Naruhiko Adachi

Positive and Negative Regulation of the Cardiovascular Transcription Factor KLF5 by p300 and the Oncogenic Regulator SET through Interaction and Acetylation on the DNA-Binding Domain

ABSTRACT Here we show a novel pathway of transcriptional regulation of a DNA-binding transcription factor by coupled interaction and modification (e.g., acetylation) through the DNA-binding domain (DBD). The oncogenic regulator SET was isolated by affinity purification of factors interacting with the DBD of the cardiovascular transcription factor KLF5. SET negatively regulated KLF5 DNA binding, transactivation, and cell-proliferative activities. Down-regulation of the negative regulator SET was seen in response to KLF5-mediated gene activation. The coactivator/acetylase p300, on the other hand, interacted with and acetylated KLF5 DBD, and activated its transcription. Interestingly, SET inhibited KLF5 acetylation, and a nonacetylated mutant of KLF5 showed reduced transcriptional activation and cell growth complementary to the actions of SET. These findings suggest a new pathway for regulation of a DNA-binding transcription factor on the DBD through interaction and coupled acetylation by two opposing regulatory factors of a coactivator/acetylase and a negative cofactor harboring activity to inhibit acetylation.

Crystal Structure of the Human BRD2 Bromodomain INSIGHTS INTO DIMERIZATION AND RECOGNITION OF ACETYLATED HISTONE H4

The BET (bromodomains and extra terminal domain) family proteins recognize acetylated chromatin through their bromodomain and act as transcriptional activators. One of the BET proteins, BRD2, associates with the transcription factor E2F, the mediator components CDK8 and TRAP220, and RNA polymerase II, as well as with acetylated chromatin during mitosis. BRD2 contains two bromodomains (BD1 and BD2), which are considered to be responsible for binding to acetylated chromatin. The BRD2 protein specifically recognizes the histone H4 tail acetylated at Lys12. Here, we report the crystal structure of the N-terminal bromodomain (BD1, residues 74-194) of human BRD2. Strikingly, the BRD2 BD1 protein forms an intact dimer in the crystal. This is the first observation of a homodimer among the known bromodomain structures, through the buried hydrophobic core region at the interface. Biochemical studies also demonstrated BRD2 BD1 dimer formation in solution. The two acetyllysine-binding pockets and a negatively charged secondary binding pocket, produced at the dimer interface in BRD2 BD1, may be the unique features that allow BRD2 BD1 to selectively bind to the acetylated H4 tail.

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 Å 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).

A Conserved Motif Common to the Histone Acetyltransferase Esa1 and the Histone Deacetylase Rpd3

Post-translational modification of histones enables dynamic regulation of chromatin structure in eukaryotes. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) modify the N-terminal tails of histones by adding or removing acetyl groups to specific lysine residues. A particular pair of HAT (Esa1) and HDAC (Rpd3) is proposed to modify the same lysine residue in vitro and in vivo. Thus, HAT and HDAC might have similar structural and functional motifs. Here we show that HAT (Esa1 family) and HDAC (Rpd3 family) have similar amino acid stretches in the primary structures through evolution. We refer to this region as the “ER (Esa1-Rpd3) motif.” In the tertiary structure of Esa1, the ER motif is located near the active center. In Rpd3, for which the tertiary structure remains unclear, we demonstrate that the ER motif contains the same secondary structure as found in Esa1 by circular dichroism analysis. We did alanine-scanning mutagenesis and found that the ER motif regions of Esa1 or Rpd3 are required for HAT activity of Esa1 or HDAC activity of Rpd3, respectively. Our discovery of the ER motif present in the pair of enzymes (HAT and HDAC) indicates that HAT and HDAC have common structural bases, although they catalyze the reaction with opposite functions.

The Crystal Structure of CCG1/TAFII250-interacting Factor B (CIB)

The general transcription initiation factor TFIID and its interactors play critical roles in regulating the transcription from both naked and chromatin DNA. We have isolated a novel TFIID interactor that we denoted as CCG1/TAFII250-interacting factor B (CIB). We show here that CIB activates transcription. To further understand the function of this protein, we determined its crystal structure at 2.2-Å resolution. The tertiary structure of CIB reveals an α/β-hydrolase fold that resembles structures in the prokaryotic α/β-hydrolase family proteins. It is not similar in structure or primary sequence to any eukaryotic transcription or chromatin factors that have been reported to date. CIB possesses a conserved catalytic triad that is found in other α/β-hydrolases, and our in vitro studies confirmed that it bears hydrolase activity. However, CIB differs from other α/β-hydrolases in that it lacks a binding site excursion, which facilitates the substrate selectivity of the other α/β-hydrolases. Further functional characterization of CIB based on its tertiary structure and through biochemical studies may provide novel insights into the mechanisms that regulate eukaryotic transcription.

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.

Purification, crystallization and preliminary X-ray diffraction analysis of human oncoprotein SET/TAF-1β

The human oncoprotein SET/TAF-1beta has been crystallized by the sitting-drop vapour-diffusion method using ammonium sulfate as a precipitant. The crystal belongs to space group C2, with unit-cell parameters a = 119.6, b = 62.8, c = 61.0 A, beta = 89.7 degrees, and contains two molecules in the asymmetric unit. A complete data set was collected to 2.8 A resolution using synchrotron radiation.

Purification, crystallization and preliminary X-ray diffraction analysis of yeast regulatory particle non-ATPase subunit 6 (Nas6p)

The regulatory particle non-ATPase subunit, Nas6p, from Saccharomyces cerevisiae has been crystallized by the hanging-drop vapour-diffusion method using PEG 4000 as precipitant. The crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 41.43 (2), b = 61.74 (1), c = 98.09 (2) A, and contain one molecule in the asymmetric unit. A complete diffraction data set using synchrotron radiation was collected to 2.6 A resolution.

Crystal structure of the DNA‐binding domain of the LysR‐type transcriptional regulator CbnR in complex with a DNA fragment of the recognition‐binding site in the promoter region

LysR‐type transcriptional regulators (LTTRs) are among the most abundant transcriptional regulators in bacteria. CbnR is an LTTR derived from Cupriavidus necator (formerly Alcaligenes eutrophus or Ralstonia eutropha) NH9 and is involved in transcriptional activation of the cbnABCD genes encoding chlorocatechol degradative enzymes. CbnR interacts with a cbnA promoter region of approximately 60 bp in length that contains the recognition‐binding site (RBS) and activation‐binding site (ABS). Upon inducer binding, CbnR seems to undergo conformational changes, leading to the activation of the transcription. Since the interaction of an LTTR with RBS is considered to be the first step of the transcriptional activation, the CbnR–RBS interaction is responsible for the selectivity of the promoter to be activated. To understand the sequence selectivity of CbnR, we determined the crystal structure of the DNA‐binding domain of CbnR in complex with RBS of the cbnA promoter at 2.55 Å resolution. The crystal structure revealed details of the interactions between the DNA‐binding domain and the promoter DNA. A comparison with the previously reported crystal structure of the DNA‐binding domain of BenM in complex with its cognate RBS showed several differences in the DNA interactions, despite the structural similarity between CbnR and BenM. These differences explain the observed promoter sequence selectivity between CbnR and BenM. Particularly, the difference between Thr33 in CbnR and Ser33 in BenM appears to affect the conformations of neighboring residues, leading to the selective interactions with DNA.

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.