1 day in advance and prerun at 240 V for 1h) and run at 240 V for 3.45-4 hours.The gel was put on Whatman paper, covered with saran wrap and dried at 80°C for 1 h.A phosphoscreen (Biorad) was exposed to the gel for 24-72 h and imaged using a Personal Molecular Imager (PMI, Biorad).Images were processed using the Quantity one analysis software (Biorad).
) Overlay of the cytosine (green; 4C64), 5-methylcytosine (cyan; 4C63) and 5hydroxymethylcytosine (pink; 4C5X) containing dsDNA.The sites of modification are marked with arrows.(B) Sequence of the used duplex with numbering.Modified sites are underlined.

A
Titration curves of MAX (A) and USF (B) measured by electrophoretic mobility shift assays (EMSAs) with radiolabeled dsDNA probes bearing different cytosine C-5 modifications at the central CpG of the E-Box sequence.The EMSA modification state of the cytosine in the central CpG of each series is given in the legend.Experiments were performed in triplicate.For detailed experimental procedure and used sequences see Supplemental Methods.(C) The affinity of MAX for the E-Box sequences containing different cytosine modifications as determined by EMSAs 6 .In the case of mC:mC and hmC:hmC only weak / non-specific binding was observed as evidenced by a slight reduction of free probe but no formation of a distinct complex.(nd: not determined) (D) Competition of a radiolabeled unmodified E-Box-USF complex with unlabeled probes containing different modifications.The central CpG modification state is given above the lanes.Electronic Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2014 Supplementary Figure S5 Structural overlay of the hmC9 containing duplex (4C5X) with the MAX-DNA complex (A; adapted from pdb entry 1AN2 7 ) and the USF-DNA complex (B; adapted from pdb entry 1AN4 3 ) showing the potential clash of the C5 modification with the arginine (Arg35 in MAX, Arg211 in USF).Both the mayor (A; 70% occupancy) and the minor (B; 30% occupancy) of the hmC9 alcohol are depicted.Electronic Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2014 Supplementary Figure S6 Modification of the central CpG in the hypoxic response element (HRE; ACGTG) abolishes binding to hypoxia-inducible factor (HIF).Full-length HIF1 and HIF1β (ARNT) were first produced by in vitro transcription translation (IVTT) and then incubated with radiolabeled DNA probes containing a HRE (see experimental section for sequence).The obtained complexes were separated on a 5% PAGE gel.(A) EMSA with IVTT HIF and different unmodified, fully methylated or fully hydroxymethylated probes derived from the erythropoietin (EPO) promoter 6, 8 .Different modifications were introduced by substitution of dCTP with dmCTP or dhmCTP during the PCR amplification 9 .(B) Titrations of increasing amounts of IVTT translated HIF1-α/β with differently modified probes.Electronic Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2014 Supplemental Figure S7.Structural overlay of the hmC9 containing duplex (4C5X) with the CLOCK-DNA complex (A) and the BMAL1-DNA complex (B; both adapted from pdb entry 4H10 5 ) showing the potential clash of the C5 modification with the arginine (Arg47 in CLOCK, Arg85 in BMAL1).Both the mayor (A; 70% occupancy) and the minor (B; 30% occupancy) of the hmC9 alcohol are depicted.
Electronic Supplementary Material (ESI) for Chemical CommunicationsThis journal is © The Royal Society of Chemistry 2014 10pplementary TableS2.Crystallographic data collection, processing and structure refinement statistics.*Note:Thehighredundancydatafor the 5mC structure contributes to the exaggerated high Rmeas in the high resolution bin -the I/σI value in this case is a more reasonable measure of the data quality.Supplementary TableS4Sugar pucker values and conformations for different duplexes calculated with 3DNA10.The differently modified base is highlighted.