Human JMJD2C tudor domain

Target ID:

JMJD2CA

Aliases:

bA146B14.1FLJ25949GASC1JHDM3CJMJD2CKDM4CKIAA0780

Deposition Date:

Thursday 6th May 2010

Families:

Oxidoreductases

Related Diseases:

CancerChromatin and epigenetics

Structure type:

apo

Download Coordinates:

2XDP

Authors:

W.W.Yue,C.Gileadi,T.Krojer,H.Weisbach,E.Ugochukwu,M.Daniel,C.Phillips,A.Chaikuad,F.vonDelft,C.Allerston,C.Arrowsmith,J.Weigelt,A.Edwards,C.Bountra,U.Oppermann

Site:

Oxford

2XDP

tabs

Structure Details

Post-translational modification of histone tails plays a key role in the regulation of functional chromatin organization and constitutes the "histone code" critical in epigenetic regulation. Methylation of lysine residues (Lys) of histones H3 and H4 is critical to such biological processes as heterochromatin formation, X chromosome inactivation, genome imprinting, DNA repair and transcriptional regulation. The effect of histone Lys modification is context dependent and relies on the particular residue and the methylation state (mono, di or tri), which confers specificity for the recruitment of chromatin remodeling components and adaptor molecules that dictate the downstream biological effects.

Histone Lys methylation has long been considered as a stable epigenetic mark, until the recent discovery of demethylase activity from members of the Jumonji C domain-containing, Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases, such as the JMJD2 family members (JMJD2A-JMJD2F), which demethylate the tri- and di-methylated states of histone H3K9 and H3K36. In addition to the Jumonji C domain which forms the hallmark of the demethylase core, certain members of the JMJD2 family (e.g. JMJD2A, JMJD2B, JMJD2C) contain a double-tudor domain which has been shown to recognize tri-methylated histone peptides. However there is a limited understanding of how these effector domains recognize histone residues in a site-specific and state-specific manner.

Here we have determined the crystal structure of the double-tudor domain of JMJD2C at 1.65 Å resolution. JMJD2C was originally identified by high-throughput retroviral tagging, as gene amplified in esophageal squamous carcinoma GASC1. Jmjd2c knockdown reduced tumour cell proliferation, while its overexpression upregulates Mdm2 oncogene expression dependent on its demethylase activity, suggesting a role of JMJD2C in transcriptional regulation and cancer development. The human jmjd2c gene is located at chromosome position 9p24.1.

Materials and Methods

Entry Clone Source: Origene

Entry Clone Accession: NM_015061 variant

SGC Construct ID: JMJD2CA-c705

GenBank GI number: gi|24307987

Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]

Amplified construct sequence:
CATATGCACCATCATCATCATCATTCTTCT
GGTGTAGATCTGGGTACCGAGAACCTGTAC
TTCCAATCCATGTGCGAGAAGGTCATTTCC
GTGGGTCAAACGGTCATCACGAAGCATCGG
AACACCCGGTATTACAGTTGCAGAGTGATG
GCTGTGACATCGCAGACCTTCTATGAGGTC
ATGTTTGATGATGGCTCCTTTAGCAGAGAC
ACATTTCCTGAGGATATCGTGAGCCGAGAC
TGTCTGAAGCTGGGCCCACCTGCTGAGGGA
GAAGTCGTCCAAGTCAAGTGGCCCGATGGC
AAACTCTATGGAGCAAAATATTTTGGATCA
AATATTGCCCACATGTACCAGGTTGAGTTT
GAAGATGGATCCCAGATAGCAATGAAGAGA
GAGGACATCTACACTTTAGATGAAGAGTTA
CCCAAGAGAGTGAAATGACAGTAAAGGTGG
ATACGGATCCGAA

Final protein sequence:
SMCEKVISVGQTVITKHRNTRYYSCRVMAV
TSQTFYEVMFDDGSFSRDTFPEDIVSRDCL
KLGPPAEGEVVQVKWPDGKLYGAKYFGSNI
AHMYQVEFEDGSQIAMKREDIYTLDEELPK
R

Tags and additions: N-terminal, TEV cleavable hexahistidine tag

Host: Trichoplusia Ni (High five)

Expression strain: BL-21(DE3)-R3-Rosetta (A homemade phage resistant version of BL21(DE3) containing the pRARE2 plasmid from Rosetta II (DE3) cells).
Transformation: The construct DNA was transformed into competent cells of the expression strain by a standard heat shock procedure.
Glycerol stock preparation: A number of colonies were used to inoculate 1 ml of LB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol, which was incubated in a 37°C shaker overnight. The next day glycerol stocks were prepared from this overnight culture.
Expression: 10 µl of a glycerol stock was used to inoculate 120 ml of LB containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol, which was incubated at 37°C overnight. 15ml starter culture was used per litre TB, containing 50 µg/ml kanamycin. The culture was incubated at 37°C until OD~1.5, when the temperature of the incubator was reduced to 18°C. At 18°C, expression was induced with 0.1 mM IPTG and the culture continued o/n.
Cell harvest: Cells were pelleted at 6238x g for 15 min at 4°C, and stored at -80°C. The yield was 12g cells / litre culture.

Cell Lysis: Lysis Buffer: 50mM HEPES pH 7.4, 500mM NaCl, 5% glycerol, 10 mM Imidazole pH 7, 1tbl Complete (mixture of proteinase inhibitors -EDTA).
The pellets were resuspended in lysis buffer. They were passed 4 times through an Emulsiflex C5 high-pressure homogeniser, collecting a final volume of approximately 25 ml/ litre culture. Cell debris and DNA were spun down at 45000x g, 60 min ( Beckman JA 18 17500 rpm). The supernatant was collected to which Benzonase was added with a 30-60 min incubation on ice.

Buffers:
Wash Buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 30 mM Imidazole pH 7.4
Elution Buffer:50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 250 mM Imidazole pH 7.4
Gel Filtration Buffer: 50 mM Hepes pH 7.4, 500 mM NaCl , 5% glycerol

Purification: Purification was performed in an Akta Express system (GE Healthcare) with an automated program for Ni-affinity (HisTrap FF) and gel filtration (HiLoad 16/60 S75) chromatography steps.
Step 1: Ni-affinity, HisTrap Crude FF, 5 ml. The clarified cell extract was loaded on the column at 5 ml/min on the ÄKTA-express system. The column was washed with 15 cv of binding buffer, 10 cv of wash buffer, and then eluted with elution buffer at 4 ml/min. The eluted peak of A₂₈₀ was automatically collected.
Step 2: Gel filtration, Hiload 16/60 Superdex S75 prep grade, 120 ml. The eluted fraction from the Ni-affinity column was loaded on the gel filtration column, pre-equilibrated in GF buffer, at 1.2 ml/min. Eluted proteins were collected in 2 ml fractions and analyzed on SDS-PAGE.

TEV protease digestion: Peak fractions from the gel filtration containing JMJD2CA were pooled and TEV protease was added at a molar ratio of 1:30. The digestion was left overnight at 4 °C. SDS-PAGE confirmed complete digestion. His-TEV and contaminating proteins were removed by binding to Ni resin, pre-equilibrated in GF buffer. The flow through containing TEV-cleaved protein was collected and concentrated using a centricon centrifugal device with a 10kDa MWCO. The concentrated protein was centrifuged 20 min 14000rpm, 4°C. The final concentration of protein was 20 mg/ml and yield 3.3 mg/litre culture. The protein was flash frozen and stored at -80°C in 70µl aliquots.

Mass spectrometry characterization: Measured: 13908.9; Expected: 13907.7. MSMS confirmed that the protein was the correct target.

Crystallisation: Crystals were grown at 4°C by vapour diffusion in sitting drops mixing protein (20mg/ml) and precipitant solution (0.2M (NH₄)₂SO₄; 0.1M TRIS pH 8.5; 25% PEG 3350) in a 1:2 ratio. Crystals were cryo-protected in 25% glycerol and flash frozen in liquid nitrogen.

Data collection: Data was collected to a resolution of 1.65 Å at Diamond beamline IO3.