Masami Horikoshi

Chromosomal gradient of histone acetylation established by Sas2p and Sir2p functions as a shield against gene silencing

Genes located in chromosomal regions near telomeres are transcriptionally silent, whereas those located in regions away from telomeres are not. Here we show that there is a gradient of acetylation of histone H4 at lysine 16 (H4–Lys16) along a yeast chromosome; this gradient ranges from a hypoacetylated state in regions near the telomere to a hyperacetylated state in more distant regions. The hyperacetylation is regulated by Sas2p, a member of the MYST-type family of histone acetylases, whereas hypoacetylation is under the control of Sir2p, a histone deacetylase. Loss of hyperacetylation is accompanied by an increase in localization of the telomere protein Sir3p and the inactivation of gene expression in telomere-distal regions. Thus, the Sas2p and Sir2p function in concert to regulate transcription in yeast, by acetylating and deacetylating H4–Lys16 in a mechanism that may be common to all eukaryotes.

Genome-wide Expression Analysis of Mouse Liver Reveals CLOCK-regulated Circadian Output Genes

CLOCK is a positive component of a transcription/translation-based negative feedback loop of the central circadian oscillator in the suprachiasmatic nucleus in mammals. To examine CLOCK-regulated circadian transcription in peripheral tissues, we performed microarray analyses using liver RNA isolated from Clock mutant mice. We also compared expression profiles with those of Cryptochromes (Cry1 and Cry2) double knockout mice. We identified more than 100 genes that fluctuated from day to night and of which expression levels were decreased in Clock mutant mice. In Cry-deficient mice, the expression levels of most CLOCK-regulated genes were elevated to the upper range of normal oscillation. Most of the screened genes had a CLOCK/BMAL1 binding site (E box) in the 5′-flanking region. We found that CLOCK was absolutely concerned with the circadian transcription of one type of liver genes (such as DBP, TEF, and Usp2) and partially with another (such as mPer1, mPer2, mDec1, Nocturnin, P450 oxidoreductase, and FKBP51) because the latter were damped but remained rhythmic in the mutant mice. Our results showed that CLOCK and CRY proteins are involved in the transcriptional regulation of many circadian output genes in the mouse liver. In addition to being a core component of the negative feedback loop that drives the circadian oscillator, CLOCK also appears to be involved in various physiological functions such as cell cycle, lipid metabolism, immune functions, and proteolysis in peripheral tissues.

Factors involved in specific transcription by mammalian RNA polymerase II: purification, genetic specificity, and TATA box-promoter interactions of TFIID.

Selective and accurate transcription of purified genes by RNA polymerase II requires multiple factors. The factor designated TFIID was purified extensively from HeLa cell nuclear extracts by using a simple and novel complementation assay. Thus, TFIID was preferentially inactivated by mild heat treatment of a nuclear extract, and supplementation of the heat-treated extract with TFIID-containing fractions restored adenovirus major late (ML) promoter-dependent transcription. By using this assay, TFIID was purified approximately 300-fold by conventional chromatographic methods. The most purified TFIID fraction was demonstrated to be required for transcription of a number of other cellular and viral class II genes. This factor showed specific interactions with both the adenovirus ML promoter and a human heat shock 70 (hsp-70) promoter. On the ML promoter, the DNase I-protected region extended from around position -40 to position +35, although some discontinuities (and associated hypersensitive sites) were apparent near the initiation site and near position +27; the upstream and downstream boundaries of the TFIID-binding site were also confirmed by exonuclease III digestion experiments. In contrast to these results, the DNase I-protected regions on the human hsp-70 promoter were confined to a smaller area that extended from positions -35 to -19. DNase I hypersensitive sites were observed in both the adenovirus ML and hsp-70 promoters, most notably in the region at position -47. These results indicate either that there are different forms of TFIID or that a single TFIID can interact differently with distinct promoters.

Transcription Factor ATF Interacts with the TATA Factor to Facilitate Establishment of a Preinitiation Complex

The mammalian activator protein ATF stimulates transcription from the adenovirus E4 promoter by binding to multiple upstream promoter and enhancer elements. DNAase footprint analyses have revealed that there are cooperative interactions between ATF and TFIID (the mammalian TATA factor) when both are bound simultaneously to the promoter and that these interactions in turn facilitate promoter recognition by RNA polymerase II and the general initiation factors TFIIB and TFIIE. However, the complex of TFIID and the other general factors is stable following oligonucleotide-mediated dissociation of ATF from the complete preinitiation complex. These results indicate that TFIID is a direct target for ATF, that these interactions facilitate assembly of a complete preinitiation complex, and that the role of ATF might be transient.

Structure and function of the histone chaperone CIA/ASF1 complexed with histones H3 and H4.

CIA (CCG1-interacting factor A)/ASF1, which is the most conserved histone chaperone among the eukaryotes, was genetically identified as a factor for an anti-silencing function (Asf1) by yeast genetic screening. Shortly after that, the CIA–histone-H3–H4 complex was isolated from Drosophila as a histone chaperone CAF-1 stimulator. Human CIA-I/II (ASF1a/b) was identified as a histone chaperone that interacts with the bromodomain—an acetylated-histone-recognizing domain—of CCG1, in the general transcription initiation factor TFIID. Intensive studies have revealed that CIA/ASF1 mediates nucleosome assembly by forming a complex with another histone chaperone in human cells and yeast, and is involved in DNA replication, transcription, DNA repair and silencing/anti-silencing in yeast. CIA/ASF1 was shown as a major storage chaperone for soluble histones in proliferating human cells. Despite all these biochemical and biological functional analyses, the structure–function relationship of the nucleosome assembly/disassembly activity of CIA/ASF1 has remained elusive. Here we report the crystal structure, at 2.7 Å resolution, of CIA-I in complex with histones H3 and H4. The structure shows the histone H3–H4 dimers mutually exclusive interactions with another histone H3–H4 dimer and CIA-I. The carboxy-terminal β-strand of histone H4 changes its partner from the β-strand in histone H2A to that of CIA-I through large conformational change. In vitro functional analysis demonstrated that CIA-I has a histone H3–H4 tetramer-disrupting activity. Mutants with weak histone H3–H4 dimer binding activity showed critical functional effects on cellular processes related to transcription. The histone H3–H4 tetramer-disrupting activity of CIA/ASF1 and the crystal structure of the CIA/ASF1–histone-H3–H4 dimer complex should give insights into mechanisms of both nucleosome assembly/disassembly and nucleosome semi-conservative replication.

Novel Substrate Specificity of the Histone Acetyltransferase Activity of HIV-1-Tat Interactive Protein Tip60

Tip60, originally isolated as an HIV-1-Tat interactive protein, contains an evolutionarily conserved domain with yeast silencing factors. We demonstrate here direct biochemical evidence that this domain of Tip60 has histone acetyltransferase activity. The purified recombinant effectively acetylates H2A, H3, and H4 but not H2B of core histone mixtures. This substrate specificity has not been observed among histone acetyltransferases analyzed to date. These results indicate that Tip60 is a histone acetyltransferase with a novel property, suggesting that Tip60 and its related factors may introduce a distinct alteration on chromatin.

Tip60 acetylates six lysines of a specific class in core histones in vitro.

Background: Tip60, an HIV‐1‐Tat interactive protein, is a nuclear histone acetyltransferase (HAT) with unique histone substrate specificity. Since the acetylation of core histones at particular lysines mediates distinct effects on chromatin assembly and gene regulation, the identification of lysine site specificity of the HAT activity of Tip60 is an initial step in the analysis of its molecular function.

Histone chaperones: 30 years from isolation to elucidation of the mechanisms of nucleosome assembly and disassembly

Abstract.Some three decades have passed since the discovery of nucleosomes in 1974 and the first isolation of a histone chaperone in 1978. While various types of histone chaperones have been isolated and functionally analyzed, the elementary processes of nucleosome assembly and disassembly have been less well characterized. Recently, the tertiary structure of a hetero-trimeric complex composed of the histone chaperone CIA/ASF1 and the histone H3-H4 dimer was determined, and this complex was proposed to be an intermediate in nucleosome assembly and disassembly reactions. In addition, CIA alone was biochemically shown to dissociate the histone (H3-H4)2 tetramer into two histone H3-H4 dimers. This activity suggested that CIA regulates the semi-conservative replication of nucleosomes. Here, we provide an overview of prominent histone chaperones with the goal of elucidating the mechanisms that preserve and modify epigenetic information. We also discuss the reactions involved in nucleosome assembly and disassembly.

Regulation of interaction of the acetyltransferase region of p300 and the DNA‐binding domain of Sp1 on and through DNA binding

The coactivator p300 acts as a transcriptional adaptor for many DNA‐binding activators. The finding that p300 possesses intrinsic acetyltransferase activity which, by chemically modifying histone tails affects the nucleosomal environment and transcription, has greatly advanced our understanding of its function. Subsequent recent studies have shown that non‐histone proteins are also acetylated. However, one central question which has remained unanswered is how the coactivator/acetyltransferase interacts with DNA‐binding activators to modulate their actions.

Mechanism of action of a yeast activator: Direct effect of GAL4 derivatives on mammalian TFIID-promoter interactions

We have analyzed interactions between the mammalian TATA factor (TFIID) and derivatives of the yeast activator GAL4. The interaction of the TATA factor on the adenovirus E4 promoter with GAL4 binding sites adjacent to the TATA site was qualitatively altered in response to GAL4 binding. Alterations in the TFIID interactions were observed with two GAL4 derivatives that stimulated hybrid E4 promoter activity in vitro but not with a third derivative that bound to DNA but showed no activation. These results indicate that TFIID is a direct target for a GAL4 activation domain and suggest a simple general model for the activation mechanism.