Addition Of Acetyl Groups Research Articles

Histone acetyltransferases and histone deacetyl transferases play crucial role during oogenesis and early embryo development.

Dynamic epigenetic regulation is critical for proper oogenesis and early embryo development. During oogenesis, fully grown germinal vesicle oocytes develop to mature Metaphase II oocytes which are ready for fertilization. Fertilized oocyte proliferates mitotically until blastocyst formation and the process is called early embryo development. Throughout oogenesis and early embryo development, spatio-temporal gene expression takes place, and this dynamic gene expression is controlled with the aid of epigenetics. Epigenetic means that gene expression can be altered without changing DNA itself. Epigenome is regulated through DNA methylation and histone modifications. While DNA methylation generally ends up with repression of gene expression, histone modifications can result in expression or repression depending on type of modification, type of histone protein and its specific residue. One of the modifications is histone acetylation which generally ends up with gene expression. Histone acetylation occurs through the addition of acetyl group onto amino terminal of the core histone proteins by histone acetyltransferases (HATs). Contrarily, histone deacetylation is associated with repression of gene expression, and it is catalyzed by histone deacetylases (HDACs). This review article focuses on what is known about alterations in the expression of HATs and HDACs and emphasizes importance of HATs and HDACs during oogenesis and early embryo development.

Chaetocin—A histone methyltransferase inhibitor—Impairs proliferation, arrests cell cycle and induces nucleolar disassembly in Trypanosoma cruzi

The Trypanosomatidae family includes pathogenic species of medical and veterinary interest. Chagas disease is endemic in Latin America, and about 8 million people are infected worldwide. There is a need for more effective drugs for the acute, undetermined and chronic phases of the disease that, in addition, do not cause side effects, stimulating the search for identification of new drug targets, as well as new chemotherapeutic targets. Trypanosomatids contain characteristic structures, such as the nucleus that undergoes a closed mitosis without chromosome formation and variations of chromatin packing in the different protozoa developmental stages. The nuclear DNA is condensed by histones that suffer post-translational modifications, such as addition of methyl groups by histone methyltransferases (MHT) and addition of acetyl groups by acetyltransferases. These processes modulate gene expression and chromatin organization, which are crucial to transcription, replication, repair and recombination. In the present study, the effects of chaetocin, a HMT inhibitor, on T. cruzi epimastigote proliferation, viability, ultrastructure and cell cycle were investigated. Results indicate that chaetocin promoted irreversible inhibition of protozoa growth, evident unpacking of nuclear heterochromatin and intense nucleolus fragmentation, which is associated with parasite cell cycle arrest and RNA transcription blockage. Taken together, data obtained with chaetocin treatment stimulate the use of histone methyltransferase inhibitors against pathogenic trypanosomatids.

Using Histone Deacetylase Inhibitors to Analyze the Relevance of HDACs for Translation.

Gene expression is regulated in part through the reversible acetylation of histones, by the action of histone acetyltransferases (HAT) and histone deacetylases (HDAC). HAT activity results in the addition of acetyl groups on the lysine residues of histone tails leading to decondensation of the chromatin, and increased gene transcription in general, whereas HDACs remove these acetyl groups, thus leading to an overall suppression of gene transcription. Recent evidence has elucidated that histones are not the only components of the proteome that are targeted by HATs and HDACs. A large number of nonhistone proteins undergo posttranslational acetylation. They include proteins involved in mRNA stability, protein localization and degradation, as well as protein-protein and protein-DNA interactions. In recent years, numerous studies have discovered increased HDAC expression and/or activity in numerous disease states, including cancer, where the upregulation of HDAC family members leads to dysregulation of genes and proteins involved in cell proliferation, cell cycle regulation, and apoptosis. These observations have pushed HDAC inhibitors (HDACi) to the forefront of therapeutic development of oncological conditions. HDACi, such as Vorinostat (Suberoylanilide hydroxamic acid (SAHA)), affect cancer cells in part by suppressing the translation of key proteins linked to tumorigenesis, such as cyclin D1 and hypoxia inducible factor 1 alpha (HIF-1α). Herein we describe methodologies to analyze the impact of the HDACi Vorinostat on HIF-1α translational regulation and downstream effectors.

Opposing Actions of Extracellular Signal-regulated Kinase (ERK) and Signal Transducer and Activator of Transcription 3 (STAT3) in Regulating Microtubule Stabilization during Cardiac Hypertrophy

Excessive proliferation and stabilization of the microtubule (MT) array in cardiac myocytes can accompany pathological cardiac hypertrophy, but the molecular control of these changes remains poorly characterized. In this study, we examined MT stabilization in two independent murine models of heart failure and revealed increases in the levels of post-translationally modified stable MTs, which were closely associated with STAT3 activation. To explore the molecular signaling events contributing to control of the cardiac MT network, we stimulated cardiac myocytes with an α-adrenergic agonist phenylephrine (PE), and observed increased tubulin content without changes in detyrosinated (glu-tubulin) stable MTs. In contrast, the hypertrophic interleukin-6 (IL6) family cytokines increased both the glu-tubulin content and glu-MT density. When we examined a role for ERK in regulating cardiac MTs, we showed that the MEK/ERK-inhibitor U0126 increased glu-MT density in either control cardiac myocytes or following exposure to hypertrophic agents. Conversely, expression of an activated MEK1 mutant reduced glu-tubulin levels. Thus, ERK signaling antagonizes stabilization of the cardiac MT array. In contrast, inhibiting either JAK2 with AG490, or STAT3 signaling with Stattic or siRNA knockdown, blocked cytokine-stimulated increases in glu-MT density. Furthermore, the expression of a constitutively active STAT3 mutant triggered increased glu-MT density in the absence of hypertrophic stimulation. Thus, STAT3 activation contributes substantially to cytokine-stimulated glu-MT changes. Taken together, our results highlight the opposing actions of STAT3 and ERK pathways in the regulation of MT changes associated with cardiac myocyte hypertrophy.

Small Molecule Modulators of Histone Acetyltransferase p300

Histone acetyltransferases (HATs) are a group of enzymes that play a significant role in the regulation of gene expression. These enzymes covalently modify the N-terminal lysine residues of histones by the addition of acetyl groups from acetyl-CoA. Dysfunction of these enzymes is often associated with the manifestation of several diseases, predominantly cancer. Here we report that anacardic acid from cashew nut shell liquid is a potent inhibitor of p300 and p300/CBP-associated factor histone acetyltranferase activities. Although it does not affect DNA transcription, HAT-dependent transcription from a chromatin template was strongly inhibited by anacardic acid. Furthermore, we describe the design and synthesis of an amide derivative N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide (CTPB) using anacardic acid as a synthon, which remarkably activates p300 HAT activity but not that of p300/CBP-associated factor. Although CTPB does not affect DNA transcription, it enhances the p300 HAT-dependent transcriptional activation from in vitro assembled chromatin template. However, it has no effect on histone deacetylase activity. These compounds would be useful as biological switching molecules for probing into the role of p300 in transcriptional studies and may also be useful as new chemical entities for the development of anticancer drugs.

Oh, Behave! SASsy protein team's paradoxical effects on gene silencing still confound researchers (Chromatin structure/Gene regulation)

A fickle protein continues to taunt researchers with its paradoxical behavior, but some newly discovered molecular associations are beginning to shed light on its activities. Subtle changes in the way DNA and proteins pack together can turn off genes and prevent DNA from breaking and rejoining in new combinations. This so-called gene silencing process plays a role in regulating aging: High doses of the silencing protein SIR2 extend the life-spans of yeast and nematodes (see Kaeberlein Perspective). According to new work, several proteins involved in silencing act in concert and perhaps shut down newly copied DNA. The results reveal previously unknown molecular contacts, but the mechanisms by which the proteins influence silencing--and the reasons they turn genes on in some situations and off in others--are still unclear. Considerable evidence suggests that the removal of acetyl groups from histones--the protein scaffolds that wind DNA into a compact form known as chromatin--instigates silencing of genes near the chemical modifications. Removal of any one of three yeast genes known as SAS2 , SAS4 , and SAS5 can restore defective silencing at a particular chromosomal location, known as HMR. This observation suggests that the normal versions of the SAS proteins inhibit silencing. Consistent with that role, the SAS2 protein resembles acetyltransferases, enzymes that attach acetyl groups to protein side chains. But in other genomic regions, a nonfunctional version of SAS2 further reduces silencing, hinting that at those sites addition of acetyl groups might hush DNA. Acetyltransferases frequently work as part of molecular teams, so identifying SAS2's co-conspirators could illuminate how SAS2 both enhances and hinders silencing. In the new work, two groups independently sought protein partners that bind to SAS2. Using antibodies that recognize this protein, they pulled it from a mix of yeast cell contents. But it did not emerge alone: SAS4 and SAS5 accompanied SAS2, presumably because they had attached themselves to it. The result supports previous genetic experiments suggesting that the three genes act in the same pathway. Further studies by the two teams hint that the SAS conglomeration sticks to other proteins that assist new DNA in twisting properly around histones. Osada and colleagues discovered that a protein called Asf1 also joins the SAS party; Meijsing and Ehrenhofer-Murray showed that the CacI protein attaches to the SAS trio as well. Previous work indicates that Asf1 and CacI help histones load onto freshly copied DNA to restore chromatin structure. Moreover, defects in either protein impair silencing. The new results suggest that DNA-histone assembly machinery brings the SAS conglomeration to newly replicated DNA, where SAS2 perhaps modifies histones to influence silencing. This idea is consistent with previous results indicating that silencing states must be reset after DNA is duplicated and repacked around histones. Additional results suggest that SAS2 acetylates a specific amino acid constituent of a histone protein known as H4. Meijsing and Ehrenhofer-Murray found that a mutation in H4 that locks it into a deacetylated state produces changes in silencing similar to those exhibited by SAS2 mutations: The alteration restores defective silencing at HMR but reduces it in other areas of the genome. No one has demonstrated SAS2's enzymatic activity directly, however; SAS2 might modify an as-yet-unidentified protein, which in turn modulates silencing. The SAS amalgam hinders silencing at a region implicated in aging. Meijsing and Ehrenhofer-Murray showed that removing any one of the three SAS proteins results in strong silencing of ribosomal DNA, the chromosomal site most important for SIR2's influence on aging--and SIR2 is required for this effect. Whether the SAS complex also antagonizes SIR2's ability to extend life-span is not known. SAS functions have been but a gleam in a biochemist's eye: Researchers have known that the SAS genes influence silencing, but the mechanism has remained mysterious. The new identification of molecular connections made by SAS proteins will help scientists understand how acetylation contributes to silencing. Such insight could reveal the machinations behind SAS's two-faced behavior. --R. John Davenport S. Osada, A. Sutton, N. Muster, C. E. Brown, J. R. Yates III, R. Sternglanz, J. L. Workman, The yeast SAS (something about silencing) protein complex contains a MYST-type putative acetyltransferase and functions with chromatin assembly factor ASF1. Genes Dev. 15 , 3155-3168 (2001). [Abstract] [Full Text] S. H. Meijsing and A. E. Ehrenhofer-Murray, The silencing complex SAS-I links histone acetylation to the assembly of repressed chromatin by CAF-I and Asf1 in Saccharomyces cerevisiae. Genes Dev. 15 , 3169-3182 (2001). [Abstract] [Full Text]

Identification of algI and algJ in the Pseudomonas aeruginosa alginate biosynthetic gene cluster which are required for alginate O acetylation

Mucoid strains of Pseudomonas aeruginosa overproduce alginate, a linear exopolysaccharide Of D-mannuronate and variable amounts of L-guluronate. The mannuronate residues undergo modification by C-5 epimerization to form the L-guluronates and by the addition of acetyl groups at the 0-2 and 0-3 positions. Through genetic analysis, we previously identified algF, located upstream of algA in the 18-kb alginate biosynthetic operon, as a gene required for alginate acetylation. Here, we show the sequence of a 3.7-kb fragment containing the open reading frames termed algI, algJ, and algF. An algI::Tn5O1 mutant, which was defective in algIJFA because of the polar nature of the transposon insertion, produced alginate when algA was provided in trans. This indicated that the algIJF gene products were not required for polymer biosynthesis. To examine the potential role of these genes in alginate modification, mutants were constructed by gene replacement in which each gene (algI, algJ, or algF) was replaced by a polar gentamicin resistance cassette. Proton nuclear magnetic resonance spectroscopy showed that polymers produced by strains deficient in algIJF still contained a mixture of D-mannuronate and L-guluronate, indicating that C-5 epimerization was not affected. Alginate acetylation was evaluated by a colorimetric assay and Fourier transform-infrared spectroscopy, and this analysis showed that strains deficient in algIJF produced nonacetylated alginate. Plasmids that supplied the downstream gene products affected by the polar mutations were introduced into each mutant. The strain defective only in algF expression produced an alginate that was not acetylated, confirming previous results. Strains missing only algJ or algI also produced nonacetylated alginates. Providing the respective missing gene (algI, algJ, or algF) in trans restored alginate acetylation. Mutants defective in algI or algJ, obtained by chemical and transposon mutagenesis, were also defective in their ability to acetylate alginate. Therefore, algI and algJ represent newly identified genes that, in addition to algF, are required for alginate acetylation.

Patterns of histone acetylation

The N-terminal domains of all four core histones are subject to reversible acetylation at certain lysine residues. This modification has been functionally linked to transcription, histone deposition at replication and to histone removal during spermatogenesis. To increase understanding of the significance of this modification we have studied the specificity of site utilisation in the monoacetyl, diacetyl and triacetyl forms of histones H3, H4 and H2B (histone H2A has only a single modification site), using pig thymus and HeLa cells as the source of histones. The HeLa histones were extracted from cells grown both with and without butyrate treatment. It is found that for histone H3 there is a fairly strict order of site occupancy: Lys14, followed by Lys23, followed by Lys18 in both pig and HeLa histones. Since the order and specificity is the same when butyrate is added to the HeLa cell cultures, we conclude that addition of the fatty acid does not scramble the specificity of site utilisation, but merely allows more of the natural forms of modified histone to accumulate. For histone H4, the monoacetyl form is exclusively modified at Lys16, but further addition of acetyl groups is less specific and progresses through sites 12, 8 and 5 in an N-terminal direction. Similar results were obtained for H4 from both pig thymus and butyrate-treated HeLa cells. Histone H2B could be studied in detail only from butyrate-treated HeLa cells and a much lower level of site specificity was found: sites 12 and 15 were preferred to the more N- and C-terminal sites at Lys5 and Lys20. The data reinforces the view that lysine acetylation in core histones is a very specific phenomenon that plays several functionally distinct roles. The high degree of site specificity makes it unlikely that the structural effects of acetylation are mediated merely by a generalised reduction of charge in the histone N-terminal domains.