Biochem. J. (2011) 436, 83–90 (Printed in Great Britain)
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doi:10.1042/BJ20110004
Solution structure of the Taf14 YEATS domain and its roles in cell growth of Saccharomyces cerevisiae Wen ZHANG*, Jiahai ZHANG*, Xuecheng ZHANG†, Chao XU‡ and Xiaoming TU*1 *Hefei National Laboratory for Physical Sciences at Microscale, School of Life Science, University of Science and Technology of China, Hefei, Anhui 230026, China, †School of Life Sciences, Anhui University, Hefei, Anhui 230039, China, and ‡Structural Genomics Consortium, University of Toronto, 101 College St, Toronto, Ontario, Canada M5G 1L7
Chromatin modifications play important roles in cellular biological process. A novel conserved domain family, YEATS, has been discovered in a variety of eukaryotic species ranging from yeasts to humans. Taf14, which is involved in a few protein complexes of chromatin remodelling and gene transcription, and is essential for keeping chromosome stability, regular cell growth and transcriptional regulation, contains a YEATS domain at its N-terminus. In the present study, we determined the solution structure of the Taf14 YEATS domain using NMR spectroscopy. The Taf14 YEATS domain adopts a global fold of an elongated β-sandwich, similar to the Yaf9 YEATS domain. However, the Taf14 YEATS domain differs significantly from the Yaf9 YEATS domain in some aspects, which might indicate different structural
classes of the YEATS domain family. Functional studies indicate that the YEATS domain is critical for the function of Taf14 in inhibiting cell growth under stress conditions. In addition, our results show that the C-terminus of Taf14 is responsible for its interaction with Sth1, which is an essential component of the RSC complex. Taken together, this implies that Taf14 is involved in transcriptional activation of Saccharomyces cerevisiae and the YEATS domain of Taf14 might play a negative role in cell growth.
INTRODUCTION
[4]. Thereafter, Taf14 was reported to be important for chromosome stability [5] and was associated with several chromatin remodelling and transcription complexes as follows: Swi/Snf (switch/sucrose non-fermentable), which has DNAstimulated ATPase activity and activates transcription by helping transcription factors access their binding sites [6–8]; RSC, which is essential for cell cycle progression [9,10]; INO80, which has helicase activity and is involved in transcription, replication and DNA repair [11–13]; and NuA3, which stimulates transcription or replication elongation through nucleosomes by providing a coupled acetyltransferase activity [14]. Besides roles in chromatin remodelling, Taf14 is involved in complexes related to transcription. Taf14 is a subunit of the transcription factors TFIID [15] and TFIIF [6,16] in S. cerevisiae. A two-hybrid screen revealed that Taf14 interacts with Tsm1 in TFIID complexes and Tfg1 in TFIIF complexes [17]. Moreover, Taf14 is associated with transcription initiation. There is evidence that Taf14 is recruited to the promoter region in the ADH1 gene [18] and the GAL1 gene, suggesting its role in the assembly of the RNA polymerase II preinitiation complex [17]. Taf14 is also involved in cell cycle regulation by interaction with Sth1, which plays an important role in the RSC complex. In the present study, we determined the solution structure of the Taf14 YEATS domain using NMR. The Taf14 YEATS domain exhibits a β-sandwich structure that contains two parallel β-sheets. Structural comparison between the Taf14 YEATS domain and Yaf9 YEATS domain revealed significant differences. In vivo, deletion of the Taf14 YEATS domain enhanced cell growth under stress conditions. In addition, our results indicate that the C-terminus of Taf14 has an important role in the interaction of Taf14 with Sth1.
Chromatin modification is a general biological process in many eukaryotes. The covalent modification of core histones, as a primary style of chromatin modification, regulates different states of gene expression. The covalent modifications of histone tails include acetylation, methylation, phosphorylation, SUMOylation, ubiquitination and so on. These histone modifications are carried out by a variety of protein complexes, for example NuA4, RSC and INO80. In these multi-subunit protein complexes, an increasing number of conserved domains have been identified to be crucial for establishing the different chromatin modifications. The YEATS domain is distributed widely in 59 different eukaryotes [1] and was discovered recently as one of the domains related to chromatin modification and transcription [2]. However, the structures and functions of YEATS domains are far from clear. Interestingly, unlike other domains involved in chromatin modification complexes, the YEATS domain is present in a single copy in YEATS-domain-containing proteins, always located at the N-terminus. In addition, most YEATS-domain-containing proteins do not have any other annotated domain [2]. This implies that the YEATS domain might play critical roles in the functions of YEATS-domain-containing proteins. YEATS-domain-containing proteins are usually involved in chromatin remodelling complexes. Among these proteins, Taf14 is the only one which is involved in both chromatin remodelling and transcription regulation. Taf14 was first identified to be involved in actin cytoskeletal function in Saccharomyces cerevisiae and therefore was named ANC1 [3]. Although Taf14 is a non-essential gene, cells devoid of Taf14 were thermoand osmo-sensitive and had defects in actin organization
Key words: chromatin remodelling, NMR, Saccharomyces cerevisiae, Taf14, transcriptional regulation, YEATS domain.
Abbreviations used: 3D, three-dimensional; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser enhancement spectroscopy; RMSD, root mean square deviation; SPR, surface plasmon resonance; SSE, secondary structure element; YC-URA, synthetic medium lacking uracil. 1 To whom correspondence should be addressed (email
[email protected]). The structural co-ordinates reported for the Taf14 YEATS domain will appear in the PDB under accession code 2L7E. c The Authors Journal compilation c 2011 Biochemical Society
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EXPERIMENTAL Cloning, expression and purification of recombinant Taf14 YEATS domain
The Taf14 YEATS domain was obtained from the S. cerevisiae DNA gene library by PCR and was cloned into the NdeI/XhoIcleaved plasmid pET22b(+) (Novagen). The recombinant vector was transformed into the expression host BL21(DE3). The recombinant Taf14 YEATS domain was expressed and purified as described previously [19]. Uniformly 15 N- and 13 C-labelled protein was prepared with medium containing 0.5 g/l 99 % ammonium chloride and 2.5 g/l 99 % 13 C-glucose as the sole nitrogen and carbon source respectively. The NMR sample contained 0.8 mM Taf14 YEATS domain, 20 mM sodium phosphate (pH 6.5), 100 mM sodium chloride and 1.5 mM dithiothreitol in either 90 % H2 O/10 % 2 H2 O or 100 % 2 H2 O. NMR experiments and structure calculations
All NMR data were collected at 298 K on a Bruker DMX500 spectrometer. A set of standard triple-resonance spectra was recorded for backbone and side-chain assignments. NOE (nuclear Overhauser effect) distance restraints were obtained from 3D (three-dimensional) 15 N- and 13 C-edited NOESY (nuclear Overhauser enhancement spectroscopy) spectra acquired with a mixing time of 130 ms. After these experiments, the sample was lyophilized and redissolved in 99.96 % 2 H2 O. A series of 15 N-HSQC (15 N-heteronuclear single-quantum coherence) experiments were performed to monitor the disappearance of NH signals to obtain the hydrogen-bond information. NMR data were processed with NMRPipe and analysed with Sparky 3 software. Chemical shift index was carried out for Cα , Cβ , C and Hα . The information on the ϕ and ψ backbone dihedral angles was obtained using the TALOS program [20]. Hydrogen-bond restraints were obtained by assignment of slow-exchange amide protons located in regular SSEs (secondary structure elements). The CNS program [21] was used to calculate the 3D structure of the Taf14 YEATS domain by distance restraints using the ARIA setup and protocols. Short-range NOEs and long-range NOEs were first used to determine the SSEs of the Taf14 YEATS domain. ϕ and ψ backbone dihedral angles and hydrogen-bond restraints were added in consecutive steps to constrain the 3D structure better. The 20 structures with the lowest energy were analysed with MOLMOL [22]. The Ramachandran plot was analysed with PROCHECK [23]. 15 N Longitudinal (T1) and transverse (T2) relaxation times and the heteronuclear 1 H-15 N NOE were detected at 298 K on a Bruker DMX500 spectrometer. For the T1 measurements, eight time points were collected with delays of 11.15, 61.30, 141.54, 241.84, 362.20, 522.68, 753.37 and 1144.54 ms. For T2, seven time points with delays of 0, 17.60, 35.20, 52.80, 70.40, 105.60 and 140.80 ms were obtained. The heteronuclear 1 H-15 N NOE was measured from duplicate pairs of 1 H-15 N spectra recorded with and without amide proton saturation. Yeast strains, plasmids, media and genetic methods
Yeast strains used in the present study were BY4742 (wildtype) and BY4742 (Taf14). Plasmids used in the present study are listed in Supplementary Table S1 (at http://www.BiochemJ. org/bj/436/bj4360083add.htm). Media such as YPD [1 % (w/v) yeast extract/2 % (w/v) peptone/2 % (w/v) glucose] and synthetic complete (YC) were made according to standard procedures [24]. Formamide was added at a final concentration of 2 %. Hydroxyurea was added at a concentration of 30 μg/ml. Yeast c The Authors Journal compilation c 2011 Biochemical Society
cells were transformed using lithium acetate [25]. For the growth studies on plates, cells in exponential phase were diluted. Five concentrations with D600 of 10−1 , 10−2 , 10−3 , 10−4 and 10−5 were made. The same volume of cells was applied on to YC-URA (synthetic medium lacking uracil) plates. Cells were grown at 30 ◦ C. SPR (surface plasmon resonance)
Real-time interactions of Sth1 with Taf14, Taf14 YEATS domain and the C-terminus of Taf14 were measured on a Biacore 3000 system. Taf14 and the C-terminus of Taf14 were cloned, expressed and purified in the same way as the Taf14 YEATS domain. Sth1 was cloned into the NdeI/XhoI-cleaved plasmid pGEX-4T-1 and was expressed and purified by standard affinity chromatography procedures. The CM5 (carboxymethylated dextran) chip was activated using an amine coupling-reagent mixture containing 0.4 M EDC [N-ethyl-N-(3-dimethylaminopropyl)carbodi-imide] and 0.1 M NHS (N-hydroxysuccinimide) at a flow rate of 5 μl/min for 10 min. Sth1 was diluted to a concentration of 2 mg/ml with running buffer (20 mM NaH2 PO4 , pH 7.0, containing 100 mM NaCl) and immobilized on the chip at a flow rate of 5 μl/min for 10 min. Ethanolamine (1.0 M, pH 8.5) was used to neutralize unbound activated sites on the chip at a flow rate of 5 μl/min for 10 min. The chip was washed twice with regeneration buffer (40 mM NaOH) and then with running buffer. Purified Taf14, Taf14 YEATS domain and the C-terminus of Taf14 were diluted with running buffer. The kinetic analysis of interaction between Sth1 and Taf14 was performed at six concentrations of Taf14 (7.00 μM, 3.50 μM, 1.75 μM, 0.88 μM, 0.44 μM and 0 μM) at a flow rate of 10 μl/min for 2 min. Kinetic analysis of interaction between Sth1 and C-terminus of Taf14 was also performed at six concentrations of the C-terminus of Taf14 (5.30 μM, 2.65 μM, 1.33 μM, 0.67 μM, 0.34 μM and 0 μM) at a flow rate of 10 μl/min for 2 min. Regeneration was performed at a flow rate of 30 μl/min for 2 min. The analysis was performed three times for each concentration. Kinetic analysis of SPR data was performed using BIAevaluation 4.1 (Biacore). Curves were fitted to the 1:1 (Langmuir) binding model. The dissociation constant (K d ) was derived from the kinetic analysis. RESULTS Sequence analysis of Taf14 and Taf14 YEATS domain
Taf14, 244 residues in length, can be divided into two parts: a YEATS domain at the N-terminus which is evolutionarily conserved from yeasts to humans, a C-box and another region at the C-terminus (Figure 1A). The Taf14 YEATS domain shares 18–44 % sequence identity and 40–65 % sequence similarity with other YEATS domain family members. Amino acid sequence analysis for the YEATS-domain-containing proteins shows YEATS domains contain a few conserved residues and have similar secondary structure patterns composed of eight β-strands. Interestingly, the sequence in the eighth β-strand and the loop connecting the seventh and the eighth β-strands are not conserved (Figure 1B). The highly divergent sequence in this region might imply the existence of different classes within the YEATS domain family. Solution structure of the Taf14 YEATS domain
The Taf14 YEATS domain, containing residues 1–123 of the protein, was recombinantly expressed and purified. The solution structure of the Taf14 YEATS domain was calculated
Taf14 YEATS domain structure and role in yeast growth
Figure 1
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The Taf14 YEATS domain
(A) Schematic representation of the Taf14 YEATS domain and other elements. (B) Sequence alignment of the Taf14 YEATS domain with other YEATS domains by ClustalW [30] and annotated using ESPript [31].
Table 1
NMR and structural statistics
+ i, and j are random atoms. + − values are − S.D. NMR restraints in the structure calculation
Value
Intraresidue Sequential (|i−j| = 1) Medium-range (|i−j|
