Chandrima Das

CBP/p300-mediated acetylation of histone H3 on lysine 56.

Acetylation within the globular core domain of histone H3 on lysine 56 (H3K56) has recently been shown to have a critical role in packaging DNA into chromatin following DNA replication and repair in budding yeast. However, the function or occurrence of this specific histone mark has not been studied in multicellular eukaryotes, mainly because the Rtt109 enzyme that is known to mediate acetylation of H3K56 (H3K56ac) is fungal-specific. Here we demonstrate that the histone acetyl transferase CBP (also known as Nejire) in flies and CBP and p300 (Ep300) in humans acetylate H3K56, whereas Drosophila Sir2 and human SIRT1 and SIRT2 deacetylate H3K56ac. The histone chaperones ASF1A in humans and Asf1 in Drosophila are required for acetylation of H3K56 in vivo, whereas the histone chaperone CAF-1 (chromatin assembly factor 1) in humans and Caf1 in Drosophila are required for the incorporation of histones bearing this mark into chromatin. We show that, in response to DNA damage, histones bearing acetylated K56 are assembled into chromatin in Drosophila and human cells, forming foci that colocalize with sites of DNA repair. Furthermore, acetylation of H3K56 is increased in multiple types of cancer, correlating with increased levels of ASF1A in these tumours. Our identification of multiple proteins regulating the levels of H3K56 acetylation in metazoans will allow future studies of this critical and unique histone modification that couples chromatin assembly to DNA synthesis, cell proliferation and cancer.

Elevated Histone Expression Promotes Life Span Extension

Changes to the chromatin structure accompany aging, but the molecular mechanisms underlying aging and the accompanying changes to the chromatin are unclear. Here, we report a mechanism whereby altering chromatin structure regulates life span. We show that normal aging is accompanied by a profound loss of histone proteins from the genome. Indeed, yeast lacking the histone chaperone Asf1 or acetylation of histone H3 on lysine 56 are short lived, and this appears to be at least partly due to their having decreased histone levels. Conversely, increasing the histone supply by inactivation of the histone information regulator (Hir) complex or overexpression of histones dramatically extends life span via a pathway that is distinct from previously known pathways of life span extension. This study indicates that maintenance of the fundamental chromatin structure is critical for slowing down the aging process and reveals that increasing the histone supply extends life span.

The histone shuffle: histone chaperones in an energetic dance

Our genetic information is tightly packaged into a rather ingenious nucleoprotein complex called chromatin in a manner that enables it to be rapidly accessed during genomic processes. Formation of the nucleosome, which is the fundamental unit of chromatin, occurs via a stepwise process that is reversed to enable the disassembly of nucleosomes. Histone chaperone proteins have prominent roles in facilitating these processes as well as in replacing old histones with new canonical histones or histone variants during the process of histone exchange. Recent structural, biophysical and biochemical studies have begun to shed light on the molecular mechanisms whereby histone chaperones promote chromatin assembly, disassembly and histone exchange to facilitate DNA replication, repair and transcription.

Histone exchange and histone modifications during transcription and aging

The organization of the eukaryotic genome into chromatin enables DNA to fit inside the nucleus while also regulating the access of proteins to the DNA to facilitate genomic functions such as transcription, replication and repair. The basic repeating unit of chromatin is the nucleosome, which includes 147 bp of DNA wrapped 1.65 times around an octamer of core histone proteins comprising two molecules each of H2A, H2B, H3 and H4. Each nucleosome is a highly stable unit, being maintained by over 120 direct protein-DNA interactions and several hundred water mediated ones. Accordingly, there is considerable interest in understanding how processive enzymes such as RNA polymerases manage to pass along the coding regions of our genes that are tightly packaged into arrays of nucleosomes. Here we present the current mechanistic understanding of this process and the evidence for profound changes in chromatin dynamics during aging. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly.

FACT and the Proteasome Promote Promoter Chromatin Disassembly and Transcriptional Initiation

The packaging of the eukaryotic genome into chromatin represses gene expression by blocking access of the general transcription machinery to the underlying DNA sequences. Accordingly, eukaryotes have developed a variety of mechanisms to disrupt, alter, or disassemble nucleosomes from promoter regions and open reading frames to allow transcription to occur. Although we know that chromatin disassembly from the yeast PHO5 promoter is triggered by the Pho4 activator, the mechanism is far from clear. Here we show that the Pho4 activator can occupy its nucleosome-bound DNA binding site within the PHO5 promoter. In contrast to the role of Saccharomyces cerevisiae FACT (facilitates chromatin transcription) complex in assembling chromatin within open reading frames, we find that FACT is involved in the disassembly of histones H2A/H2B from the PHO5 promoter during transcriptional induction. We have also discovered that the proteasome is required for efficient chromatin disassembly and transcriptional induction from the PHO5 promoter. Mutants of the degradation function of the proteasome have a defect in recruitment of the Pho4 activator, whereas mutants of the ATPase cap of the proteasome do recruit Pho4 but are still delayed for chromatin assembly. Finally, we rule out the possibility that the proteasome or ATPase cap is driving chromatin disassembly via a potential ATP-dependent chromatin remodeling activity.

Sanguinarine Interacts with Chromatin, Modulates Epigenetic Modifications, and Transcription in the Context of Chromatin

DNA-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.

Transcriptional regulation by the acetylation of nonhistone proteins in humans -- a new target for therapeutics.

Gene expression from the dynamic chromatin template is regulated by certain key cellular players that cause post‐translational modifications of both histones and nonhistone proteins. The acetyltransferases and deacetylases are two such key groups of enzymes that play crucial roles in maintaining the reversible acetylation status of histones and nonhistone proteins. Emerging evidence suggests that acetylation of nonhistone protein is equally important in the transcription regulation as the histone acetylation. Since dysfunction of HATs and HDACs leads to several diseases, aberrant acetylation of nonhistone protein is also associated with diseases. Small molecule modulators of these enzymes, which may help in maintaining the normal cellular acetylation status of these proteins, have important therapeutic implications.IUBMB Life, 57: 137‐149, 2005

Reversible Acetylation Of Non Histone Proteins

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

Binding of the histone chaperone ASF1 to the CBP bromodomain promotes histone acetylation.

Significance The Creb-binding protein (CBP) transcriptional coactivator contains a histone acetyl transferase domain and a bromodomain. Bromodomains bind to acetylated lysines, and their function as previously understood was limited to mediating recruitment to chromatin via binding to acetylated proteins. Here we show that the acetyl lysine-binding activity of the CBP bromodomain has unexpected roles in CBP-mediated acetylation of nonchromatin bound histones, and we show that the interaction between a bromodomain and acetyl lysine is stimulated by autoacetylation. Furthermore, we find that the histone chaperone anti-silencing function 1 binds to the bromodomain of CBP to present free histones correctly for efficient acetylation. Through a combination of structural, biochemical, and cell-based analyses, these studies enhance our understanding of bromodomain function and regulation. The multifunctional Creb-binding protein (CBP) protein plays a pivotal role in many critical cellular processes. Here we demonstrate that the bromodomain of CBP binds to histone H3 acetylated on lysine 56 (K56Ac) with higher affinity than to its other monoacetylated binding partners. We show that autoacetylation of CBP is critical for the bromodomain–H3 K56Ac interaction, and we propose that this interaction occurs via autoacetylation-induced conformation changes in CBP. Unexpectedly, the bromodomain promotes acetylation of H3 K56 on free histones. The CBP bromodomain also interacts with the histone chaperone anti-silencing function 1 (ASF1) via a nearby but distinct interface. This interaction is necessary for ASF1 to promote acetylation of H3 K56 by CBP, indicating that the ASF1–bromodomain interaction physically delivers the histones to the histone acetyl transferase domain of CBP. A CBP bromodomain mutation manifested in Rubinstein–Taybi syndrome has compromised binding to both H3 K56Ac and ASF1, suggesting that these interactions are important for the normal function of CBP.

Human Positive Coactivator 4 Controls Heterochromatinization and Silencing of Neural Gene Expression by Interacting with REST/NRSF and CoREST

The highly abundant, multifunctional transcriptional positive coactivator 4 (PC4) plays important roles in transcription, replication and DNA repair. Our recent work showed that PC4 is a bona fide non-histone component of chromatin. Here, we report that knockdown of PC4 dramatically alters heterochromatin organization of the genome, accompanied by increased H3K9 (histone H3 at lysine residue 9)/14 acetylation, H3K4 trimethylation and reduction in the level of H3K9 dimethylation. These posttranslational modifications of histone H3 result in overexpression of normally silenced genes (e.g., neural genes) located in heterochromatin. The results of ChIP (chromatin immunoprecipitation) and re-ChIP assays showed that overexpression of a neuronal-specific gene is accompanied by histone hyperacetylation. We further show that PC4 interacts with heterochromatin protein 1alpha, REST/NRSF (RE1-silencing transcription factor/neuron-restrictive silencer factor) and CoREST to establish the repressed state of neural genes in nonneuronal cells. Thus, PC4 plays a crucial role in maintaining a dynamic chromatin state and heterochromatin gene silencing.