Histone acetyltransferases: Preparation of substrates and assay procedures

Mizzen, Craig A.; Brownell, James E.; Cook, Richard G.; Allis, C. David

doi:10.1016/s0076-6879(99)04041-0

Cited by 61 publications

(43 citation statements)

References 32 publications

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“…HAT assays were performed as previously described (49). Immunoprecipitates were incubated at 30°C for 30 min with 2 g of calf thymus total histones (type IIA; Sigma) and 0.3 Ci of […”

Section: Methodsmentioning

confidence: 99%

Three Yeast Proteins Related to the Human Candidate Tumor Suppressor p33^ING1 Are Associated with Histone Acetyltransferase Activities

Loewith

Meijer

Lees‐Miller

et al. 2000

Molecular and Cellular Biology

145

185

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Three Saccharomyces cerevisiae proteins (Yng1/YOR064c, Yng2/YHR090c, and Pho23) and two Schizosaccharomyces pombe proteins (Png1/CAA15917 and Png2/CAA21250) share significant sequence identity with the human candidate tumor suppressor p33 ING1 in their C-terminal regions. The homologous regions contain PHD finger domains which have been implicated in chromatin-mediated transcriptional regulation. We show that GFP-Yng2, like human Ing1, is localized in the nucleus. Deletion of YNG2 results in several phenotypes, including an abnormal multibudded morphology, an inability to utilize nonfermentable carbon sources, heat shock sensitivity, slow growth, temperature sensitivity, and sensitivity to caffeine. These phenotypes are suppressed by expression of either human Ing1 or S. pombe Png1, suggesting that the yeast and human proteins are functionally conserved. Yng1-and Pho23-deficient cells also share some of these phenotypes. We demonstrated by yeast two-hybrid and coimmunoprecipitation tests that Yng2 interacts with Tra1, a component of histone acetyltransferase (HAT) complexes. We further demonstrated by coimmunoprecipitation that HAYng1, HA-Yng2, HA-Pho23, and HA-Ing1 are associated with HAT activities in yeast. Genetic and biochemical evidence indicate that the Yng2-associated HAT is Esa1, suggesting that Yng2 is a component of the NuA4 HAT complex. These studies suggest that the yeast Ing1-related proteins are involved in chromatin remodeling. They further suggest that these functions may be conserved in mammals and provide a possible mechanism for the human Ing1 candidate tumor suppressor. Several observations suggest that mammalian p33ING1 is involved in the regulation of cell proliferation and apoptosis (18,21,28). NIH 3T3 cells transformed by infection with a retrovirus containing a region of the Ing1 cDNA in the antisense orientation exhibit anchorage-independent growth in soft agar, and they form tumors in nude mice. Furthermore, microinjection of constructs that express Ing1 in the sense orientation results in inhibition of DNA synthesis and cell cycle progression in human diploid fibroblasts. Ing1 levels are also increased upon the induction of apoptosis in P19 cells by serum deprivation, and overexpression of Ing1 in P19 and rodent fibroblasts enhances Myc-dependent apoptosis (28). Evidence indicates that expression of Ing1 is repressed in a majority of breast and lymphoid cancer cell lines and glioblastomas and is mutated in some neuroblastoma cell lines, breast cancers, and brain tumors (21,52,74). Together, these observations suggest that Ing1 acts as a tumor suppressor and that it is involved in regulating apoptosis. This is further supported by reports that Ing1 and the p53 tumor suppressor form a complex and functionally cooperate to control cell growth (20, 83).The carboxyl-terminal 70 amino acid residues of Ing1 contain the Cys 4 -His-Cys 3 sequence of a PHD finger domain. This evolutionarily conserved domain is predicted to chelate two Zn 2ϩ ions and is similar to, but distinct from, other zinc...

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“…HAT assays were performed as previously described (49). Immunoprecipitates were incubated at 30°C for 30 min with 2 g of calf thymus total histones (type IIA; Sigma) and 0.3 Ci of […”

Section: Methodsmentioning

confidence: 99%

Three Yeast Proteins Related to the Human Candidate Tumor Suppressor p33^ING1 Are Associated with Histone Acetyltransferase Activities

Loewith

Meijer

Lees‐Miller

et al. 2000

Molecular and Cellular Biology

145

185

View full text Add to dashboard Cite

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“…The supernatant was collected, dialyzed for 1 h against HAT buffer (HB: 50 mM Tris.HCL at pH 8.0, 10% glycerol, 10 mM butyric acid, 1 mM DTT, 1 mM PMSF), and stored in aliquots at −80°C. In vitro HAT reactions were performed for 1 h at 30°C (25 µL reactions containing 100 ng histone substrates, 2 µM acetyl CoA, and 2 or 5 µL of immunopurified HAT complexes; conditions derived from Mizzen et al 1999), resolved by 15% SDS-PAGE, and specific acetylation of Htz1-K14 determined by immunoreactivity with anti-K14 Ac .…”

Section: In Vitro Hat Assaysmentioning

confidence: 99%

The Saccharomyces cerevisiae histone H2A variant Htz1 is acetylated by NuA4

Keogh¹,

Mennella²,

Sawa³

et al. 2006

Genes Dev.

225

228

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The histone H2A variant H2A.Z (Saccharomyces cerevisiae Htz1) plays roles in transcription, DNA repair, chromosome stability, and limiting telomeric silencing. The Swr1-Complex (SWR-C) inserts Htz1 into chromatin and shares several subunits with the NuA4 histone acetyltransferase. Furthermore, mutants of these two complexes share several phenotypes, suggesting they may work together. Here we show that NuA4 acetylates Htz1 Lys 14 (K14) after the histone is assembled into chromatin by the SWR-C. K14 mutants exhibit specific defects in chromosome transmission without affecting transcription, telomeric silencing, or DNA repair. Functionspecific modifications may help explain how the same component of chromatin can function in diverse pathways. Two classes of enzymes have been implicated in regulating chromatin structure and access to the underlying DNA template. The ATP-dependent chromatin remodeling enzymes use ATP hydrolysis to induce nucleosome mobility or disrupt histone-DNA interactions. The second class of enzymes covalently modify (e.g., lysine acetylation, serine phosphorylation, lysine and arginine methylation, ubiquitylation, or ADP ribosylation) various histones, usually on their N-terminal tails (Strahl and Allis 2000; Jenuwein and Allis 2001). Acetylation is carried out by histone acetyltransferases (HATs), which in Saccharomyces cerevisiae include the Gcn5-containing ADA and SAGA complexes, Hat1, Elongator, NuA3, and NuA4 (for review, see Bottomley 2004). These typically have specificity for distinct lysine residues on certain histone N-terminal tails. The acetylation of lysine residues on the N-terminal tails of histones H3 and H4 neutralizes their positive charge, possibly decreasing their affinity for DNA and facilitating chromatin decompaction and disassembly (Eberharter and Becker 2002). Perhaps more important than simple charge neutralization is the specific pattern of acetylation at individual lysine residues, at least some of which recruit bromodomain-containing proteins (Matangkasombut and Buratowski 2003, and references therein).Further chromatin specialization can be introduced by incorporation of variant histones. The major histones are assembled during DNA replication, but can be replaced by variants at specific locations (for review, see The SWR-C shares several subunits with the NuA4 HAT complex (Kobor et al. 2004;Krogan et al. 2004;Mizuguchi et al. 2004), and expression microarray analysis shows the two complexes have common regulatory targets (Krogan et al. 2004). NuA4 is required for the majority of histone H4 acetylation on Lys 5 (K5), K8, and K12 and some on histone H2A K7 Allard et al. 1999). Htz1, SWR-C, and NuA4 have each been implicated in the maintenance of chromosome stability (Krogan et al. 2004). This function for H2A.Z is conserved in the fission yeast Schizosaccharomyces pombe (Carr et al. 1994) and metazoans (Rangasamy et al. 2004).How NuA4 and SWR-C are functionally connected remains unclear. Htz1 incorporation into chromatin is dependent on SWR-C, but independent of NuA...

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“…After 4 h, beads were collected and washed four times with 500 mM KCl immunoprecipitation wash buffer (20 mM Tris at pH 7.9, 10% glycerol, 0.05% NP-40, 1 mM MgCl 2 , 1 mM EDTA at pH 8, 1 mM DTT, 1 mM PMSF), followed by two washes with 100 mM KCl immunoprecipitation wash buffer, and then two washes with HAT assay buffer (50 mM Tris at pH 8, 10% glycerol, 10 mM butyric acid, 1 mM PMSF, 1 mM DTT). HAT assays were then performed using Calf Thymus IIA histones (Sigma) and 14 C-acetyl-CoA (Amersham) substrates as described previously (Mizzen et al 1999). SDS-PAGE and fluorography were used to detect acetylated histone products.…”

mentioning

confidence: 99%

Conserved role of SIRT1 orthologs in fasting-dependent inhibition of the lipid/cholesterol regulator SREBP

Walker¹,

Yang²,

Jiang³

et al. 2010

Genes Dev.

314

260

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The sterol regulatory element-binding protein (SREBP) transcription factor family is a critical regulator of lipid and sterol homeostasis in eukaryotes. In mammals, SREBPs are highly active in the fed state to promote the expression of lipogenic and cholesterogenic genes and facilitate fat storage. During fasting, SREBP-dependent lipid/cholesterol synthesis is rapidly diminished in the mouse liver; however, the mechanism has remained incompletely understood. Moreover, the evolutionary conservation of fasting regulation of SREBP-dependent programs of gene expression and control of lipid homeostasis has been unclear. We demonstrate here a conserved role for orthologs of the NAD + -dependent deacetylase SIRT1 in metazoans in down-regulation of SREBP orthologs during fasting, resulting in inhibition of lipid synthesis and fat storage. Our data reveal that SIRT1 can directly deacetylate SREBP, and modulation of SIRT1 activity results in changes in SREBP ubiquitination, protein stability, and target gene expression. In addition, chemical activators of SIRT1 inhibit SREBP target gene expression in vitro and in vivo, correlating with decreased hepatic lipid and cholesterol levels and attenuated liver steatosis in dietinduced and genetically obese mice. We conclude that SIRT1 orthologs play a critical role in controlling SREBPdependent gene regulation governing lipid/cholesterol homeostasis in metazoans in response to fasting cues. These findings may have important biomedical implications for the treatment of metabolic disorders associated with aberrant lipid/cholesterol homeostasis, including metabolic syndrome and atherosclerosis. Lipids and sterols play key roles in diverse biological processes in eukaryotes, such as membrane biosynthesis, intra-and extracellular signaling, and energy storage. In humans, aberrant lipid and cholesterol homeostasis has been linked to a number of diseases prevalent in the developed world, including metabolic syndrome-a constellation of conditions and diseases that includes obesity, insulin resistance, liver steatosis, and hypertension, as well as type 2 diabetes, cardiovascular disease, and cancers (Cornier et al. 2008). An improved understanding of the molecular mechanisms governing lipid/cholesterol homeostasis might lead to novel therapeutic strategies to ameliorate such diseases.Fasting (short-term food deprivation) produces a rapid metabolic shift from lipid/cholesterol synthesis and fat storage to mobilization of fat, and recent studies have suggested that fasting may improve conditions associated with metabolic syndrome (Varady and Hellerstein 2008;Fontana et al. 2010). There is thus keen interest in determining the mechanism of fasting-dependent regulation of lipid/cholesterol metabolism to facilitate the development

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Histone acetyltransferases: Preparation of substrates and assay procedures

Cited by 61 publications

References 32 publications

Three Yeast Proteins Related to the Human Candidate Tumor Suppressor p33^ING1 Are Associated with Histone Acetyltransferase Activities

Three Yeast Proteins Related to the Human Candidate Tumor Suppressor p33^ING1 Are Associated with Histone Acetyltransferase Activities

The Saccharomyces cerevisiae histone H2A variant Htz1 is acetylated by NuA4

Conserved role of SIRT1 orthologs in fasting-dependent inhibition of the lipid/cholesterol regulator SREBP

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Histone acetyltransferases: Preparation of substrates and assay procedures

Cited by 61 publications

References 32 publications

Three Yeast Proteins Related to the Human Candidate Tumor Suppressor p33ING1 Are Associated with Histone Acetyltransferase Activities

Three Yeast Proteins Related to the Human Candidate Tumor Suppressor p33ING1 Are Associated with Histone Acetyltransferase Activities

The Saccharomyces cerevisiae histone H2A variant Htz1 is acetylated by NuA4

Conserved role of SIRT1 orthologs in fasting-dependent inhibition of the lipid/cholesterol regulator SREBP

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Three Yeast Proteins Related to the Human Candidate Tumor Suppressor p33^ING1 Are Associated with Histone Acetyltransferase Activities

Three Yeast Proteins Related to the Human Candidate Tumor Suppressor p33^ING1 Are Associated with Histone Acetyltransferase Activities