Leading role of TBP in the Establishment of Complexity in Eukaryotic Transcription Initiation Systems.

While both archaeal and eukaryotic transcription initiation systems utilize TBP (TATA box-binding protein) and TFIIB (transcription factor IIB), eukaryotic systems include larger numbers of initiation factors. It remains uncertain how eukaryotic transcription initiation systems have evolved. Here, we investigate the evolutionary development of TBP and TFIIB, each of which has an intramolecular direct repeat, using two evolutionary indicators. Inter-repeat sequence dissimilarity (dDR, distance between direct repeats) indicates that the asymmetry of two repeats in TBP and TFIIB has gradually increased during evolution. Interspecies sequence diversity (PD, phylogenetic diversity) indicates that the resultant asymmetric structure, which is related to the ability to interact with multiple factors, diverged in archaeal TBP and archaeal/eukaryotic TFIIB during evolution. Our findings suggest that eukaryotic TBP initially acquired multiple Eukarya-specific interactors through asymmetric evolution of the two repeats. After the asymmetric TBP generated the complexity of the eukaryotic transcription initiation systems, its diversification halted and its asymmetric structure spread throughout eukaryotic species.

Uncovering ancient transcription systems with a novel evolutionary indicator.

TBP and TFIIB are evolutionarily conserved transcription initiation factors in archaea and eukaryotes. Information about their ancestral genes would be expected to provide insight into the origin of the RNA polymerase II-type transcription apparatus. In obtaining such information, the nucleotide sequences of current genes of both archaea and eukaryotes should be included in the analysis. However, the present methods of evolutionary analysis require that a subset of the genes should be excluded as an outer group. To overcome this limitation, we propose an innovative concept for evolutionary analysis that does not require an outer group. This approach utilizes the similarity in intramolecular direct repeats present in TBP and TFIIB as an evolutionary measure revealing the degree of similarity between the present offspring genes and their ancestors. Information on the properties of the ancestors and the order of emergence of TBP and TFIIB was also revealed. These findings imply that, for evolutionarily early transcription systems billions of years ago, interaction of RNA polymerase II with transcription initiation factors and the regulation of its enzymatic activity was required prior to the accurate positioning of the enzyme. Our approach provides a new way to discuss mechanistic and system evolution in a quantitative manner.

Interaction of the Jhd2 Histone H3 Lys-4 Demethylase with Chromatin Is Controlled by Histone H2A Surfaces and Restricted by H2B Ubiquitination.

Histone H3 lysine 4 (H3K4) methylation is a dynamic modification. In budding yeast, H3K4 methylation is catalyzed by the Set1-COMPASS methyltransferase complex and is removed by Jhd2, a JMJC domain family demethylase. The catalytic JmjC and JmjN domains of Jhd2 have the ability to remove all three degrees (mono-, di-, and tri-) of H3K4 methylation. Jhd2 also contains a plant homeodomain (PHD) finger required for its chromatin association and H3K4 demethylase functions. The Jhd2 PHD finger associates with chromatin independent of H3K4 methylation and the H3 N-terminal tail. Therefore, how Jhd2 associates with chromatin to perform H3K4 demethylation has remained unknown. We report a novel interaction between the Jhd2 PHD finger and histone H2A. Two residues in H2A (Phe-26 and Glu-57) serve as a binding site for Jhd2 in vitro and mediate its chromatin association and H3K4 demethylase functions in vivo. Using RNA sequencing, we have identified the functional target genes for Jhd2 and the H2A Phe-26 and Glu-57 residues. We demonstrate that H2A Phe-26 and Glu-57 residues control chromatin association and H3K4 demethylase functions of Jhd2 during positive or negative regulation of transcription at target genes. Importantly, we show that H2B Lys-123 ubiquitination blocks Jhd2 from accessing its binding site on chromatin, and thereby, we have uncovered a second mechanism by which H2B ubiquitination contributes to the trans-histone regulation of H3K4 methylation. Overall, our study provides novel insights into the chromatin binding dynamics and H3K4 demethylase functions of Jhd2.

Roles of common subunits within distinct multisubunit complexes.

Currently, there is no method to distinguish between the roles of a subunit in one multisubunit protein complex from its roles in other complexes in vivo. This is because a mutation in a common subunit will affect all complexes containing that subunit. Here, we describe a unique method to discriminate between the functions of a common subunit in different multisubunit protein complexes. In this method, a common subunit in a multisubunit protein complex is genetically fused to a subunit that is specific to that complex and point mutated. The resulting phenotype(s) identify the specific function(s) of the subunit in that complex only. Histone H2B is a common subunit in nucleosomes containing H2A/H2B or Htz1/H2B dimers. The H2B was fused to H2A or Htz1 and point mutated. This strategy revealed that H2B has common and distinct functions in different nucleosomes. This method could be used to study common subunits in other multisubunit protein complexes.

Histone acetylation: from code to web and router via intrinsically disordered regions.

Structural changes of chromatin, which consists of nucleosomes and nucleosome-associated factors, lead to functional changes that are important determinants of eukaryotic gene regulation. These structural changes are regulated by modifications of histones and DNA, both of which are components of nucleosomes, as well as by replacement of histone variants and the actions of noncoding RNAs. In studies of chromatin modifications, a great deal of attention has been paid to histone acetylation. Progress in understanding this subject has been extensive, including i) elucidation of the relationship of histone acetylation and gene activity; ii) the first isolation of a histonemodifying enzyme; iii) the first identification of a factor that recognizes a modified site; iv) elucidation of the mechanism by which histone modification leads to structural changes in nucleosomes; and v) elucidation of the mechanism of border formation between euchromatin and heterochromatin. Histone acetylation is considered to be fundamental in several fields, including studies of a) the role of chromatin and epigenetics in higher-order biochemical systems such as transcription, DNA replication, and repair; b) biological phenomena such as cell proliferation and differentiation; and c) cancer and aging, potentially leading to clinical applications. In this review, I will discuss the histone code hypothesis, at one time believed to represent a unified theory regarding the functions of histone modification. In addition, I will describe the "modification web theory, " by which the problems in the histone code hypothesis can be overcome, as well as the "signal router theory, " which explains the mechanisms of formation, development, and evolution of the modification web from a structural viewpoint. Lastly, I will illustrate how these novel theories partially explain the robustness of biological systems against various perturbations, and elucidate the strategy that a cell employs to avoid fatal fragility.

Nucleosome surface containing nucleosomal DNA entry/exit site regulates H3-K36me3 via association with RNA polymerase II and Set2.

A nucleosome is composed of intrinsically disordered histone tails and a structured nucleosome core surrounded by DNA. A variety of modifiable residues on the intrinsically disordered histone tails have been identified in the last decade. Mapping of the functional residues on the structured nucleosome core surface was recently initiated by global analysis of a comprehensive histone point mutant library (histone-GLibrary). It stands to reason that a functional relationship exists between modifiable residues on the intrinsically disordered histone tails and functional residues on the structured nucleosome core; however, this matter has been poorly explored. During transcription elongation, trimethylation of histone H3 at lysine 36 (H3-K36me3) is mediated by histone methyltransferase Set2, which binds to RNA polymerase II. Here, we used a histone-GLibrary that encompasses the nucleosomal DNA entry/exit site to show that six residues (H2A-G107, H2A-I112, H2A-L117, H3-T45, H3-R49 and H3-R52) form a surface on the structured nucleosome core and regulate H3-K36me3. Trimethylation at H3-K4 introduced by histone methyltransferase Set1 was not affected by the mutation of any of the six residues. Chromatin immunoprecipitation analysis showed that most of these residues are critical for the chromatin association of RNA polymerase II and Set2, suggesting that these components regulate H3-K36me3 through functional interactions with the structured nucleosome core surface.

Roles of histone chaperone CIA/Asf1 in nascent DNA elongation during nucleosome replication.

The nucleosome, which is composed of DNA wrapped around a histone octamer, is a fundamental unit of chromatin and is duplicated during the eukaryotic DNA replication process. The evolutionarily conserved histone chaperone cell cycle gene 1 (CCG1) interacting factor A/anti-silencing function 1 (CIA/Asf1) is involved in histone transfer and nucleosome reassembly during DNA replication. CIA/Asf1 has been reported to split the histone (H3-H4)(2) tetramer into histone H3-H4 dimer(s) in vitro, raising a possibility that, in DNA replication, CIA/Asf1 is involved in nucleosome disassembly and the promotion of semi-conservative histone H3-H4 dimer deposition onto each daughter strand in vivo. Despite numerous studies on the functional roles of CIA/Asf1, its mechanistic role(s) remains elusive because of lack of biochemical analyses. The biochemical studies described here show that a V94R CIA/Asf1 mutant, which lacks histone (H3-H4)(2) tetramer splitting activity, does not form efficiently a quaternary complex with histones H3-H4 and the minichromosome maintenance 2 (Mcm2) subunit of the Mcm2-7 replicative DNA helicase. Interestingly, the mutant enhances nascent DNA strand synthesis in a cell-free chromosomal DNA replication system using Xenopus egg extracts. These results suggest that CIA/Asf1 in the CIA/Asf1-H3-H4-Mcm2 complex, which is considered to be an intermediate in histone transfer during DNA replication, negatively regulates the progression of the replication fork.

The histone chaperone facilitates chromatin transcription (FACT) protein maintains normal replication fork rates.

Ordered nucleosome disassembly and reassembly are required for eukaryotic DNA replication. The facilitates chromatin transcription (FACT) complex, a histone chaperone comprising Spt16 and SSRP1, is involved in DNA replication as well as transcription. FACT associates with the MCM helicase, which is involved in DNA replication initiation and elongation. Although the FACT-MCM complex is reported to regulate DNA replication initiation, its functional role in DNA replication elongation remains elusive. To elucidate the functional role of FACT in replication fork progression during DNA elongation in the cells, we generated and analyzed conditional SSRP1 gene knock-out chicken (Gallus gallus) DT40 cells. SSRP1-depleted cells ceased to grow and exhibited a delay in S-phase cell cycle progression, although SSRP1 depletion did not affect the level of chromatin-bound DNA polymerase α or nucleosome reassembly on daughter strands. The tracking length of newly synthesized DNA, but not origin firing, was reduced in SSRP1-depleted cells, suggesting that the S-phase cell cycle delay is mainly due to the inhibition of replication fork progression rather than to defects in the initiation of DNA replication in these cells. We discuss the mechanisms of how FACT promotes replication fork progression in the cells.

Global analysis of core histones reveals nucleosomal surfaces required for chromosome bi-orientation.

The attachment of sister kinetochores to microtubules from opposite spindle poles is essential for faithful chromosome segregation. Kinetochore assembly requires centromere-specific nucleosomes containing the histone H3 variant CenH3. However, the functional roles of the canonical histones (H2A, H2B, H3, and H4) in chromosome segregation remain elusive. Using a library of histone point mutants in Saccharomyces cerevisiae, 24 histone residues that conferred sensitivity to the microtubule-depolymerizing drugs thiabendazole (TBZ) and benomyl were identified. Twenty-three of these mutations were clustered at three spatially separated nucleosomal regions designated TBS-I, -II, and -III (TBZ/benomyl-sensitive regions I-III). Elevation of mono-polar attachment induced by prior nocodazole treatment was observed in H2A-I112A (TBS-I), H2A-E57A (TBS-II), and H4-L97A (TBS-III) cells. Severe impairment of the centromere localization of Sgo1, a key modulator of chromosome bi-orientation, occurred in H2A-I112A and H2A-E57A cells. In addition, the pericentromeric localization of Htz1, the histone H2A variant, was impaired in H4-L97A cells. These results suggest that the spatially separated nucleosomal regions, TBS-I and -II, are necessary for Sgo1-mediated chromosome bi-orientation and that TBS-III is required for Htz1 function.

Global analysis for functional residues of histone variant Htz1 using the comprehensive point mutant library.

Histone variants perform unique functions and are deposited onto DNA by mechanisms distinct from those of canonical histones. The H2A variant, H2A.Z, also known as Htz1 in Saccharomyces cerevisiae, is not uniformly distributed across the genome but facilitates transcriptional activation at target gene promoters and anti-silencing at heterochromatin loci. Htz1 is also involved in DNA replication, DNA repair, chromosome segregation and cell cycle control. Its sequence identity to canonical H2A is only ∼60%, and it is likely that the nonconserved residues are responsible for Htz1-specific functions. However, precise roles of these variant-specific residues are not well understood. To gain insights into the molecular basis underlying the functional differences between canonical and variant histones, 117 alanine-scanning point mutants of Htz1 were constructed for this study, and chemical genetic screens were carried out. Consequently, seven Htz1 residues that conferred one or more abnormal phenotypes when mutated were identified. Based on primary sequence and functional conservation between H2A and Htz1, two of these residues (F32 and I109) appear to have an Htz1-specific role, whereas the rest seem to have functions shared between H2A and Htz1. This study provides a useful resource for future investigations into functional convergence and divergence between canonical and variant histones.

Biphasic chromatin binding of histone chaperone FACT during eukaryotic chromatin DNA replication.

The facilitates chromatin transcription (FACT) complex affects nuclear DNA transactions in a chromatin context. Though the involvement of FACT in eukaryotic DNA replication has been revealed, a clear understanding of its biochemical behavior during DNA replication still remains elusive. Here, we analyzed the chromatin-binding dynamics of FACT using Xenopus egg extract cell-free system. We found that FACT has at least two distinct chromatin-binding phases: (1) a rapid chromatin-binding phase at the onset of DNA replication that did not involve origin licensing and (2) a second phase of chromatin binding that initiated after origin licensing. Intriguingly, early-binding FACT dissociated from chromatin when DNA replication was blocked by the addition of Cdc6 in the licensed state before origin firing. Cdc6-induced removal of FACT was blocked by the inhibition of origin licensing with geminin, but not by suppressing the activity of DNA polymerases, CDK, or Cdc7. Furthermore, chromatin transfer experiments revealed that impairing the later binding of FACT severely compromises DNA replication activity. Taken together, we propose that even though FACT has rapid chromatin-binding activity, the binding pattern of FACT on chromatin changes after origin licensing, which may contribute to the establishment of its functional link to the DNA replication machinery.

Chromatin dynamics mediated by histone modifiers and histone chaperones in postreplicative recombination.

Eukaryotic chromatin is regulated by chromatin factors such as histone modification enzymes, chromatin remodeling complexes and histone chaperones in a variety of DNA-dependent reactions. Among these reactions, transcription in the chromatin context is well studied. On the other hand, how other DNA-dependent reactions, including postreplicative homologous recombination, are regulated in the chromatin context remains elusive. Here, histone H3 Lys56 acetylation, mediated by the histone acetyltransferase Rtt109 and the histone chaperone Cia1/Asf1, is shown to be required for postreplicative sister chromatid recombination. This recombination did not occur in the cia1/asf1-V94R mutant, which lacks histone binding and histone chaperone activities and which cannot promote the histone acetyltransferase activity of Rtt109. A defect in another histone chaperone, CAF-1, led to an increase in acetylated H3-K56 (H3-K56-Ac)-dependent postreplicative recombination. Some DNA lesions recognized by the putative ubiquitin ligase complex Rtt101-Mms1-Mms22, which is reported to act downstream of the H3-K56-Ac signaling pathway, seem to be increased in CAF-1 defective cells. Taken together, these data provide the framework for a postreplicative recombination mechanism controlled by histone modifiers and histone chaperones in multiple ways.

Global analysis of functional relationships between histone point mutations and the effects of histone deacetylase inhibitors.

Comprehensive analyses of the histone-GLibrary in previous studies showed that most mutants of modification sites in the histone core regions show phenotypes, whereas those with modifications in the histone N-terminal unstructured tail regions (N-tails) do not. One possible reason is that modifications in N-tails are linked to each other to form a scale-free network termed histone 'modification web'. In the network, the compensatory pathways are created to acquire the robustness against the any defects. Because of this robustness, it is difficult to determine the significance of the individual histone modifications in N-tails in vivo. To overcome this problem, we used a strategy using drugs coordinately to inhibit modification enzymes and observed the mutant phenotypes when the compensatory pathways are largely interrupted. We analyzed histone-GLibrary using inhibitors of histone deacetylases (HDACs) and identified novel phenotypic mutants. We also examined the phenotypic changes through the combined use of an HDAC inhibitor and an inhibitor of DNA-mediated reactions. Mutation of modifiable sites H3-K4 and H4-K16 in histone N-tails, which are presumed to be the 'hubs' of the network, resulted in identifiable phenotypes. The data obtained provide valuable information for speculation on novel relationships between histone modification in N-tails and biological function and for predicting unknown modification sites in core histones.

Structure of the histone chaperone CIA/ASF1-double bromodomain complex linking histone modifications and site-specific histone eviction.

Nucleosomes around the promoter region are disassembled for transcription in response to various signals, such as acetylation and methylation of histones. Although the interactions between histone-acetylation-recognizing bromodomains and factors involved in nucleosome disassembly have been reported, no structural basis connecting histone modifications and nucleosome disassembly has been obtained. Here, we determined at 3.3 A resolution the crystal structure of histone chaperone cell cycle gene 1 (CCG1) interacting factor A/antisilencing function 1 (CIA/ASF1) in complex with the double bromodomain in the CCG1/TAF1/TAF(II)250 subunit of transcription factor IID. Structural, biochemical, and biological studies suggested that interaction between double bromodomain and CIA/ASF1 is required for their colocalization, histone eviction, and pol II entry at active promoter regions. Furthermore, the present crystal structure has characteristics that can connect histone acetylation and CIA/ASF1-mediated histone eviction. These findings suggest that the molecular complex between CIA/ASF1 and the double bromodomain plays a key role in site-specific histone eviction at active promoter regions. The model we propose here is the initial structure-based model of the biological signaling from histone modifications to structural change of the nucleosome (hi-MOST model).

Global analysis of mutual interaction surfaces of nucleosomes with comprehensive point mutants.

The surfaces of core histones in nucleosome are exposed as required for factor recognition, or buried for histone-DNA and histone-histone interactions. To understand the mechanisms by which nucleosome structure and function are coordinately altered in DNA-mediated reactions, it is essential to define the roles of both exposed and buried residues and their functional relationships. For this purpose, we developed GLASP (GLobal Analysis of Surfaces by Point mutation) and GLAMP (GLobal Analysis of Mutual interaction surfaces of multi-subunit protein complex by Point mutation) strategies, both of which are comprehensive analyses by point mutagenesis of exposed and buried residues in nucleosome, respectively. Four distinct DNA-mediated reactions evaluated by Ty suppression (the Spt(-) phenotype), and sensitivities to 6-azauracil (6AU), hydroxyurea (HU), and methyl methanesulfonate (MMS), require common and different GLAMP residues. Mutated GLAMP residues at the interface between histones H2A and H2B mainly affect the Spt(-) phenotype but not HU and MMS sensitivities. Interestingly, among the mutated GLAMP residues surrounding the histone H3-H3' interface, some equally affect the Spt(-) phenotype, and HU and MMS sensitivities, whereas others differentially affect the Spt(-) phenotype, and HU and MMS sensitivities. Based on these and other results, the functional relationships among chromatin factors and GLASP and GLAMP residues provide insights into nucleosome disassembly/assembly processes in DNA-mediated reactions.

Theoretical framework for the histone modification network: modifications in the unstructured histone tails form a robust scale-free network.

A rapid increase in research on the relationship between histone modifications and their subsequent reactions in the nucleus has revealed that the histone modification system is complex, and robust against point mutations. The prevailing theoretical framework (the histone code hypothesis) is inadequate to explain either the complexity or robustness, making the formulation of a new theoretical framework both necessary and desirable. Here, we develop a model of the regulatory network of histone modifications in which we encode histone modifications as nodes and regulatory interactions between histone modifications as links. This network has scale-free properties and subnetworks with a pseudo-mirror symmetry structure, which supports the robustness of the histone modification network. In addition, we show that the unstructured tail regions of histones are suitable for the acquisition of this scale-free property. Our model and related insights provide the first framework for an overall architecture of a histone modification network system, particularly with regard to the structural and functional roles of the unstructured histone tail region. In general, the post-translational "modification webs" of natively unfolded regions (proteins) may function as signal routers for the robust processing of the large amounts of signaling information.

Crystal structure of Methanococcus jannaschii TATA box-binding protein.

As the archaeal transcription system consists of a eukaryotic-type transcription apparatus and bacterial-type regulatory transcription factors, analyses of the molecular interface between the transcription apparatus and regulatory transcription factors are critical to reveal the evolutionary change of the transcription system. TATA box-binding protein (TBP), the central components of the transcription apparatus are classified into three groups: eukaryotic, archaeal-I and archaeal-II TBPs. Thus, comparative functional analysis of these three groups of TBP is important for the study of the evolution of the transcription system. Here, we present the first crystal structure of an archaeal-II TBP from Methanococcus jannaschii. The highly conserved and group-specific conserved surfaces of TBP bind to DNA and TFIIB/TFB, respectively. The phylogenetic trees of TBP and TFIIB/TFB revealed that they evolved in a coupled manner. The diversified surface of TBP is negatively charged in the archaeal-II TBP, which is completely different from the case of eukaryotic and archaeal-I TBPs, which are positively charged and biphasic, respectively. This difference is responsible for the diversification of the regulatory functions of TBP during evolution.

Effect of leucine-to-methionine substitutions on the diffraction quality of histone chaperone SET/TAF-Ibeta/INHAT crystals.

One of the most frequent problems in crystallization is poor quality of the crystals. In order to overcome this obstacle several methods have been utilized, including amino-acid substitutions of the target protein. Here, an example is presented of crystal-quality improvement by leucine-to-methionine substitutions. A variant protein with three amino-acid substitutions enabled improvement of the crystal quality of the histone chaperone SET/TAF-Ibeta/INHAT when combined with optimization of the cryoconditions. This procedure improved the resolution of the SET/TAF-Ibeta/INHAT crystals from around 5.5 to 2.3 A without changing the crystallization conditions.

Novel structural and functional mode of a knot essential for RNA binding activity of the Esa1 presumed chromodomain.

Chromodomains are methylated histone binding modules that have been widely studied. Interestingly, some chromodomains are reported to bind to RNA and/or DNA, although the molecular basis of their RNA/DNA interactions has not been solved. Here we propose a novel binding mode for chromodomain-RNA interactions. Essential Sas-related acetyltransferase 1 (Esa1) contains a presumed chromodomain in addition to a histone acetyltransferase domain. We initially determined the solution structure of the Esa1 presumed chromodomain and showed it to consist of a well-folded structure containing a five-stranded beta-barrel similar to the tudor domain rather than the canonical chromodomain. Furthermore, the domain showed no RNA/DNA binding ability. Because the N-terminus of the protein forms a helical turn, we prepared an N-terminally extended construct, which we surprisingly found to bind to poly(U) and to be critical for in vivo function. This extended protein contains an additional beta-sheet that acts as a knot for the tudor domain and binds to oligo(U) and oligo(C) with greater affinity compared with other oligo-RNAs and DNAs examined thus far. The knot does not cause a global change in the core structure but induces a well-defined loop in the tudor domain itself, which is responsible for RNA binding. We made 47 point mutants in an esa1 mutant gene in yeast in which amino acids of the Esa1 knotted tudor domain were substituted to alanine residues and their functional abilities were examined. Interestingly, the knotted tudor domain mutations that were lethal to the yeast lost poly(U) binding ability. Amino acids that are related to RNA interaction sites, as revealed by both NMR and affinity binding experiments, are found to be important in vivo. These findings are the first demonstration of how the novel structure of the knotted tudor domain impacts on RNA binding and how this influences in vivo function.

Promoter region-specific histone incorporation by the novel histone chaperone ANP32B and DNA-binding factor KLF5.

Regulation of chromatin in eukaryotic transcription requires histone-modifying enzymes, nucleosome remodeling complexes, and histone chaperones. Specific regulation of histone incorporation/eviction by histone chaperones on the promoter (e.g., region specific) is still poorly understood. In the present study, we show that direct and functional interaction of histone chaperone and DNA-binding transcription factor leads to promoter region-specific histone incorporation and inhibition of histone acetylation. We report here that the DNA-binding transcription factor Krüppel-like factor 5 (KLF5) interacts with the novel histone chaperone acidic nuclear phosphoprotein 32B (ANP32B), leading to transcriptional repression of a KLF5-downstream gene. We further show that recruitment of ANP32B onto the promoter region requires KLF5 and results in promoter region-specific histone incorporation and inhibition of histone acetylation by ANP32B. Extracellular stimulus (e.g., phorbol ester) regulates this mechanism in the cell. Collectively, we have identified a novel histone chaperone, ANP32B, and through analysis of the actions of this factor show a new mechanism of promoter region-specific transcriptional regulation at the chromatin level as mediated by the functional interaction between histone chaperone and DNA-binding transcription factor.