Chang-Hui Shen

Global Regulation by the Yeast Spt10 Protein Is Mediated through Chromatin Structure and the Histone Upstream Activating Sequence Elements

ABSTRACT The yeast SPT10 gene encodes a putative histone acetyltransferase (HAT) implicated as a global transcription regulator acting through basal promoters. Here we address the mechanism of this global regulation. Although microarray analysis confirmed that Spt10p is a global regulator, Spt10p was not detected at any of the most strongly affected genes in vivo. In contrast, the presence of Spt10p at the core histone gene promoters in vivo was confirmed. Since Spt10p activates the core histone genes, a shortage of histones could occur in spt10Δ cells, resulting in defective chromatin structure and a consequent activation of basal promoters. Consistent with this hypothesis, the spt10Δ phenotype can be rescued by extra copies of the histone genes and chromatin is poorly assembled in spt10Δ cells, as shown by irregular nucleosome spacing and reduced negative supercoiling of the endogenous 2μm plasmid. Furthermore, Spt10p binds specifically and highly cooperatively to pairs of upstream activating sequence elements in the core histone promoters [consensus sequence, (G/A)TTCCN6TTCNC], consistent with a direct role in histone gene regulation. No other high-affinity sites are predicted in the yeast genome. Thus, Spt10p is a sequence-specific activator of the histone genes, possessing a DNA-binding domain fused to a likely HAT domain.

Remodeling of Yeast CUP1 Chromatin Involves Activator-Dependent Repositioning of Nucleosomes over the Entire Gene and Flanking Sequences

ABSTRACT The yeast CUP1 gene is activated by the copper-dependent binding of the transcriptional activator, Ace1p. An episome containing transcriptionally active or inactiveCUP1 was purified in its native chromatin structure from yeast cells. The amount of RNA polymerase II on CUP1 in the purified episomes correlated with its transcriptional activity in vivo. Chromatin structures were examined by using the monomer extension technique to map translational positions of nucleosomes. The chromatin structure of an episome containing inactive CUP1 isolated from ace1Δ cells is organized into clusters of overlapping nucleosome positions separated by linkers. Novel nucleosome positions that include the linkers are occupied in the presence of Ace1p. Repositioning was observed over the entire CUP1 gene and its flanking regions, possibly over the entire episome. Mutation of the TATA boxes to prevent transcription did not prevent repositioning, implicating a chromatin remodeling activity recruited by Ace1p. These observations provide direct evidence in vivo for the nucleosome sliding mechanism proposed for remodeling complexes in vitro and indicate that remodeling is not restricted to the promoter but occurs over a chromatin domain including CUP1 and its flanking sequences.

Targeted Histone Acetylation at the Yeast CUP1 Promoter Requires the Transcriptional Activator, the TATA Boxes, and the Putative Histone Acetylase Encoded by SPT10

ABSTRACT The relationship between chromatin remodeling and histone acetylation at the yeast CUP1 gene was addressed. CUP1 encodes a metallothionein required for cell growth at high copper concentrations. Induction of CUP1 with copper resulted in targeted acetylation of both H3 and H4 at the CUP1 promoter. Nucleosomes containing upstream activating sequences and sequences farther upstream were the targets for H3 acetylation. Targeted acetylation of H3 and H4 required the transcriptional activator (Ace1p) and the TATA boxes, suggesting that targeted acetylation occurs when TATA-binding protein binds to the TATA box or at a later stage in initiation. We have shown previously that induction results in nucleosome repositioning over the entire CUP1 gene, which requires Ace1p but not the TATA boxes. Therefore, the movement of nucleosomes occurring on CUP1 induction is independent of targeted acetylation. Targeted acetylation of both H3 and H4 also required the product of the SPT10 gene, which encodes a putative histone acetylase implicated in regulation at core promoters. Disruption of SPT10 was lethal at high copper concentrations and correlated with slower induction and reduced maximum levels of CUP1 mRNA. These observations constitute evidence for a novel mechanism of chromatin activation at CUP1, with a major role for the TATA box.

Neuroprotective role of taurine during aging

Aging of the brain is characterized by several neurochemical modifications involving structural proteins, neurotransmitters, neuropeptides and related receptors. Alterations of neurochemical indices of synaptic function are indicators of age-related impairment of central functions, such as locomotion, memory and sensory performances. Several studies demonstrate that ionotropic GABA receptors, glutamate decarboxylase (GAD), and somatostatinergic subpopulations of GABAergic neurons are markedly decreased in experimental animal brains during aging. Additionally, levels of several neuropeptides co-expressed with GAD decrease during aging. Thus, the age-related decline in cognitive functions could be attributable, at least in part, to decrements in GABA inhibitory neurotransmission. In this study, we showed that chronic supplementation of taurine to aged mice significantly ameliorated the age-dependent decline in spatial memory acquisition and retention. We also demonstrated that concomitant with the amelioration in cognitive function, taurine caused significant alterations in the GABAergic and somatostatinergic system. These changes included (1) increased levels of the neurotransmitters GABA and glutamate, (2) increased expression of both isoforms of GAD (65 and 67) and the neuropeptide somatostatin, (3) decreased hippocampal expression of the β3 subunits of the GABAA receptor, (4) increased expression in the number of somatostatin-positive neurons, (5) increased amplitude and duration of population spikes recorded from CA1 in response to Schaefer collateral stimulation and (6) enhanced paired pulse facilitation in the hippocampus. These specific alterations of the inhibitory system caused by taurine treatment oppose those naturally occurring in the aging brain, suggesting a protective role of taurine in this process. An increased understanding of age-related neurochemical changes in the GABAergic system will be important in elucidating the underpinnings of the functional changes of aging. Taurine supplementation might help forestall the age-related decline in cognitive functions through interaction with the GABAergic system.

Activator-dependent recruitment of SWI/SNF and INO80 during INO1 activation

Transcriptional activation of yeast INO1 requires SWI/SNF and INO80 for nucleosome disruption at the promoter. However, the cooperative interplay among remodelers and their recruitment dynamics in activation have thus far been vague. Here, we showed, using chromatin immunoprecipitation, that both SWI/SNF and INO80 are present at the promoter and are restricted to the promoter, indicating that they directly participate in localized INO1 chromatin remodeling. Furthermore, both SWI/SNF and INO80 are absent at the INO1 promoter in ino2Delta cells, suggesting that these are activator-dependent remodelers. We have also found that the presence of INO80 is required for SWI/SNF recruitment, indicating that INO80 arrives first at the promoter followed by SWI/SNF. In light of these findings, we proposed a model which describes the order of events in INO1 activation.

Downregulation of GABA A β Subunits is Transcriptionally Controlled by Fmr1p

Fragile X mental retardation syndrome is caused by the transcriptional silence of FMR1. Here, a quantitative PCR technique was used to examine the effect of Fmr1p on the expression of GABAA β subunits in different mouse brain regions. Our results demonstrated the reduction of GABAA β2 mRNA in all brain regions assessed, and the reduction of GABAA β3 mRNA in the cortex, suggesting that the expression of GABAA β subunits is transcriptionally regulated by Fmr1p. This finding may help to establish the link between the transcriptional profile of the GABAergic inhibitory system and the development of fragile X mental retardation syndrome.

The DNA-binding domain of the yeast Spt10p activator includes a zinc finger that is homologous to foamy virus integrase.

The yeast SPT10 gene encodes a putative histone acetyltransferase that binds specifically to pairs of upstream activating sequence (UAS) elements found only in the histone gene promoters. Here, we demonstrate that the DNA-binding domain of Spt10p is located between residues 283 and 396 and includes a His2-Cys2 zinc finger. The binding of Spt10p to the histone UAS is zinc-dependent and is disabled by a zinc finger mutation (C388S). The isolated DNA-binding domain binds to single histone UAS elements with high affinity. In contrast, full-length Spt10p binds with high affinity only to pairs of UAS elements with very strong positive cooperativity and is unable to bind to a single UAS element. This implies the presence of a “blocking” domain in full-length Spt10p, which forces it to search for a pair of UAS elements. Chromatin immunoprecipitation experiments indicate that, unlike wild-type Spt10p, the C388S protein does not bind to the promoter of the gene encoding histone H2A (HTA1) in vivo. The C388S mutant has a phenotype similar to that of the spt10Δ mutant: poor growth and global aberrations in gene expression. Thus, the C388S mutation disables the DNA-binding function of Spt10p in vitro and in vivo. The zinc finger of Spt10p is homologous to that of foamy virus integrase, perhaps suggesting that this integrase is also a sequence-specific DNA-binding protein.

INO1 induction requires chromatin remodelers Ino80p and Snf2p but not the histone acetylases

Transcriptional co-activators contribute to gene expression through different mechanisms. We used various biochemical tools available for Saccharomyces cerevisiae to examine the mechanism of INO1 expression. INO1 encodes inositol-3-phosphate synthase, which catalyzes the rate-limiting step in the synthesis of inositol, a key player in phospholipid biosynthesis. Herein, we had demonstrated that the recruitment of histone acetylases Gcn5p and Esa1p mainly relied on the presence of transcriptional activator Ino2p during INO1 activation. However, the presence of the chromatin remodelers, Ino80p and Snf2p, may contribute to the additive effect of Gcn5p recruitment. We also showed that the recruitment of chromatin remodelers, Ino80p and Snf2p, is independent of the presence of histone acetylases. Furthermore, INO1 expression can be activated exclusively by the activator and chromatin remodelers, suggesting a dispensable role of histone acetylases in INO1 induction. Therefore, our data provide a mechanism for cross talk within transcriptional co-activators during INO1 activation.

Gene-wide histone acetylation at the yeast INO1 requires the transcriptional activator Ino2p

The relationship between histone acetylation and transcriptional activation at the yeast INO1 gene was addressed. INO1 encodes a key enzyme required for the de novo synthesis of phosphatidylinositol. Induction of INO1 resulted in acetylation of both histones H3 and H4 at the INO1 promoter and sequences farther downstream in the coding region, suggesting a gene-wide acetylation in response to transcriptional activation. Such chromatin remodeling activity requires the presence of transcriptional activator Ino2p. This indicates that histone acetylation is an activator-dependent event. Furthermore, the increase of histone acetylation is due to the increase of acetylation levels per nucleosome rather than the increase of nucleosome density. Therefore, these observations constitute evidence for the molecular mechanism of the correlation between histone acetylation and INO1 activity.

The effects of serotonin1A receptor on female mice body weight and food intake are associated with the differential expression of hypothalamic neuropeptides and the GABAA receptor.

Both common eating disorders anorexia nervosa and bulimia nervosa are characteristically diseases of women. To characterize the role of the 5-HT1A receptor (5-HT1A-R) in these eating disorders in females, we investigated the effect of saline or 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) treatment on feeding behavior and body weight in adult WT female mice and in adult 5-HT1A-R knockout (KO) female mice. Our results showed that KO female mice have lower food intake and body weight than WT female mice. Administration of 8-OH-DPAT decreased food intake but not body weight in WT female mice. Furthermore, qRT-PCR was employed to analyze the expression levels of neuropeptides, γ-aminobutyric acid A receptor subunit β (GABAA β subunits) and glutamic acid decarboxylase in the hypothalamic area. The results showed the difference in food intake between WT and KO mice was accompanied by differential expression of POMC, CART and GABAA β2, and the difference in body weight between WT and KO mice was associated with significantly different expression levels of CART and GABAA β2. As such, our data provide new insight into the role of 5-HT1A-R in both feeding behavior and the associated expression of neuropeptides and the GABAA receptor.