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1 Laboratory of Developmental Biology, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
2 Horikoshi Gene Selector Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency (JST), 5-9-6 Tokodai, Tsukuba, Ibaraki 300-2635, Japan
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Abstract |
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 Top Abstract IntroductionResultsIdentification of MMS-sensitive...DiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsIdentification of MMS-sensitive...DiscussionExperimental proceduresReferences |
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Methods that exploit mutagenesis can be roughly classified into two groups. One is naturally occurring or artificially introduced random mutagenesis (Morgan 1910), and the other is the site-directed mutagenesis technique improved during last 20 years on the basis of Smith and colleagues’ concepts (Smith 1985). The random mutagenesis approach is good for the identification and rough characterization of proteins in a particular system, but it is not useful for identifying the functional roles of residues on the surfaces of a full-length protein. On the other hand, site-directed mutagenesis allows particular residues in a system to be characterized, and it is generally applied to domains of interest. There are two main methods for site-directed mutagenesis, deletion and point mutagenesis. Deletion mutagenesis is powerful for the domain-based functional analysis of proteins. However, when the deleted domain is necessary for the maintenance of tertiary protein structure or for complex formation, it is difficult to analyze the mutant protein in its native conformation. On the other hand, point mutagenesis is useful for the functional analysis of a specific domain of interest, and it generally has little effect on overall protein structure. For example, point mutation analyses of TBP succeeded in identifying the residues involved in protein–protein/DNA interactions and/or transcriptional activation (Lee et al. 1992; Yamamoto et al. 1992; Hisatake et al. 1993; Kim et al. 1994). Residues on the TBP surface based on structural information were also characterized (Bryant et al. 1996; Tang et al. 1996).
We thus aimed to explore a functional interaction network by analyzing a hub-like protein that forms a scale-free structure. A scale-free network such as a computer network contains centrally located "hubs," which are connected with vast numbers of other constituents and dramatically influence the way the network operates. These hub-like factors are usually evolutionarily conserved (Hoffmann et al. 1990). Also, their functionally important residues are often highly conserved, which in turn indicates that highly conserved residues are likely to be functionally important. This notion indicates that most of the amino acid residues in the hubs of a network could be effectively analyzed by a comprehensive point mutagenesis approach. We thus, adopted a strategy called GLobal Analysis of Surfaces by Point mutation (GLASP), which entails individually mutagenizing each of the residues that comprises the surface of an evolutionarily conserved factor. The GLASP strategy allows identification of all of the residues involved in a particular protein function both in vivo and in vitro.
In this study, we selected core histones as highly conserved factors for characterization by GLASP. In eukaryotes, genomic DNA is wrapped around a core histone octamer, composed of a single (H3–H4)2 tetramer and two H2A–H2B dimers, forming a nucleosome (Luger et al. 1997). Core histones are highly conserved; for example, the amino acid sequence of histone H4 is 92% identical between yeast and human. Therefore, the control of gene function through core histone structure and function would also be expected to be highly conserved in eukaryotes. Since genomic DNA is negatively regulated by the nucleosome, activation of DNA-mediated reactions such as transcription, DNA replication or DNA repair requires altering the structure and function of the nucleosome. The core histones are thus involved in the reactivation of DNA-mediated reactions through being targeted by chromatin-associated factors.
Three types of chromatin-associated factors have been well characterized to date: (i) ATP-independent histone chaperones such as nucleoplasmin, which facilitate assembly and disassembly of the nucleosome (Laskey et al. 1978); (ii) chromatin remodelling ATPases such as SWI/SNF and NURF, which take part in sliding of the nucleosome (Côté et al. 1994; Tsukiyama et al. 1994); and (iii) histone modification enzymes such as histone acetyltransferase and histone deacetylase, which mediate reversible functional changes in core histones (Kleff et al. 1995; Brownell et al. 1996; Taunton et al. 1996). The functional roles of these chromatin-associated factors have been characterized in the regulation of transcription, but their roles in DNA replication and DNA repair have hardly been studied.
Numerous studies on core histones for analyzing mutants on focused regions such as the N-terminal tails reveal that their chemical modification are crucial for activation or silencing of gene expression (Grunstein 1990; Turner et al. 1992). These studies also revealed that chemical modification of the N-terminal tails were targeted by various chromatin-associated factors, leading to the hypothesis of the histone code, which holds that patterns of histone modifications regulate downstream biochemical and biological reactions (Strahl & Allis 2000). The central core regions of core histones are also post-translationally modified (Cosgrove et al. 2004) and mutations in core regions induce Sin (Swi-independent) phenotype and loss of transcriptional silencing (Kruger et al. 1995; Park et al. 2002; Hyland et al. 2005). In most previous studies, functional characterization of core histone residues focused primarily on covalently modified residues of core histones and on chromatin-associated factors. Therefore, several problems still remain: (i) functional characterization of the residues of core histones are restricted to particular ones; (ii) histone-mediated reactions other than transcription are poorly understood; and (iii) how the surfaces of core histones are utilized in different DNA-mediated reactions such as transcription, DNA replication, or DNA repair has not been solved.
Since the interactions between histones and various chromatin-associated factors are the driving forces behind broad nucleosomal reactions, it is essential for elucidation of these problems to analyze not only the roles of covalently-modified residues of core histones and chromatin-associated factors but also the roles of all the surface residues of core histones including unmodified residues in each DNA-mediated reaction. In order to tackle the fundamental basis of chromatin-mediated reactions as well as to understand the global framework of functional core histone surface at single amino acid level, we mutagenized each of the histone residues located on the protein surface, and investigated the effects of each mutation on a variety of nuclear reactions in vivo.
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Results |
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TopAbstractIntroductionResultsIdentification of MMS-sensitive...DiscussionExperimental proceduresReferences |
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To evaluate the functional role of each amino acid that constitutes the core histones, we individually mutated all 320 residues in histones H2A, H2B, H3, and H4 to alanine, with the exception of endogenous alanine residues and those amino acids not located on the surface of the nucleosome. A histone surface residue was defined as that, at least, a part of the residue is exposed outside when crystal structure of the nucleosome was displayed at the electrostatic surface presentation (Fig. 1A–D) (White et al. 2001). The flanking tail sequences of the N- and C-terminal regions were also mutated. Alanine was chosen for this mutagenesis to minimize structural effects on conformation of the histone subunit and on the histone core octamer structure. No structural abnormalities have been shown on X-ray crystallography of Sin mutant nucleosomes (Muthurajan et al. 2004), which bear mutations on the molecular surfaces of core histones. Given that each histone subunit is essential for viability, the mutant histone proteins were assumed to form functional conformation, and some of the mutated proteins were confirmed not to be degraded (Downs et al. 2000; Kimura et al. 2002).
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1 to L1 of histone H4, they are assumed to function in the formation, maintenance and alternation of the nucleosome entry site. The other region composed of H2A-Y58, -E62, -D91 and H2B-L109 is located at the acidic patch, and its nearby region, of the nucleosome (Fig. 1G, right). In the crystal structure of Xenopus nucleosome, this acidic patch mediates interaction with the N-terminal tail of the histone H4 in the adjacent nucleosome (Luger et al. 1997), and also interacts with the LANA protein of the Kaposi's sarcoma herpesvirus (Barbera et al. 2006). Since these residues responsible for lethality are completely conserved between yeast and Xenopus, they are also likely to be targeted by several chromatin-associated factors in yeast. Using viable point mutants, we investigated the suppressor of Ty (Spt) phenotype (Silverman & Fink 1984), 6-azauracil (6AU) sensitivity (Exinger & Lacroute 1992), hydroxyurea (HU) sensitivity (Rosenkranz & Levy 1965) and methyl-methanesulfonate (MMS) sensitivity (Schwartz 1989). These assay systems are well characterized and have been widely used to investigate the components and mechanisms involved in particular DNA-mediated reactions. We have identified the functional molecular surfaces of core histones in each reaction as described in the following sections.
Identification of Spt phenotypic residues on the histone surface
The Spt assay evaluates the effect of mutations on transcription (Silverman & Fink 1984) and it takes advantage of a transcription inhibition that is caused by insertion of a Ty- or delta-element at the 5' end of a gene, disturbing its normal transcription initiation. There are several genes isolated whose mutations display Spt- phenotypes. For example, genes encoding transcription/chromatin-related factors such as TBP (Eisenmann et al. 1989) and subunits of the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex (Winston et al. 1984) have been isolated by the Spt assay. In addition, SPT11 and SPT12, which encode histone H2A and H2B, respectively (Clark-Adams et al. 1988), and 11 residues of histones produce Spt- phenotypes (Santisteban et al. 1997; Duina & Winston 2004). In a screen of the 312 histone point mutants, 42 showed Spt- phenotypes (Fig. 2A). Although it is not clear whether these Spt phenotypic residues directly act on the nucleosome, the affected residues mainly clustered at three different regions (regions SPT-I, -II, and -III) on the surfaces of core histones (Fig. 2B).
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N to 1 of histone H3, and the 1 to 2 of histone H4 (Fig. 2B–D). There are 6 positively-charged residues out of 13 Spt phenotypic residues in the SPT-I region, all of which are accessible to DNA. This region also contains the H3-K56 and -L61 residues, which are responsible for the recruitment of Snf5, a subunit of the SWI/SNF nucleosome remodelling complex, to promoter regions (Duina & Winston 2004; Xu et al. 2005). Consistent with a previous report showing an Spt- phenotype for H3-L61W, the H3-L61A mutant was also Spt- (Duina & Winston 2004). Furthermore, we discovered that alanine substitution of H3-K56 and other nearby residues caused the Spt- phenotype. These data suggest that the surface defined by H3-K56 and -L61 in the SPT-I region might be targeted and remodelled by the SWI/SNF complex. In addition, five other Spt phenotypic residues (H4-R35, -R36, -G48, -L49 and -Y51) belong to the SPT-I region, and are positioned at the boundary of the bromodomain factor 1 (Bdf1) binding domain which was identified by two-hybrid assay (Pamblanco et al. 2001). This result supports previous data showing that the disruption of BDF1 induces an Spt- phenotype (Chimura et al. 2002). Thus, these residues might mediate binding between histone H4 and Bdf1 in transcription. The SPT-II region (affecting H3-K115, -V117 and -Q120) is located at the L2 loop of histone H3 (Fig. 2B, D). It corresponds to a domain interacting with human histone chaperone CCG1-interacting factor A (CIA) (Munakata et al. 2000), whose counterpart in Saccharomyces cerevisiae, Asf1, shows an Spt- phenotype when disrupted (Chimura et al. 2002). The SPT-I and -II regions are in close proximity to each other, and Bdf1 and CIA bind to the SPT-I and -II regions, respectively (Munakata et al. 2000; Pamblanco et al. 2001). In addition, Bdf1 functionally and directly interacts with Asf1 (Chimura et al. 2002). These data suggest that Bdf1 and CIA/Asf1 cooperatively modulate nucleosome structure through the SPT-I and -II regions.
The SPT-III region is restricted to one side of the C of histone H2B and contains nine residues responsible for the Spt- phenotype (Fig. 2B). H2B-E108 interacts with the adjacent nucleosome as revealed in the crystal structure of yeast nucleosome (White et al. 2001) and other Spt phenotypic residues in the SPT-III region (from H2B-L105 to H2B-S125) converge near H2B-E108 (Fig. 2E–G). The SPT-III region is therefore suggested to regulate nucleosome–nucleosome interactions, as discussed in the following section.
Identification of 6AU-sensitive residues on the histone surface
6AU is known to block transcription elongation by disturbing the balance of intracellular nucleotide concentrations (Exinger & Lacroute 1992). Several genes, encoding S-II (Hubert et al. 1983), subunits of FACT (Formosa et al. 2001), Spt4–Spt5 (Hartzog et al. 1998) and RNA polymerase II (Archambault et al. 1992) are responsible for the 6AU sensitivity when mutated. Recently, the mutation to H2B-K123 has been reported to introduce 6AU sensitivity (Xiao et al. 2005), and mutation to H3-K36 has been reported to introduce 6AU resistancy (Kizer et al. 2005).
In a screen of the histone point mutant library, we discovered eight histone point mutants that show 6AU sensitivity (Fig. 3A). Surprisingly, all residues are located on the one side of the nucleosome (Fig. 3B). Furthermore, seven of the eight 6AU-sensitive residues are located in only histone H2A or H2B (Fig. 3A–D). This is consistent with increased RNA polymerase II binding to the H2A/H2B-deficient nucleosome (Baer & Rhodes 1983) and transcription facilitation by the loss of one H2A–H2B dimer (Gonzalez & Palacian 1989).
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N, 2 and L2 of histone H3, and L1 of histone H4 (Fig. 3E). These in vivo effects might be caused by change(s) in the step(s) of the formation, maintenance or alternation of the nucleosome structure. Importance of this region is also suggested by its close location to N of histone H3 which involves several residues responsible for lethality (Fig. 1G, left). H2B-K123A and H2A-E65A showed the strongest sensitivity to 6AU. Based on their positions, these residues may mediate nucleosome–nucleosome interactions on crystal structure (White et al. 2001) (Fig. 3F). Rad6, the ubiquitin-conjugating enzyme for H2B-K123 (Robzyk et al. 2000), causes 6AU sensitivity when deleted and interacts with several transcription elongation factors in vivo and in vitro (Xiao et al. 2005). These facts suggest that ubiquitination of histone H2B-K123 is involved in transcription elongation through disruption of the nucleosome–nucleosome interactions.
It is striking that no overlapping residues were found, when we compared the positions of 6AU-sensitive residues with those of the Spt phenotypic residues (Fig. 3G,H). Though there is the possibility that the 6AU sensitivity was induced by indirect effects of point mutation, such as change in expression of the genes involved in the transcription elongation, one possible explanation is that the reactions of transcription initiation and elongation take place along different surfaces of the nucleosome, which in turn suggests different nucleosome remodelling mechanisms for these two systems.
Identification of HU-sensitive residues on the histone surface
HU inhibits ribonucleotide reductase and blocks DNA replication by impairing deoxyribonucleotide synthesis (Rosenkranz & Levy 1965). Cells become sensitive to HU when genes involved in DNA replication such as POL1 (Pavlov et al. 2001), MCM2 (Araki et al. 2003) and SNF2 (Duina & Winston 2004) are mutated. To date, 15 histone residues responsible for HU sensitivity have been identified (Duina & Winston 2004; Hyland et al. 2005; Ye et al. 2005). The screen of histone point mutants isolated 30 mutants of core histones which mediate sensitivity to HU (Fig. 4A).
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The HU-II region involves residues H113 and L126 of histone H3, which are part of the domain that is responsible for H3–H3' interaction (White et al. 2001). Although it is not clear how alteration of the H3–H3' interaction triggers HU sensitivity, either formation, maintenance or disruption of the H3–H3' interaction is supposed to be essential for DNA replication, as discussed in the following sections.
HU-III region is composed of HU-sensitive residues that are located in the nucleosome acidic region, the H2A C-terminal region, and the H2BC (Fig. 4B). Although some factors involved in acidic region of the nucleosome were known, the function of acidic patch has been unknown (Luger et al. 1997). Our results suggest for the first time its participation of DNA replication. In addition, H2A C-terminal tail is adjacent to the proximity of the nucleosomal entry site (HU-I) and the H33 region (HU-II), indicating functional importance of this region (Fig. 4H).
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Identification of MMS-sensitive residues on the histone surface |
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TopAbstractIntroductionResultsIdentification of MMS-sensitive...DiscussionExperimental proceduresReferences |
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The MMS-I region, which is located at the nucleosome entry site, overlaps the HU-I and SPT-I regions (Figs 2, 4 and 5). The MMS-I region also overlaps with the Bdf1-interacting region of histone H4 which was shown by the two-hybrid assay (Pamblanco et al. 2001) and disruption of the BDF1 gene also induces MMS and HU sensitivities (Chang et al. 2002), as well as the Spt- phenotype (Chimura et al. 2002). All the MMS- and HU- sensitive residues in MMS-II and HU-II region are completely consistent with each other (Figs 4 and 5). On the other hand, although the residues composing the MMS-II (HU-II) region are totally different from those of the SPT-II region, the MMS-II (HU-II) region is closely located to the SPT-II region (Figs 2, 4 and 5). Additionally, the MMS-II region overlaps both the CIA-interacting region of histone H3 (Munakata et al. 2000; Mousson et al. 2005) and as well as the SPT-II region, and disruption of the CIA/ASF1 gene results in MMS and HU sensitivities (Bennett et al. 2001; Chang et al. 2002), as well as the Spt- phenotype (Chimura et al. 2002). These data suggest that the MMS-I (HU-I and SPT-I) region and the MMS-II (HU-II and SPT-II) region are targeted by common chromatin-associated factor(s) (ex. Bdf1 and CIA/Asf1) in the course of DNA repair, DNA replication, and transcription. We hypothesize that other distinct factors interact with Bdf1 and CIA/Asf1 to provide them with specific commitments to function in each of these DNA-mediated reactions.
The MMS-III region contains H2B-K123, and is involved in interactions with the adjacent nucleosome (White et al. 2001), as described in "Identification of 6AU-sensitive residues" section. Ubiquitination of H2B-K123 is known to be involved in transcription (Kao et al. 2004) and DNA repair of DSBs (Yamashita et al. 2004), which is consistent with our data showing both 6AU and MMS sensitivities of the H2B-K123A mutant. Considering that H2B-K123A is responsible for HU sensitivity and that disruption of the RAD6 gene shows HU sensitivity (Bennett et al. 2001), our data indicate that ubiquitination of H2B-K123 is not only involved in transcription and DNA repair but also in DNA replication.
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Discussion |
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TopAbstractIntroductionResultsIdentification of MMS-sensitive...DiscussionExperimental proceduresReferences |
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Our analysis of core histone point mutants has uncovered several intriguing issues. First, the positions of the residues that are involved in these assays are clustered on the left side of nucleosome when it is observed along the DNA superhelix axis (Figs 5G and 6A). Interestingly, when we compare the observation with the results of silencing assays, which are performed in many studies, the right side of the nucleosome is significantly covered with the residues involved in the regulation of transcriptional silencing (Park et al. 2002; Thompson et al. 2003; Hyland et al. 2005). Although we should not overlook that functional roles of H2A and H2B, which mainly exist in the left side of the nucleosome, are not well characterized in silencing assays, the observation suggests that each side of the nucleosome functions as a differently utilized unit.
Second, the positions of the residues that were affected in these assays were clustered in several specific regions of the primary structures of the core histones (Fig. 6B). This suggests that the basis of the regulatory mechanisms that alter the nucleosome structure and function in each of the DNA-mediated reactions involves the same surface regions of the core histones.
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3 of histone H3 are involved in an H3–H3' interaction required for formation of a single (H3–H4)2 tetramer and influences sensitivities to HU and MMS, but not the Spt- phenotype (left panels in Fig. 7A). These results suggest that, for the regulation of DNA replication and DNA repair, formation of a single (H3–H4)2 tetramer is more important than the H3–DNA interactions, whereas the H3–DNA interaction is more important for transcription than formation of a single (H3–H4)2 tetramer. The differences in the histone interactions mentioned above are probably one of the reasons why histone requirements for DNA replication and DNA repair are different from those required for transcription.
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C of histone H2B should be highlighted. These residues are speculated to be involved in nucleosome–nucleosome interactions (White et al. 2001). The residues responsible for the Spt- phenotypes and for sensitivities to 6AU, HU and MMS differ in this region (right panels in Fig. 7A), indicating the presence of distinct factors and diverse reactions for altering nucleosomal array through the interaction with this region in the course of transcription, DNA replication and DNA repair. Effectiveness of comprehensive histone point mutant library
In this study, we constructed a comprehensive library of histone point mutants and obtained that 88 histone point mutants which showed either an Spt- phenotype (42 residues), 6AU sensitivity (8 residues), HU sensitivity (30 residues) or MMS sensitivity (61 residues), and a number of mutants which showed multiple sensitivities in the in vivo assays. As compared with the residues identified in this study, approximately one-ninth of the residues have been only reported to be involved in the same assays. In addition, we found that a substantial number of the same residues on the surfaces of core histones are engaged in quite different DNA-mediated reactions (Fig. 5F). This study should thus lead to a better understanding of global regulation which involves, and presumably integrates, multiple DNA-mediated reactions. Moreover, given the high conservation of core histones from yeast to human, we expect that many of the corresponding residues in human core histones will also be involved in the same DNA-mediated reactions. Considering that many chromatin-associated factors are known to be linked to human disease, the availability of a comprehensive library of histone point mutants constitutes a valuable resource not alone for scientists working in the field of chromatin research but also for medical science as well.
In previously characterized residues, almost all the results obtained in this study were consistent with the known results. However, following three different observations with literatures must be referred. First, the H3-R52A mutant was reported to be lethal (Hyland et al. 2005), while the same alanine mutation did not affect cell growth in our experiment, causing Spt- phenotype, and HU and MMS sensitivities (Figs 2A, 4A and 5A). Second, H2A-T125A was reported to show Spt- phenotype at an LYS2 promoter (Wyatt et al. 2003), while our mutant at the same residue did not show the Spt- phenotype at the LYS2 promoter (data not shown). Third, H3-K115A was reportedly sensitive to 200 mM HU (Hyland et al. 2005), while our mutant at the same residue has no growth effect on a plate containing 100 mM HU (data not shown). These apparent discrepancies might be originated from the difference of strains and/or experimental conditions.
We concluded that phenotypes caused by the point mutations are biologically relevant to transcription, DNA replication and DNA repair, because the sensitive residues are concentrated in specific overlapping regions on the surfaces of the core histones, and the phenotypes caused by the point mutations in core histones are well consistent with those caused by mutations in chromatin-associated factors (Bennett et al. 2001; Chang et al. 2002; Chimura et al. 2002; Kagalwala et al. 2004). Further analyses of the residues involved in each reaction will be necessary to confirm and extend the present results, and to exclude potential artifacts that can sometimes occur in these in vivo assay systems. Surprisingly, 37 of 88 residues showed sensitivities in more than two different assays, and most of these 37 residues clustered in a few regions located at the nucleosome entry site, the surface required for nucleosome–nucleosome interactions on crystal structure or their surroundings. Thus, these data suggest that the region targeted by chromatin-associated factors and the mechanisms altering nucleosome structure in transcription, DNA replication and DNA repair are similar. The comprehensive data of potential residues responsible for these nuclear systems provides us with the first picture of how they work in a coordinated manner on core histone surfaces.This information can also be used to answer an important question of how distinct nuclear reactions are related to each other on DNA and/or chromatin.
In particular, only five post-translationally modifiable residues (H2A-K4, -S121, -S128, H2B-K123 and H3-K56) were identified in the assays performed here, although more than 20 residues (such as lysine, arginine, serine and threonine) are known to undergo post-translational modification and most residues are functionally involved in DNA-mediated reactions in yeast (Strahl & Allis 2000). This apparent contradiction suggests that modification of multiple residues of core histones may be sufficient to trigger downstream reactions, but modification of a specific residue is not always necessary. In fact, a strong sensitivity to MMS appeared only when cells carried four point mutations in lysines at the N-terminal tail of histone H4 (Bird et al. 2002). In addition, previous studies depended primarily on biochemical approaches (Strahl & Allis 2000) and it was difficult to draw conclusions from these approaches until the significance of the modified residues were confirmed genetically. In this study, 83 residues that are not considered to be covalently modified showed sensitivities, and the contributions of non-covalently modified residues must be considered no less important than those of the covalently modified residues. To date, covalent modifications of histones have been thoroughly characterized (Strahl & Allis 2000). However, residues not only subject to covalent modification but also have roles in mediating the regulatory mechanisms, which alter the nucleosome structure and function, and the data we presented here indicate that interactions between core histones and various chromatin-associated factors such as Bdf1 and CIA in a manner that does not require covalent modification (Munakata et al. 2000; Pamblanco et al. 2001) are key to controlling nucleosome structure and function.
Prospects of histone point mutant library and GLASP method
In this study, we utilized the histone point mutants at four different in vivo assays as described. Importantly, more than 70 genetic reactions have been developed to date for yeast assays in vivo, including detection of genetic defects at DNA recombination and gene silencing. It is thus significant to relate the identified positions of the point mutation with further studies utilizing other assay systems.Thus, it is possible to evaluate the functional roles of core histone surfaces more broadly by genetically utilizing the histone point mutants presented in this study. Further studies using a suppressor screen against the 88 histone point mutants that showed a phenotype in the four assays presented here will enable us to better understand the functional network of chromatin-associated factors that interact with core histones, as well as to identify novel chromatin-related factors.
By introducing point mutations into all histone surface residues, we obtained novel in vivo results and generated valuable resources for future studies of chromatin (Fig. 7B). The findings presented in this study indicate the usefulness of the GLASP strategy for understanding protein functions at the single amino acid level. GLASP is most suitable for analysis of evolutionarily conserved factors and/or hub proteins in cellular networks. The strategy allows elucidation of the structure and function of all surface residues and of functional networks composed of many interacting factors. Additionally, since GLASP is based on point mutagenesis, it is also effective for the functional analysis of multi-subunit complexes, which are difficult to characterize by deletion mutagenesis without the introduction of structural abnormalities. GLASP is similar but different in its concept with the conventional alanine scanning method in which: (i) GLASP is intended to cover the nearly completed residues composing the overall surface of a particular highly-conserved protein, which is not intended in the conventional alanine scanning method; and (ii) GLASP does not limit the mutations to alanine alone but involves substitutions to amino acids other than alanine, to obtain requisite molecular surface information. An extension of GLASP that entails the introduction of double mutations on molecular surfaces would be effective for investigating residues for which single mutation does not confer a phenotype, such as those that work in complementary ways and/or those that are less conserved. Furthermore, the GLASP method is also adaptable for high throughput applications employing robotics.
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Experimental procedures |
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TopAbstractIntroductionResultsIdentification of MMS-sensitive...DiscussionExperimental proceduresReferences |
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The genes encoding histone H2A and H2B were deleted in the host strain FY406, MATa 
(HTA1 HTB1) (HTA2 HTB2) lys2-128
his3200 ura3-52 pSAB6 [URA3 HTA1 HTB1], a gift from Dr F. Winston (Hirschhorn et al. 1995). These deletions were rescued with a plasmid carrying the wild-type H2A and H2B genes along with the selectable marker URA3 (pSAB6). We then transformed strain FY406 with plasmids carrying either the mutant H2A or H2B genes, or the corresponding wild-type gene as a control. The resultant transformants were spread on plates containing 5-fluoroorotic acid (5-FOA) to obtain cells that had lost pSAB6. Construction of histone H3 and H4 mutants employed MSY748, MAT
(HHT1 HHF1) (HHT2 HHF2) his4-912
lys2-128
leu2-3,112 ura3-52 pMS329 [URA3 HHT1 HHF1], a gift from Dr M. M. Smith (Santisteban et al. 1997), as previously described (Kimura et al. 2002).
Mutagenesis
We introduced alanine substitution mutations using a site-directed mutagenesis method. H2A or H2B were carried in a plasmid with the HIS3 selectable marker (pRS313-HTA1-HTB1) and histone H3 or H4 were on a LEU2 selectable marker bearing plasmid (pRS315-HHT1-HHF1). The mutagenesis positions are illustrated in Fig. 1E,F.
Media
Synthetic complete (SC) medium contained 0.67% (w/v) yeast nitrogen base without amino acids, 2% (w/v) glucose, and 2% (w/v) bacto agar, supplemented with adenine, uracil and appropriate amino acids. For the assay of Spt- phenotype, we prepared SC medium with or without lysine. 6AU, HU, and MMS-containing media were prepared with SC medium by supplementing it with 1 mg/mL 6AU, 100 mM HU, or 0.016% (v/v) MMS, respectively.
Spt- phenotype- and drug sensitivity assays
Three-fold serial dilutions of strains with the indicated genotypes were spotted onto SC agar medium with or without lysine (for the Spt- phenotype assay), and SC agar medium with or without each drug (for the drug sensitivity assays). Each spot contained approximately 1 x 105, 3 x 104, 1 x 104, 3 x 103, 1 x 103, 3 x 102, 1 x 102 and 3 x 101 cells, respectively. The yeast-spotted plates were incubated at 30 °C for 3–4 days for the assay of Spt- phenotype, and 3 days for the drug sensitivity assays. The experiments were performed in duplicate and were repeated multiple times.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: horikosh{at}iam.u-tokyo.ac.jp
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References |
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TopAbstractIntroductionResultsIdentification of MMS-sensitive...DiscussionExperimental proceduresReferences |
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