Human Jumonji/ARID domain-containing protein 1C

Target ID:

JARID1CA

Aliases:

DXS1272EJARID1CKDM5CMRXJMRXSJSMCXXE169

Deposition Date:

Friday 29th June 2007

Families:

Oxidoreductases

Related Diseases:

Chromatin and epigeneticsNeurobiology

Structure type:

apo

Download Coordinates:

2JRZ

Authors:

C. Koehler, S. Bishop, E.F. Dowler, A. Diehl, P. Schmieder, M. Leidert, M. Sundstrom, C.H. Arrowsmith, J. Wiegelt, A. Edwards, H. Oschkinat and L.J. Ball.

Site:

Oxford

2JRZ

tabs

Structure Details

The JAIRD1C protein (Jumonji/ARID domain-containing protein 1C, formerly known as SMCX) is a member of the recently discovered ARID (AT-rich interaction domain) family of DNA-binding proteins, found in fungi and invertebrate and vertebrate metazoans. The proteins are involved in a variety of biological processes including embryonic development, cell lineage gene regulation and cell cycle control. Although the specific roles of this domain and of ARID-containing proteins in transcriptional regulation are still unclear, they include both positive and negative transcriptional regulation and are also thought to include the modification of chromatin structure. JARID1C has been proposed to act as histone H3 lysine 4 demethylase and to be involved in transcriptional repression. Mutations in JARID1C are a known cause of X-linked mental retardation (XLMR) and epilepsy, suggesting that JARID1C plays a role in human brain function (Tahiliani et al., 2007; Tzschach et al., 2006, Jensen et al. 2005).

The overall composition of JARID proteins includes a JmjN domain, a Bright/ARID domain, a JmjC domain, a C5HC2 zinc finger domain, and PHD zinc finger domains. The basic structure of the Bright/ARID domain comprises a series of six alpha-helices separated by beta-strands, loops, or turns, but the structured region may extend to an additional helix at either or both ends of this basic fold (Iwahara et al., 2002; Yuan et al., 1998; Treisman et al, 1997). Based on primary sequence homology, they can be partitioned into three structural classes: Minimal ARID proteins that consist of a core domain formed by six alpha helices; ARID proteins that supplement the core domain with an N-terminal alpha-helix; and Extended-ARID proteins, which contain the core domain and additional alpha-helices at their N- and C-termini.

The approximately 100-residue ARID sequence is present in a series of proteins strongly implicated in the regulation of cell growth, development, and tissue-specific gene expression. Although about a dozen ARID proteins can be identified from database searches, to date, only Bright (a regulator of B-cell-specific gene expression), dead ringer (a Drosophila melanogaster gene product required for normal development), and MRF-2 (which represses expression from the cytomegalovirus enhancer) have been analyzed directly in regard to their DNA binding properties. Each binds preferentially to AT-rich sites. In contrast, the human SWI-SNF complex protein p270 shows no sequence preference in its DNA binding activity, thereby demonstrating that AT-rich binding is not an intrinsic property of ARID domains and that ARID family proteins may be involved in a wider range of DNA interactions.

Here we present the NMR solution structure of the DNA-binding Bright/ARID domain from human JARID1C.

Materials and Methods

JARID1C

PDB:2JRZ

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|11321605
Entry Clone Source:MGC
SGC Clone Accession:JARID1CA-c556
Tag:N-terminal, TEV cleavable (*) hexahistidine tag.
mhhhhhhssgvdlgtenlyfq(*)sm
Host:BL21(DE3)-R3-pRARE2

Construct

Prelude:
Sequence:smNELEAQTRVKLNYLDQIAKFWEIQGSSLKIPNVERRILDLYSLSKIVVEEGGYEAICKDRRWARVAQRLNYPPGKNIGSLLRSHYERIVYPYEMYQSGANLVCNTRPFDNEEKDK
Vector:pNIC28-Bsa4.

Growth

Medium:
Antibiotics:
Procedure:BL21(DE3)-Rosetta competent cells were transformed with the expression plasmid and plated on LB plates containing 25 µg/ml kanamycin and 34µg/ml chloramphenicol.
5 Colonies from the transformation were used to inoculate 5 times 5 ml LB containing 25 µg/ml kanamycin and 34 µg/ml chloramphenicol. The cells were then grown at 250 RPM, 37°C up to OD 0.65 then induced with 1 mM IPTG for 3 h. Expression of the 5 clones was analyzed by SDS-PAGE. The best clone was used for large scale expression in either [¹³C, ¹⁵N]-M9 medium, containing 0.5g [¹⁵N]-NH4Cl and 2g [¹³C]-glucose per L.

Starter overnight cultures of 2 x 50 ml were grown at 37°C in LB medium supplemented with 25 µg/ml kanamycin and 34 µg/ml chloramphenicol. The cells of 40 ml preculture were collected and resuspended in 10 ml M9 medium for inoculation of the large scale culture of 5 x 400 ml M9 with appropriate antibiotics in 5 x 2 L bottles (dilution for inoculation 1:80). The culture was grown at 37°C and transferred to 18°C when the OD600 reached a value of 0.6. The culture was induced with 0.5 mM IPTG and grown at 18°C overnight. The next day the cells were harvested by centrifugation, washed with ice-cold 150 mM NaCl and frozen at -80°C.

Purification

Procedure
Column 1: Ni-affinity, MC-POROS, 8 ml (Applied Biosystems)
Column 2: Superdex 75, 320 ml

The cell extract was loaded on the column at 1 ml/min on a workstation Vision (Applied Biosystems). The column was washed with 10 column volumes of start buffer and eluted with a gradient from 5 to 500 mM imidazole at a flow rate of 5 ml/min. The extinction at 280 nm was monitored and fractions were collected and analyzed by SDS-PAGE. Positive fractions were pooled and filled into a dialysis bag with 3.5 kDa MW cutoff.

Dialysis and TEV-cleavage: The His-tag was cleaved with 1 mg TEV per 40 mg target protein in a dialysis bag and dialysed with 5 L of: 20 mM Tris HCl 8.0, 500 mM NaCl, 1 mM beta-mercaptoethanol, at 15°C overnight. The digest was checked by SDS-Page, as the cleavage was completed, the sample was supplemented with 5 mM arginine and glutamine concentrated to 3 ml on a 5 kDa Amicon Ultra-15 centrifuge concentrator. Fractions containing the protein of interest was collected and concentrated.

Extraction

Procedure
Frozen cell pellets from 2 L culture (ca. 6.3 g wet mass) were resuspended in a total volume of 25 ml lysis buffer. The cells were broken by 4 passes at 16,000 psi through a high pressure homogeniser (EmulsiFlex-C3/Avestin) followed by centrifugation for 60 minutes at 60,000g. The supernatant was further clarified by filtration (0.45 µm).
Concentration:Using Amicon Ultra-15 concentrators with 5 kDa cutoff, the JARID1CA-c556 was exchanged into NMR buffer and concentrated as follows: U-[¹³C,¹⁵N]- labelled JARID1CA: protein concentration 1 mM in 50 mM Phosphate, pH 6.0, 50 mM NaCl, 5 mM d DTT, 0,02% azide 10% D2O.
Ligand
MassSpec:
Crystallization:
NMR Spectroscopy:NMR spectra were acquired at 297 K, using Bruker DRX 600 and DMX 750 spectrometers in standard configuration with triple resonance probes equipped with self-shielded triple axis gradient coils. Spectra for the resonance and NOE assignment were recorded essentially as described in the original references. A 1 mM [¹³C, [¹⁵N-labelled sample of JARID1CA in 90% H2O/10% D2O (NMR buffer; pH 6.0) was used for all H N -detected triple resonance experiments, 3D CBCA(CO)NNH, CBCANNH, CC(CO)NNH, H(CCCO)NNH, HBHA(CBCACO)NNH, HNCO, HN(CA)CO, 3D ¹⁵N-separated NOESY-HSQC, [¹⁵N T1 and ¹⁵N T2 relaxation, and heteronuclear ¹⁵N- ¹H NOE experiments and for a 3D ¹³C-separated, aliphatic-centred NOESY-HSQC spectrum. The sample was then freeze-dried and re-dissolved in 100% D2O for acquisition of 3D ¹³C-separated HMQC-NOESY, HCCH-COSY, HCCH-TOCSY spectra. Data were processed using the program XWIN-NMR (version 2.6) of Bruker BioSpin GmbH ( Rheinstetten , Germany ).
Data Collection:
Data Processing:Assignment of ¹³C, ¹⁵N and ¹H resonances was carried out using standard assignment procedures on Silicon Graphics O2 workstations and an Intel Dual Xeon 3GHz PC, with the interactive program Sparky version 3.10 ( http://www.cgl.ucsf.edu/home/sparky/ ). The assignments are deposited in the BioMagResBank (http://www.bmrb.wisc.edu/) under accession code BMRB- 15348

Assigned ¹H- ¹⁵N-HSQC spectrum of the Bright/ARID domain from human JARID1CA recorded at 297 K and 750 MHz.

Preliminary three dimensional structures of the JARID1CA Bright/ARID domain were calculated using the program CYANA v. 2.0 ( Güntert et al., (1997) J. Mol. Biol. 273 , 283-298; Herrmann et al., (2002). J. Mol. Biol. 319 , 209-227 ) and were based on the resonance assignments together with NOE peak lists from ¹³C HMQC-NOESY, 3D ¹³C-aliphatic-centred NOESY-HSQC and 3D ¹⁵N NOESY-HSQC spectra. Dihedral angle restraints were predicted based on the chemical shift data using the program TALOS (Cornilescu et al., J. Biomol. NMR, 13 (1999) 289-302; http://spin.niddk.nih.gov/NMRPipe/talos/) and were used to aid initial rounds of structure calculations. Most of these were excluded in the final rounds of calculations. A precise ensemble of structures showing the expected Bright domain fold was obtained. Hydrogen bond restraints were added on the basis of the protected amides following sample exchange into D2O. Iterative structure refinement was carried out using XPLOR-NIH v. 2.14 ( Schwieters et al., (2003) J. Magn. Reson. 160 , 66-74; http://nmr.cit.nih.gov/xplor-nih/ ). Structures were refined in explicit water using CNS ( Linge et al (2003) Proteins, 50:496-506). Structures validation was performed using WHATIF http://swift.cmbi.kun.nl/WIWWWI and the Protein Structure Validation Suite (PSVS) http://www-nmr.cabm.rutgers.edu/PSVS . The refined ensemble of the 20 lowest energy structures with no NOE violations was submitted to the PDB under code 2JRZ. Chemical shift list and the list of NMR restraints were deposited with the structures.

References

Tahiliani, M., Mei, P., Fang, R., Leonor, T., Rutenberg, M., Shimizu, F., Li, J., Rao, A & Shi, Y. (2007). The histone H3K4 demethylase SMCX links REST target genes to X-linked mental retardation. Nature 447 , 601-605
Tzschach, A. et al. ( 2006 ) Novel JARID1C/SMCX mutations in patients with X-linked mental retardation . Hum. Mutat. 27, 389-394.
Jensen, L. R. et al. ( 2005 ) Mutations in the JARID1C gene, which is involved in transcriptional regulation and chromatin remodeling, cause X-linked mental retardation . Am. J. Hum. Genet. 76 , 227-236.
Yuan YC, Whitson RH, Liu Q, Itakura K, Chen Y (1998) * A novel DNA-binding motif shares structural homology to DNA replication and repair nucleases and polymerases Nat Struct Biol.; 5: 959-64.
Treisman JE, Luk A, Rubin GM, Heberlein U. (1997) Eyelid antagonizes wingless signaling during Drosophila development and has homology to the Bright family of DNA-binding proteins. Genes Dev. 1997; 11: 1949-62
Iwahara J., Iwahara M., Daughdrill G.W., Ford J., Clubb R.T (2002). The structure of the Dead ringer-DNA complex reveals how AT-rich interaction domains. EMBO J. 21:1197-1209.