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Human transcription factor IIIC (hTFIIIC) is a multisubunit complex that directly recognizes promoter elements and recruits TFIIIB and RNA polymerase III. Here we describe the cDNA cloning and characterization of the 90-kDa subunit (hTFIIIC90) that is present within a DNA-binding subcomplex (TFIIIC2) of TFIIIC. hTFIIIC90 has no specific homology to any of the known yeast TFIIIC subunits. Immunodepletion and immunoprecipitation studies indicate that hTFIIIC90 is a bona fide subunit of TFIIIC2 and absolutely required for RNA polymerase III transcription. hTFIIIC90 shows interactions with the hTFIIIC220, hTFIIIC110, and hTFIIIC63 subunits of TFIIIC, the hTFIIIB90 subunit of TFIIIB, and the human RPC39 (hRPC39) and hRPC62 subunits of an initiation-specific subcomplex of RNA polymerase III. These interactions may facilitate both TFIIIB and RNA polymerase III recruitment to the preinitiation complex by TFIIIC. We show that hTFIIIC90 has an intrinsic histone acetyltransferase activity with a substrate specificity for histone H3.

CpG islands are stretches of DNA sequence that are enriched in the (CpG)n repeat and are present in close association with all housekeeping genes as well as some tissue-specific genes in the mammalian genome. Methylation of CpG islands strongly influences both structural organization and function of chromatin. The presence of a CpG island in a given chromosomal domain can, by itself, give rise to relatively open and active chromatin. Recently, several histone acetyltransferases, histone deacetylases, and chromatin remodeling factors have been found to be part of the transcription machinery. It is becoming increasingly clear that CpG islands and their methylation status may influence the function or recruitment of these newly discovered chromatin remodeling factors, especially the histone deacetylases. In addition, CpG islands may also play a significant role in the reorganization of chromatin during mammalian spermiogenesis.

The functional association of NPM1 with Aurora kinases is well documented. Surprisingly, although NPM1 is a well characterized phosphoprotein, it is unknown whether it is a substrate of Aurora kinases. We have found that Aurora kinases A and B can phosphorylate NPM1 at a single serine residue, Ser125, in vitro and in vivo. Phosphorylated-S125-NPM1 (pS125-NPM1) localizes to the midbody region during late cytokinesis where it colocalizes with Aurora B. The overexpression of mutant (S125A) NPM1 resulted in the deregulation of centrosome duplication and mitotic defects possibly due to cytokinesis failure. These data suggest that Aurora kinase B-mediated phosphorylation of NPM1 plays a critical role during mitosis, which could have wider implications in oncogenesis.

It is becoming increasingly clear that the functionalities of an organism are mostly derived from regulation of its gene repertoire. Specialized cell types are created from pluripotent stem cells by regulating expression of genes. In eukaryotes, genes are primarily regulated by gene regulatory networks consisting of highly sequence-specific transcription factors and epigenetic modifications. The former mode of regulation is more readily reversible and non-heritable across cell generations, whereas the latter mode is less reversible and heritable. In this article, we explore the relationship between cell differentiation and the two modes of regulation of gene expression, focusing primarily on pluripotent and multipotent stem cells. Recent studies suggest that stem cells execute different gene expression programs, probably driven by one or more gene regulatory network(s). It is now also evident that as stem cells differentiate to more specialized progeny cells, rewriting of epigenetic marks occurs in parallel with the change in the pattern of gene expression. A conceptual framework is put forward in which it is proposed that the cell fate determining gene regulatory network in a pluripotent or multipotent cell has the capability to exist in multiple stationary states with each stationary state dictating a particular pattern of gene expression. We also propose that the broad pattern of gene expression in each stationary state, termed the lineage biased state or LIBS, resembles that of a more differentiated progeny cell. The differentiation process leading to a particular progeny cell involves rewriting of epigenetic marks that result in upregulation of genes in a LIBS and silencing of genes involved in alternative LIBS; thus selecting a particular pattern of gene expression and making a lineage commitment.

The acetylation of histone and non-histone proteins controls a great deal of cellular functions, thereby affecting the entire organism, including the brain. Acetylation modifications are mediated through histone acetyltransferases (HAT) and deacetylases (HDAC), and the balance of these enzymes regulates neuronal homeostasis, maintaining the pre-existing acetyl marks responsible for the global chromatin structure, as well as regulating specific dynamic acetyl marks that respond to changes and facilitate neurons to encode and strengthen long-term events in the brain circuitry (e.g., memory formation). Unfortunately, the dysfunction of these finely-tuned regulations might lead to pathological conditions, and the deregulation of the HAT/HDAC balance has been implicated in neurological disorders. During the last decade, research has focused on HDAC inhibitors that induce a histone hyperacetylated state to compensate acetylation deficits. The use of these inhibitors as a therapeutic option was efficient in several animal models of neurological disorders. The elaboration of new cell-permeant HAT activators opens a new era of research on acetylation regulation. Although pathological animal models have not been tested yet, HAT activator molecules have already proven to be beneficial in ameliorating brain functions associated with learning and memory, and adult neurogenesis in wild-type animals. Thus, HAT activator molecules contribute to an exciting area of research.

Persistence of hepatitis C virus (HCV) infection is observed only in a subset of infected individuals and among them only some respond to treatment. Genome-wide association studies (GWAS) carried out around the world identified single nucleotide polymorphisms (SNPs) in the IL28B locus that are strongly associated with both HCV clearance and treatment response. The functional significance of these associations however, is not clear. In this report we show that an SNP rs28416813 in the distal promoter region of IL28B that is in close proximity to a non-consensus NF-κB-binding site affects downstream reporter gene expression. The effect is likely due to differential binding of NF-κB at the non-consensus site. The non-protective allele showed a reduction in luciferase reporter gene expression compared to the protective allele in HEK293T cells under different experimental conditions including treatment with tumor necrosis factor alpha (TNF-α) and 5′ triphosphorylated dsRNA. Furthermore, the HCV RNA polymerase was able to induce transcription from the IL28B promoter in a RIG-I-dependent manner. This induction was influenced by the alleles present at rs28416813. We also demonstrate strong linkage disequilibrium between rs28416813 and another important SNP rs12979860 in two ethnic populations. These results suggest possible mechanisms by which SNPs at the IL28B locus influence spontaneous clearance and treatment response in chronic HCV infections.

Constitutive activation of proinflammatory transcription factors such as STAT3 and NF-κB plays a pivotal role in the proliferation and survival of squamous cell carcinoma of the head and neck (HNSCC). Thus, the agents that can modulate deregulated STAT3 and NF-κB activation have a great potential both for the prevention and treatment of HNSCC. In the present report, we investigated the potential effects of garcinol, an active component of Garcinia indica on various inflammatory mediators involved in HNSCC progression using cell lines and xenograft mouse model. We found that garcinol inhibited constitutively activated STAT3 in HNSCC cells in a time- and dose-dependent manner, which correlated with the suppression of the upstream kinases (c-Src, JAK1, and JAK2) in HNSCC cells. Also, we noticed that the generation of reactive oxygen species is involved in STAT3 inhibitory effect of garcinol. Furthermore, garcinol exhibited an inhibitory effect on the constitutive NF-κB activation, mediated through the suppression of TGF-β–activated kinase 1 (TAK1) and inhibitor of IκB kinase (IKK) activation in HNSCC cells. Garcinol also downregulated the expression of various gene products involved in proliferation, survival, and angiogenesis that led to the reduction of cell viability and induction of apoptosis in HNSCC cells. When administered intraperitoneally, garcinol inhibited the growth of human HNSCC xenograft tumors in male athymic nu/nu mice. Overall, our results suggest for the first time that garcinol mediates its antitumor effects in HNSCC cells and mouse model through the suppression of multiple proinflammatory cascades. Cancer Prev Res; 6(8); 843–54. ©2013 AACR.

Although the brain functions of specific acetyltransferases such as the CREB-binding protein (CBP) and p300 have been well documented using mutant transgenic mice models, studies based on their direct pharmacological activation are still missing due to the lack of cell-permeable activators. Here we present a small-molecule (TTK21) activator of the histone acetyltransferases CBP/p300, which, when conjugated to glucose-based carbon nanosphere (CSP), passed the blood-brain barrier, induced no toxicity, and reached different parts of the brain. After intraperitoneal administration in mice, CSP-TTK21 significantly acetylated histones in the hippocampus and frontal cortex. Remarkably, CSP-TTK21 treatment promoted the formation of long and highly branched doublecortin-positive neurons in the subgranular zone of the dentate gyrus and reduced BrdU incorporation, suggesting that CBP/p300 activation favors maturation and differentiation of adult neuronal progenitors. In addition, mRNA levels of the neuroD1 differentiation marker and BDNF, a neurotrophin required for the terminal differentiation of newly generated neurons, were both increased in the hippocampus concomitantly with an enrichment of acetylated-histone on their proximal promoter. Finally, we found that CBP/p300 activation during a spatial training, while not improving retention of a recent memory, resulted in a significant extension of memory duration. This report is the first evidence for CBP/p300-mediated histone acetylation in the brain by an activator molecule, which has beneficial implications for the brain functions of adult neurogenesis and long-term memory. We propose that direct stimulation of acetyltransferase function could be useful in terms of therapeutic options for brain diseases.

PCAF (KAT2B) belongs to the GNAT family of lysine acetyltransferases (KAT) and specifically acetylates the histone H3K9 residue and several nonhistone proteins. PCAF is also a transcriptional coactivator. Due to the lack of a PCAF KAT-specific small molecule inhibitor, the exclusive role of the acetyltransferase activity of PCAF is not well understood. Here, we report that a natural compound of the hydroxybenzoquinone class, embelin, specifically inhibits H3Lys9 acetylation in mice and inhibits recombinant PCAF-mediated acetylation with near complete specificity in vitro. Furthermore, using embelin, we have identified the gene networks that are regulated by PCAF during muscle differentiation, further highlighting the broader regulatory functions of PCAF in muscle differentiation in addition to the regulation via MyoD acetylation.

Among the different posttranslational modifications (PTMs) that significantly regulate the protein function, lysine acetylation has become the major focus, especially to understand the epigenetic role of the acetyltransferases, in cellular physiology. Furthermore, dysfunction of these acetyltransferases is well documented under pathophysiological conditions. Therefore, it is important to understand the dynamic structure-function relationship of acetyltransferases in a relatively less complicated and faster method, which could be efficiently exploited to design and synthesis of small molecule modulators (activators/inhibitors) of these enzymes for in vivo functional analysis and therapeutic purposes. We have developed surface-enhanced Raman scattering (SERS) method, for acetyltransferases towards this goal. By employing SERS, we have not only demonstrated the autoacetylation induced structural changes of p300 enzyme but also could use this technique to characterize and design potent, specific inhibitors as well as activators of the p300. In this chapter we shall describe the methods in detail which could be highly useful for other classes of HATs and PTM enzymes.

Background Post-translational modifications (PTMs) of histones and other proteins are perturbed in tumours. For example, reduced levels of acetylated H4K16 and trimethylated H4K20 are associated with high tumour grade and poor survival in breast cancer. Drug-like molecules that can reprogram selected histone PTMs in tumour cells are therefore of interest as potential cancer chemopreventive agents. In this study we assessed the effects of the phytocompounds garcinol and curcumin on histone and p53 modification in cancer cells, focussing on the breast tumour cell line MCF7. Methods Cell viability/proliferation assays, cell cycle analysis by flow cytometry, immunodetection of specific histone and p53 acetylation marks, western blotting, siRNA and RT-qPCR. Results Although treatment with curcumin, garcinol or the garcinol derivative LTK-14 hampered MCF7 cell proliferation, differential effects of these compounds on histone modifications were observed. Garcinol treatment resulted in a strong reduction in H3K18 acetylation, which is required for S phase progression. Similar effects of garcinol on H3K18 acetylation were observed in the osteosarcoma cells lines U2OS and SaOS2. In contrast, global levels of acetylated H4K16 and trimethylated H4K20 in MCF7 cells were elevated after garcinol treatment. This was accompanied by upregulation of DNA damage signalling markers such as γH2A.X, H3K56Ac, p53 and TIP60. In contrast, exposure of MCF7 cells to curcumin resulted in increased global levels of acetylated H3K18 and H4K16, and was less effective in inducing DNA damage markers. In addition to its effects on histone modifications, garcinol was found to block CBP/p300-mediated acetylation of the C-terminal activation domain of p53, but resulted in enhanced acetylation of p53K120, and accumulation of p53 in the cytoplasmic compartment. Finally, we show that the elevation of H4K20Me3 levels by garcinol correlated with increased expression of SUV420H2, and was prevented by siRNA targeting of SUV420H2. Conclusion In summary, although garcinol and curcumin can both inhibit histone acetyltransferase activities, our results show that these compounds have differential effects on cancer cells in culture. Garcinol treatment alters expression of chromatin modifying enzymes in MCF7 cells, resulting in reprogramming of key histone and p53 PTMs and growth arrest, underscoring its potential as a cancer chemopreventive agent.

PART I: EPIGENETICS AND EVOLUTION.- 1.1 Chromatin Organization, Epigenetics and Differentiation: An evolutionary perspective.- 1.2 Secondary structures of the core histone N-terminal tails: their role in regulating chromatin structure.- 1.3 Megabase replication domains along the human genome: Relation to chromatin structure and genome organization.- 1.4 Role of DNA methyltransferases in epigenetic regulation in bacteria.- PART II: DEVELOPMENTAL EPIGENETICS.- 2.1 Metabolic aspects of epigenome: Coupling of S-adenosylmethionine synthesis and gene regulation on chromatin by SAMIT module.- 2.2 Epigenetic regulation of male germ cell differentiation.- 2.3 Epigenetic Regulation of Skeletal Muscle Development and Differentiation.- 2.4 Small changes, big effects: chromatin goes aging.- 2.5 Homeotic gene regulation: A paradigm for epigenetic mechanisms underlying organismal development.- PART III: EPIGENETICS AND TRANSCRIPTION REGULATION.- 3.1 Basic mechanisms in RNA Polymerase I transcription of the ribosomal RNA genes.- 3.2 The RNA polymerase II transcriptional machinery and its epigenetic context.- 3.3 RNA polymerase III transcriptions - regulated by chromatin structure and regulator of nuclear chromatin organization.- 3.4. The Role of DNA Methylation and Histone Modifications in Transcriptional Regulation in humans.- 3.5 Histone variants and Transcription regulation.- 3.6 Noncoding RNAs in Chromatin Organization and Transcription Regulation: An Epigenetic View.- 3.7 Chromatin structure and organization: the relation with gene expression during development and disease.- PART IV: EPIGENETICS AND DISEASE.- 4.1 Epigenetics and cancer: Development, Differentiation and Pathobiology.- 4.2 Epigenetic Regulation of Cancer Stem Cell Gene Expression.- 4.3 Role of Epigenetic Mechanisms in the Vascular Complications of Diabetes.- 4.4 Epigenetic changes in inflammatory and autoimmune diseases.- 4.5 Epigenetic regulation of HIV-1 persistence and evolving strategies for virus eradication.- 4.6 Epigenetics in Parkinson's and Alzheimer's diseases.- 4.7 Cellular Redox, Epigenetics and Diseases.- PART V: UNDERSTANDING OF EPIGENETICS: A CHEMICAL BIOLOGY APPROACH AND EPIGENETIC THERAPY.- 5.1 Stem cell plasticity in development and cancer: Epigenetic origin of cancer stem cells.- 5.2 Histone acetylation as a therapeutic target- Implication of nanodelivery.- 5.3 DNA methylation and Cancer.- 5.4 Role of epigenetics in inflammation-associated diseases.- 5.5 Plasmodium falciparum: Epigenetic control of var gene regulation and disease.

The condensed structure of chromatin limits access of cellular machinery towards template DNA. This in turn represses essential processes like transcription, replication, repair and recombination. The repression is alleviated by a variety of energy dependent processes, collectively known as “chromatin remodeling”. In a eukaryotic cell, a fine balance between condensed and de-condensed states of chromatin helps to maintain an optimum level of gene expression. DNA binding small molecules have the potential to perturb such equilibrium. We present herein the study of an oligopeptide antibiotic distamycin, which binds to the minor groove of B-DNA. Chromatin mobility assays and circular dichroism spectroscopy have been employed to study the effect of distamycin on chromatosomes, isolated from the liver of Sprague-Dawley rats. Our results show that distamycin is capable of remodeling both chromatosomes and reconstituted nucleosomes, and the remodeling takes place in an ATP-independent manner. Binding of distamycin to the linker and nucleosomal DNA culminates in eviction of the linker histone and the formation of a population of off-centered nucleosomes. This hints at a possible corkscrew type motion of the DNA with respect to the histone octamer. Our results indicate that distamycin in spite of remodeling chromatin, inhibits transcription from both DNA and chromatin templates. Therefore, the DNA that is made accessible due to remodeling is either structurally incompetent for transcription, or bound distamycin poses a roadblock for the transcription machinery to advance.

Among the different posttranslational modifications (PTMs) that significantly regulate the protein function, lysine acetylation has become the major focus, especially to understand the epigenetic role of the acetyltransferases, in cellular physiology. Furthermore, dysfunction of these acetyltransferases is well documented under pathophysiological conditions. Therefore, it is important to understand the dynamic structure-function relationship of acetyltransferases in a relatively less complicated and faster method, which could be efficiently exploited to design and synthesis of small molecule modulators (activators/inhibitors) of these enzymes for in vivo functional analysis and therapeutic purposes. We have developed surface-enhanced Raman scattering (SERS) method, for acetyltransferases towards this goal. By employing SERS, we have not only demonstrated the autoacetylation induced structural changes of p300 enzyme but also could use this technique to characterize and design potent, specific inhibitors as well as activators of the p300. In this chapter we shall describe the methods in detail which could be highly useful for other classes of HATs and PTM enzymes.

The recent developments in the field of epigenetics have changed the way the covalent modifications were perceived from mere chemical tags to important biological recruiting platforms as well as decisive factors in the process of transcriptional regulation and gene expression. Over the years, the parallel investigations in the area of epigenetics and disease have also shown the significance of the epigenetic modifications as important regulatory nodes that exhibit dysfunction in disease states. In the present scenario where epigenetic therapy is also being considered at par with the conventional therapeutic strategies, this article reviews the role of histone acetylation as an epigenetic mark involved in different biological processes associated with normal as well as abnormal gene expression states, modulation of this acetylation by small molecules and warrants the possibility of acetylation as a therapeutic target.

Genome packaging is a universal phenomenon from prokaryotes to higher mammals. Genomic constituents and forces have however, travelled a long evolutionary route. Both DNA and protein elements constitute the genome and also aid in its dynamicity. With the evolution of organisms, these have experienced several structural and functional changes. These evolutionary changes were made to meet the challenging scenario of evolving organisms. This review discusses in detail the evolutionary perspective and functionality gain in the phenomena of genome organization and epigenetics.

The protein p300 is a multifunctional transcriptional coactivator that plays pivotal role in several cellular functions. Although structures of several domains have been solved in isolation, the structures of full-length protein (p300 FL) or its complexes with transcription activators are completely unknown. Herein, we applied atomic force microscopy to visualize p300 FL. We found that it is almost prolate ellipsoidal in shape, having several bulges. We further identified the functionally significant N-terminal and C-terminal regions, by applying domain-specific antibodies and found that they are located near one end and centre of the molecule, respectively. Importantly, we have visualized the complex between p300 FL and tumor suppressor protein p53. The relevance of these data in understanding dynamics of p300 during acetylation and transcription will be mentioned.Structured summary of protein interactionsp300 and p53 bind by atomic force microscopy (View interaction)

The recent developments in the field of epigenetics have changed the way the covalent modifications were perceived from mere chemical tags to important biological recruiting platforms as well as decisive factors in the process of transcriptional regulation and gene expression. Over the years, the parallel investigations in the area of epigenetics and disease have also shown the significance of the epigenetic modifications as important regulatory nodes that exhibit dysfunction in disease states. In the present scenario where epigenetic therapy is also being considered at par with the conventional therapeutic strategies, this article reviews the role of histone acetylation as an epigenetic mark involved in different biological processes associated with normal as well as abnormal gene expression states, modulation of this acetylation by small molecules and warrants the possibility of acetylation as a therapeutic target.

Cancer is not a single disease, rather a group of abnormality generally associated with uncontrolled cell growth. The characteristic of a cancer is determined by its tissue of origin. In humans during the development of cancer the tumor tissue acquires several physiological abilities, termed as “Hallmarks of cancer”, through which cancer cells overcome the check point of cell cycle, avoid the immune surveillance system, disobey the growth regulatory signals and induce the assembly of new blood vessels in the tumor. Later these cells become metabolically hyperactive to harness the energy required for maintaining the various cancer hallmarks. However, apart from these cellular characteristics, physiologically cancer growth and progression is significantly dependant on the “tumor microenvironment”. All cancers are genetic but a very few are hereditary. Somatic mutations are considered to be the point of initiation. Nevertheless, the fine tuning of cancer progression, more precisely the establishment of a complex network among the genes expressed in a cancer cell is mediated by the epigenetic reprogramming, which could be affected by the tumor microenvironment (Fig. 17.1). In this chapter we shall discuss about the present understanding of the possible contribution of chromatin modifications and remodelling in cancer manifestation.

Genome packaging is a universal phenomenon from prokaryotes to higher mammals. Genomic constituents and forces have however, travelled a long evolutionary route. Both DNA and protein elements constitute the genome and also aid in its dynamicity. With the evolution of organisms, these have experienced several structural and functional changes. These evolutionary changes were made to meet the challenging scenario of evolving organisms. This review discusses in detail the evolutionary perspective and functionality gain in the phenomena of genome organization and epigenetics.

DNA damage in cells is often the result of constant genotoxic insult. Nevertheless, efficient DNA repair pathways are able to maintain genomic integrity. Over the past decade it has been revealed that it is not only kinase signalling pathways which play a central role in this process, but also the different post-translational modifications at lysine residues of histone (chromatin) and non-histone proteins. These lysine modifications include acetylation, methylation, ubiquitination and SUMOylation. Genomic instability is often the major cause of different diseases, especially cancer, where lysine modifications are altered and thereby have an impact on the various DNA repair mechanisms. This chapter will discuss the recent advances in our understanding of the role of different lysine modifications in DNA repair and its physiological consequences.

The Aurora A kinase belongs to serine/threonine group of kinases, well known for its role in cell cycle, especially in the regulation of mitosis. Numerous substrates of Aurora A kinase have been identified, which are predominantly related to cell cycle progression while some of them are transcription factors. Aurora A-mediated phosphorylation can either directly or indirectly regulate the function of its substrates. There are overwhelming evidences which report overexpression and gene amplification of Aurora A in several human cancers, and suggest that Aurora A could be a bona fide oncogene involved in tumorigenesis. Hence, Aurora A plays wide-ranging roles in both mitosis and its deregulation manifests in cancer progression. These observations have favored the choice of Aurora kinases as a target for cancer therapy. Recently, numerous small molecules have been discovered against Aurora kinases and many have entered clinical trials. Most of these small-molecule modulators designed are specific against either Aurora A or Aurora B, but some are dual inhibitors targeting the ATP-binding site which is highly conserved among the three human homologues of Aurora kinase. In this review, we discuss the physiological functions of Aurora A, interactions between Aurora A kinase and its cellular substrates, tumorigenesis mediated by Aurora A kinase upon overexpression, and small-molecule modulators of Aurora kinase as targets for cancer therapy.

The human transcriptional positive coactivator 4 (PC4) activates several p53-dependent genes. It has been demonstrated that this is a consequence of direct interaction with p53. Previously, we have concluded that PC4 interacts mainly with the C-terminal negative regulatory domain of p53 through its DNA binding C-terminal half. NMR chemical shift perturbation studies with peptide fragments indicated that amino acids 380–386 of p53 are crucial for interaction with PC4. This was verified by fluorescence anisotropy and sedimentation velocity studies. A peptide consisting of p53-(380–386) sequence, when attached to a cell penetration tag and nuclear localization signal, localizes to the nucleus and inhibits luciferase gene expression from a transfected plasmid carrying a Luc gene under a p53-dependent promoter. Acetylation of lysine 382/381 enhanced the binding of this peptide to PC4 by about an order of magnitude. NMR and mutagenesis studies indicated that serine 73 of PC4 is an important residue for recognition of p53. Intermolecular nuclear Overhauser effect placed aspartate 76 in the vicinity of lysine 381, indicating that the region around residues 73–76 of PC4 is important for p53 recognition. We conclude that the 380–386 region of p53 interacts with the region around residues 73–76 of PC4, and acetylation of lysine 382/381 of p53 may play an important role in modulating p53-PC4 interaction and as a consequence PC4 mediated activation of p53 target genes.

Human immunodeficiency virus type 1 (HIV-1) following integration hijacks host cell machineries where chromatinization of the viral genome regulates its latency, transcription, and replication. The cooperation among ATP-dependent chromatin remodeling factors, posttranslational modifying enzymes, and histone chaperones is well established during transcriptional activation in eukaryotes. However, the role of histone chaperones in transcription of the HIV promoter is poorly understood. Previous studies from our group have established the role of the human histone chaperone nucleophosmin (NPM1) in the acetylation-dependent chromatin transcription. NPM1 is known to interact with HIV-Tat. Here, we report that infection by HIV-1 induces the acetylation of histone chaperone NPM1. Acetylation of NPM1 was found to be critical for nuclear localization of Tat as well as Tat-mediated transcription alluding to the critical role for the host factor towards viral pathogenesis. Furthermore, knockdown experiments mediated by small interfering RNA identified the critical role played by the chaperone NPM1 in transcriptional activation of the integrated provirus. These results shed further insights into the possible role of histone chaperone NPM1 acetylation in viral gene transcription, which could be a potential therapeutic target.

Lectin pattern of Vigna glabrescens, a naturally occurring allotetraploid, was studied as a guide to determine its probable parents. Lectin patterns of a few of the synthetic allotetraploids and their known parents were also studied for purposes of comparison with V. glabrescens. Specific activities of lectins, obtained by the dissociation of agglutinated erythrocytes, were compared. Similarity index values of the proteins present in crude seed extracts were determined by polyacrylamide gel electrophoresis. Sugar inhibition and divalent cation activation of lectins were also investigated as markers for phylogenetic relationships in Vigna. From these studies it has been concluded that V. radiata, V. umbellata and V. trilobata are the probable parents of V. glabrescens and the evolution of V. glabrescens is perhaps the result of trigenomic interactions.

Neurodegenerative diseases, such as polyglutamine-related diseases, amyotrophic lateral sclerosis, and Alzheimer's disease are accompanied by transcriptional dysfunctions, leading to neuronal death. It is becoming more evident that the chromatin acetylation status is impaired during the lifetime of neurons, by a common mechanism related to the loss of function of histone acetyltransferase (HAT) activity. Notably, the HAT termed cAMP response element binding protein (CREB)-binding protein (CBP) was shown to display neuroprotective functions. Several other HATs have now been shown to participate in basic but vital neuronal functions. In addition, there is increasing evidence of several HATs (including CBP), as essential regulators of neuronal plasticity and memory formation processes. In order to counteract neuronal loss and/or memory deficits in neurodegenerative diseases, the current therapeutic strategies involve the use of small molecules antagonizing histone deacetylase (HDAC) activity (i.e. HDAC inhibitors). Although this strategy lacks specificity, some of these molecules display promising therapeutic properties. With the rapidly evolving literature on HATs and their respective functions in neuronal survival and memory formation, it seems essential to envisage direct stimulation of the acetyltransferase function as a new therapeutic tool in neurodegenerative diseases. In this review, we will highlight the present understanding and the future prospects of such therapeutic approach.

Reversible lysine acetylation is an important modification involved in the regulation of gene expression. Acetyl-CoA and NAD + are major determinants of this modification, NAD + levels being regulated by the cellular redox status. Recent reports have shown that lysine acetylation also regulates metabolic processes, thus linking the central metabolic process to gene expression.

Chromatin modifications have gained immense significance in the past few decades as key regulators of gene expression. The enzymes responsible for these modifications along with the other non-histone proteins, remodeling factors and small RNAs modulate the chromatin dynamicity, which in turn directs the chromatin function. A concerted action of different modifying enzymes catalyzes these modifications, which are read by effector modules and converted to functional outcomes by various protein complexes. Several small molecules in the physiological system such as acetyl CoA, NAD(+), and ATP are actively involved in regulating these functional outcomes. Recent understanding in the field of epigenetics indicate the possibility of the existence of a network, 'the epigenetic language' involving cross talk among different modifications that could regulate cellular processes like transcription, replication and repair. Hence, these modifications are essential for the cellular homeostasis, and any alteration in this balance leads to a pathophysiological condition or disease manifestation. Therefore, it is becoming more evident that modulators of these modifying enzymes could be an attractive therapeutic strategy, popularly referred to as 'Epigenetic therapy.' Although this field is currently monopolized by DNA methylation and histone deacetylase inhibitors, this review highlights the modulators of the other modifications namely histone acetylation, lysine methylation and arginine methylation and argues in favor of their therapeutic potential.

The Aurora A kinase belongs to serine/threonine group of kinases, well known for its role in cell cycle, especially in the regulation of mitosis. Numerous substrates of Aurora A kinase have been identified, which are predominantly related to cell cycle progression while some of them are transcription factors. Aurora A-mediated phosphorylation can either directly or indirectly regulate the function of its substrates. There are overwhelming evidences which report overexpression and gene amplification of Aurora A in several human cancers, and suggest that Aurora A could be a bona fide oncogene involved in tumorigenesis. Hence, Aurora A plays wide-ranging roles in both mitosis and its deregulation manifests in cancer progression. These observations have favored the choice of Aurora kinases as a target for cancer therapy. Recently, numerous small molecules have been discovered against Aurora kinases and many have entered clinical trials. Most of these small-molecule modulators designed are specific against either Aurora A or Aurora B, but some are dual inhibitors targeting the ATP-binding site which is highly conserved among the three human homologues of Aurora kinase. In this review, we discuss the physiological functions of Aurora A, interactions between Aurora A kinase and its cellular substrates, tumorigenesis mediated by Aurora A kinase upon overexpression, and small-molecule modulators of Aurora kinase as targets for cancer therapy.

The chromatin is comprised of repeating subunits that make up the nucleosome which is composed of an octamer of histones: H3, H4, H2A, and H2B. The replication-dependent and -independent nucleosome assembly occurs in an ordered fashion and is aided by cellular proteins such as histone chaperones and chromatin remodelers. Previously, we found that the histone chaperone NPM1 activates transcription from the chromatin template. Here we report that NPM3, a member of the nucleophosmin/nucleoplasmin family, lacks intrinsic histone chaperone activity, inhibits histone assembly activity of NPM1 in vitro, and dramatically enhances transcription in a cellular system.

The hallmark of mammalian spermiogenesis is the dramatic chromatin remodeling process wherein the nucleosomal histones are replaced by the transition proteins TP1, TP2, and TP4. Subsequently these transition proteins are replaced by the protamines P1 and P2. Hyperacetylation of histone H4 is linked to their replacement by transition proteins. Here we report that TP2 is acetylated in vivo as detected by anti-acetylated lysine antibody and mass spectrometric analysis. Further, recombinant TP2 is acetylated in vitro by acetyltransferase KAT3B (p300) more efficiently than by KAT2B (PCAF). In vivo p300 was demonstrated to acetylate TP2. p300 acetylates TP2 in its C-terminal domain, which is highly basic in nature and possesses chromatin-condensing properties. Mass spectrometric analysis showed that p300 acetylates four lysine residues in the C-terminal domain of TP2. Acetylation of TP2 by p300 leads to significant reduction in its DNA condensation property as studied by circular dichroism and atomic force microscopy analysis. TP2 also interacts with a putative histone chaperone, NPM3, wherein expression is elevated in haploid spermatids. Interestingly, acetylation of TP2 impedes its interaction with NPM3. Thus, acetylation of TP2 adds a new dimension to its role in the dynamic reorganization of chromatin during mammalian spermiogenesis.

Nucleophosmin (NPM1) is a multifunctional protein involved in the regulation of centrosome duplication, ribosome biogenesis, genomic stability, histone chaperone function, and transcription. Overexpression of NPM1 is associated with cancers of diverse histological origins. Here, we have found that p300-mediated acetylation of NPM1 modulates its subcellular localization and augments its oncogenic potential. Acetylated NPM1 is predominantly localized in the nucleoplasm, where it associates with transcriptionally active RNA polymerase II. Deacetylation of NPM1 is brought about by human SIRT1 and reduces its transcriptional activation potential. Remarkably, increased levels of acetylated NPM1 were found in grade II and III oral squamous cell carcinoma (OSCC) patient samples. Small interfering RNA (siRNA)-mediated knockdown of NPM1 in an OSCC cell line, followed by microarray analysis and chromatin immunoprecipitation experiments, revealed that some of the genes involved in oral cancer malignancy are regulated by NPM1 and have acetylated NPM1 localized at their promoters. Either suppression of p300 by siRNA or mutation of acetylatable lysine residues of NPM1 resulted in reduced occupancy of acetylated NPM1 on the target gene promoter concomitant with its decreased transcript levels. These observations suggest that acetylated NPM1 transcriptionally regulates genes involved in cell survival and proliferation during carcinogenesis.

Lysine acetyltransferases (KATs), p300 (KAT3B), and its close homologue CREB-binding protein (KAT3A) are probably the most widely studied KATs with well documented roles in various cellular processes. Hence, the dysfunction of p300 may result in the dysregulation of gene expression leading to the manifestation of many disorders. The acetyltransferase activity of p300/CREB-binding protein is therefore considered as a target for new generation therapeutics. We describe here a natural compound, plumbagin (RTK1), isolated from Plumbago rosea root extract, that inhibits histone acetyltransferase activity potently in vivo. Interestingly, RTK1 specifically inhibits the p300-mediated acetylation of p53 but not the acetylation by another acetyltransferase, p300/CREB-binding protein -associated factor, PCAF, in vivo. RTK1 inhibits p300 histone acetyltransferase activity in a noncompetitive manner. Docking studies and site-directed mutagenesis of the p300 histone acetyltransferase domain suggest that a single hydroxyl group of RTK1 makes a hydrogen bond with the lysine 1358 residue of this domain. In agreement with this, we found that indeed the hydroxyl group-substituted plumbagin derivatives lost the acetyltransferase inhibitory activity. This study describes for the first time the chemical entity (hydroxyl group) required for the inhibition of acetyltransferase activity.

Histone chaperones are a group of histone-interacting proteins, involved in several important cellular functions. These chaperones are essential to facilitate ordered assembly of nucleosomes, both in replication dependent and independent manner. Replication independent function of histone chaperone is necessary for histone eviction during transcriptional initiation and elongation. In this chapter we have discussed a method to evaluate the role of histone chaperone NPM1 (the only known chaperone to get acetylated with functional consequence) in the transcriptional activation which is acetylation dependent.

Histone chaperones are a group of histone-interacting proteins, involved in several important cellular functions. These chaperones are essential to facilitate ordered assembly of nucleosomes, both in replication dependent and independent manner. Replication independent function of histone chaperone is necessary for histone eviction during transcriptional initiation and elongation. In this chapter we have discussed a method to evaluate the role of histone chaperone NPM1 (the only known chaperone to get acetylated with functional consequence) in the transcriptional activation which is acetylation dependent.

We report a study of dynamics with a dsDNA-specific dye called PicoGreen bound to plasmid DNA (3.4 kb), and show that at low dye/DNA phosphate ratios (1 : 100 and below), PicoGreen dynamics reflect the motional dynamics of dsDNA. We further evaluated the usefulness of this probe by measuring the time-resolved fluorescence dynamics of PicoGreen bound to dsDNA in the presence of cationic reagents that affect DNA dynamics [MgCl2 and polyethyleneimine (PEI)] and also with plasmid DNA in different topological states. Among these conditions, MgCl2, PEI and the supercoiled form of plasmid resulted in increases in the very short component (0.2-0.4 ns) of the rotational correlation time. Separately, HMGB1 protein enhanced DNA dynamics, as observed from the rotational correlation times of very short (0.2-0.4 ns) and short (2-4 ns) rotational correlation timescale components. By studying the effect of specific deletion mutants HMGB1-DeltaA (deletion of 98 N-terminal amino acids) and HMGB1-DeltaC (deletion of 30 C-terminal amino acids), we show that the acidic C-terminal tail is required for enhancement of DNA dynamics. We then discuss the possible mechanisms and implications of HMGB1-mediated flexibility of DNA in the context of formation of large nucleoprotein complexes.

SummaryDNA-binding anticancer agents cause alteration in chromatin structure and dynamics. We report the dynamic interaction of the DNA intercalator and potential anticancer plant alkaloid, sanguinarine (SGR), with chromatin. Association of SGR with different levels of chromatin structure was enthalpy driven with micromolar dissociation constant. Apart from DNA, it binds with comparable affinity with core histones and induces chromatin aggregation. The dual binding property of SGR leads to inhibition of core histone modifications. Although it potently inhibits H3K9 methylation by G9a in vitro, H3K4 and H3R17 methylation are more profoundly inhibited in cells. SGR inhibits histone acetylation both in vitro and in vivo. It does not affect the in vitro transcription from DNA template but significantly represses acetylation-dependent chromatin transcription. SGR-mediated repression of epigenetic marks and the alteration of chromatin geography (nucleography) also result in the modulation of global gene expression. These data, conclusively, show an anticancer DNA binding intercalator as a modulator of chromatin modifications and transcription in the chromatin context.

Reversible acetylation of histone and non-histone proteins plays an important role in the regulation of gene expression and cellular homeostasis. A balance between acetylation and deacetylation of these proteins are maintained by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Among different HATs, p300/CBP is the most widely studied chromatin modifying enzymes. p300 is involved in several physiological processes like cell growth, regulation of gene expression, development, and tumor suppressor, and therefore its dysfunction causes different diseases. The autoacetylation of p300 is one of the key regulators of its catalytic activity. Mechanistically, autoacetylation induced structural changes in the p300 HAT domain acts as a master switch. In this report, we have shown that the natural HAT inhibitor garcinol could potently inhibit the autoacetylation activity. Furthermore, for the first time, we demonstrate that indeed autoacetylation induces structural changes in p300 HAT domain, as probed by surface-enhanced Raman scattering. Presumably, SERS will be a very useful tool to find out the structural changes in the other self-modifying enzymes like kinases and methyltransferases.

Histone acetyltransferases form crucial links in transducing extrinsic signals to actual initiation of transcription. A multitude of stress signal integrations occur through the interaction of p300 with p53 phosphorylated at different residues of the transactivation domain. How such interactions activate different gene expression programs remains largely unknown. p300 contains at least five domains that are known to interact with p53, but their role in transcription regulation is not known. We measured the binding affinity of various phosphorylated transactivation domains towards several p53 binding domains of p300 by fluorescence anisotropy. The binding affinities of different phosphorylated transactivation domains of p53 towards different domains of p300 vary by several orders of magnitude, indicating that interactions of different post-translationally modified forms of p53 may occur through different domains of p300. Thus, different post-translationally modified p53 fragments may form transcription-initiating complexes of different configurations, leading to the activation of different promoters and pathways.

SummaryReversible acetylation of histone and nonhistone proteins plays pivotal role in cellular homeostasis. Dysfunction of histone acetyltransferases (HATs) leads to several diseases including cancer, neurodegenaration, asthma, diabetes, AIDS, and cardiac hypertrophy. We describe the synthesis and characterization of a set of p300-HAT-specific small-molecule inhibitors from a natural nonspecific HAT inhibitor, garcinol, which is highly toxic to cells. We show that the specific inhibitor selectively represses the p300-mediated acetylation of p53 in vivo. Furthermore, inhibition of p300-HAT down regulates several genes but significantly a few important genes are also upregulated. Remarkably, these inhibitors were found to be nontoxic to T cells, inhibit histone acetylation of HIV infected cells, and consequently inhibit the multiplication of HIV.

Post-translational modifications of nonhistone proteins play a significant role in regulating the chromatin structure, dynamics and thereby gene regulation. Among the different posttranslational modifications, reversible acetylation of non-histone proteins has profound functional implications on wide range of cellular processes. The acetylation status of these proteins is regulated by several cellular and non-cellular factors like viruses, physiological stresses, DNA damaging agents and ROS. Mutations found in the acetylation sites of these proteins and aberrant acetylation are related to imbalances in different cellular pathways and various diseases. Several factor acetyltransferases and deacetylases are known to regulate the acetylation of the nonhistone proteins. Modulators of these enzymes derived from natural as well as synthetic sources can thus have important therapeutic implications. Designing strategies to specifically target the acetylation of these proteins can be used as a valuable tool for new generation drugs

Histone chaperones are the histone interacting factors that stimulate histone transfer reaction without being a part of the final product. They are involved in the histone storage, histone translocation to the nucleus, and histone exchange and histone deposition onto the DNA for replication dependent chromatin assembly. Interestingly, they have also been demonstrated to possess the histone removal activity. While the involvement of the histone chaperones in chromatin transcription is undisputed, the question of their local versus global involvement is under scrutiny. This review enumerates the role played by various histone chaperones in the establishment of chromatin structure and regulation of chromatin transcription. The role of histone chaperones in disease manifestation is not very clear, preliminary results with few histone chaperones suggest that expression and function of these factors dramatically alters in carcinogenesis. This review will also focus on the possible role of histone chaperones in cancer diagnosis and progression

Altered gene expression resulting from changes in the post-translational modification patterns of the histones and DNA is collectively termed epigenetics. Such changes are inherited albeit there are no alterations in the DNA sequence. Epigenetic regulation of gene expression is implemented by a wide repertoire of histone and DNA modifying enzymes including the acetyltransferases and deacetylases, the methyltransferases and kinases among others. Therefore, a regulation of these enzyme activities affords a tighter regulation of gene expression. Conversely, aberrant enzymatic activities lead to unregulated gene expression, resulting in several diseases such as RTS (loss of CBP HAT activity) and Spinal and Bulbar muscular atrophy (HATs and HMTases), apart from several forms of cancers, particularly myeloid leukemia (RAR-PML or RAR-PLZF fusion proteins resulting in the mistargeting of HDACs). Thus these enzymes have emerged as novel targets for the design of therapeutics. In this direction, several small molecule modulators (activators and inhibitors) of HATs, HDACs and HMTases are being reported in literature. This chapter introduces the different histone modifying enzymes involved in gene regulation, their connection to disease manifestation and focuses on the role of small molecule modulators in understanding enzyme function and also the design and the evolution of chromatin therapeutics

Reversible acetylation of nucleosomal histones and nonhistone proteins play pivotal roles in the regulation of all the DNA templated phenomenon. Dysfunction of the enzymes involved in the acetylation/deacetylation leads to several diseases. Therefore, these enzymes are the targets for new generation therapeutics. Here, we report the synthesis of trifluoromethyl phenyl benzamides and their effect on histone acetyltransferase (HAT) activity of p300. One of these benzamides, CTPB (N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide), was discovered as a potent activator of the p300 HAT activity. We have found that pentadecyl hydrocarbon chain of CTPB is required to activate the HAT only under certain context. Furthermore, our results show that the relative position of -CF 3 and -Cl in CTB (N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-benzamide) is also very critical for the activation. Surface-enhanced Raman spectroscopy (SERS) of p300 and the HAT activator complexes evidently suggest that the activation of HAT activity is achieved by the alteration of p300 structure. Therefore, apart from elucidating the chemical basis for small molecule mediated activation of p300, this report also describes, for the first time, Raman spectroscopic analysis of the complexes of histone-modifying enzymes and their modulators, which may be highly useful for therapeutic applications.

PART I: Chromatin Structure, Dynamics and Disease 1 Structural organization of dynamic chromatin in humans. Kohji Hizume, Shige H. Yoshimura and Kunio Takeyasu 2 Chromatin Remodeling: Action and Reaction. Parul Choudhary and Patrick Varga-Weisz 3 Regulation of Chromatin Structure and Transcription by Poly (ADP- Ribose) Polymerase- I: Possible targets for drug based therapies. David A. Wacker, Kristine M. Frizzell, and W. Lee Kraus 4 Histone variant nucleosomes: structure, function and implication in disease. Mathieu Boulard, Philippe Bouvet, Tapas K Kundu and Stefan Dimitrov 5 Histone variants and complexes involved in their exchange. Thomas Kusch and Jerry L. Workman 6 Histone chaperones in chromatin dynamics: Implication in disease manifestation. Jayasha Shandilya, Shrikanth Gadad, V.Swaminathan and Tapas K. Kundu 7 Function of the histone chaperone Nucleolin in diseases. Sebastien Storck, Manu Shukla, Philippe Bouvet 8 Chromatin as a target for the DNA binding anti-cancer drugs. Parijat Majumder, Suman K. Pradhan, Pukhrambam Grihanjali Devi, Sudipta Pal and Dipak Dasgupta PART II: Nonhistone Proteins, Sites for Epigenetic Marks: link to Disease. 9 Reversible acetylation of Non Histone proteins: Role in cellular function and disease. Kiran Batta, Chandrima Das, Shrikanth Gadad, Jayasha Shandilya and Tapas K. Kundu 10 MARs and MARBPs: Key modulators of gene regulation and disease manifestation. Samit Chattopadhyay and Lakshminarasimhan Pavithra PART III : Epigenetic Modifications of Chromatin : Implication in Disease and Therapeutics. 11 Aberrant forms of acetyltransferases in human diseases. Olivier van Beekum and Eric Kalkhoven 12 Chromatin acetylation status in the manifestation of neurodegenerative diseases: HDAC inhibitors as therapeutic tools? Anne-Laurence Boutillier, CarolineRouaux, Panteleeva 13 Function of MYST family Histone acetyltransferases and their link to diseases. Nikita Avvakumov and Jacques Cote 14 Role of histone phosphorylation in chromatin dynamics and its implication in diseases. Masaya Oki, Hitoshi Aihara and Takashi Ito 15 Function of euchromatic histone H3K9 methyltransferases, G9a/GLP. Yoichi Shinkai 16 Histone acetylation and methylation: Combinatorial players for transcriptional regulation Woojin An 17.Chromatin modifying enzymes and HIV gene expression. Vincent Quivy, Stephane de Walque, and Carine Van Lint 18.Small Molecule Modulators in Epigenetics: Implication in gene expression and therapeutics. V.Swaminathan, B.A.Ashok Reddy, Selvi R, Sukanya.M.S. and Tapas K. Kundu Index