Kinetochores are large protein assemblies that connect chromosomes to microtubules of the mitotic and meiotic spindles in order to distribute the replicated genome from a mother cell to its daughters. Kinetochores also control feedback mechanisms responsible for the correction of incorrect microtubule attachments, and for the coordination of chromosome attachment with cell cycle progression. Finally, kinetochores contribute to their own preservation, across generations, at the specific chromosomal loci devoted to host them, the centromeres. They achieve this in most species by exploiting an epigenetic, DNA-sequence-independent mechanism; notable exceptions are budding yeasts where a specific sequence is associated with centromere function. In the last 15 years, extensive progress in the elucidation of the composition of the kinetochore and the identification of various physical and functional modules within its substructure has led to a much deeper molecular understanding of kinetochore organization and the origins of its functional output. Here, we provide a broad summary of this progress, focusing primarily on kinetochores of humans and budding yeast, while highlighting work from other models, and present important unresolved questions for future studies.

A Molecular View of Kinetochore Assembly and Function.

Since their discovery in the 1980s and 1990s, the human protein lysine acetyltransferase encoded by the paralogous p300 and CBP genes has received much interest. p300/CBP functions in regulating the expression of genes controlling several basic cellular processes, such as proliferation and homeostasis, and plays a role in a variety of human diseases, particularly solid tumors.

Numerous natural product and synthetic inhibitors of the acetyltransferase activity have been identified and used to generate a crystal structure of the active site, probe the p300/CBP enzyme kinetics, and interrogate p300/CBP cellular functions. These studies revealed that acetyl-CoA binds in a tunnel enclosed by a unique loop, while the substrate protein transiently associates with an acidic patch, following a hit-and-run kinetic mechanism. p300/CBP acetylates histones as well as other proteins including other epigenetic enzymes and transcription factors. Studies with inhibitor compounds in cells and animals have confirmed that the p300/CBP acetylation activity has roles in diverse functions including cell migration and invasion, maintenance of the differentiated state, tau-mediated neurodegeneration, and learning and memory.

Also, important components of p300/CBP are the domains flanking the acetyltransferase domain, including three cysteine/histidine-rich domains and a bromodomain. Protein ligands of these have been identified. Their roles in regulating the acetyltransferase activity and substrate specificity, as well as identification of compounds that can block or mimic ligand binding, are topics of ongoing study.

Biochemical investigation of protein acetylation has posed unique challenges, due in part to its dynamic reversibility, the weak affinity of binding modules that recognize it, and the complexity of the multiprotein interaction networks. Therefore, diverse techniques in biochemistry, molecular biology, and proteomics have coevolved with our understanding of the p300/CBP enzyme. In this Review, part of the thematic issue on Epigenetics, we summarize the current understanding of p300/CBP including the novel technologies developed for these studies.

Protein lysine acetylation by p300/CBP.

Editorial Manager(tm) for Developmental Cell Manuscript Draft Manuscript Number: DC-D-06-00013R1 Title: Mislocalization of the Drosophila centromere-specific histone CID promotes formation of functional ectopic kinetochores Article Type: Research Article Section/Category: Keywords: centromere CID CENP-A kinetochore mitosis aneuploidy Corresponding Author: Dr. Gary H. Karpen, Corresponding Author's Institution: Lawrence Berkeley National Lab/UC Berkeley First Author: Patrick Heun, PhD Order of Authors: Patrick Heun, PhD; Sylvia Erhardt, PhD; Michael D Blower, PhD; Samara Weiss, BA; Andrew D Skora, BA; Gary H Karpen, PhD Manuscript Region of Origin: Abstract: The centromere-specific histone variant CENP-A (CID in Drosophila) is a structural and functional foundation for kinetochore formation and chromosome segregation. Here, we show that overexpressed CID is mislocalized into normally non-centromeric regions in Drosophila tissue culture cells and animals. Analysis of mitoses in living and fixed cells reveals that mitotic delays, anaphase bridges, chromosome fragmentation, and cell and organismal lethality are all direct consequences of CID mislocalization. In addition, proteins that are normally restricted to endogenous kinetochores assemble at a subset of ectopic

Mislocalization of the Drosophila centromere-specific histone CID promotes formation of functional ectopic kinetochores

Bromodomain protein 4 (BRD4) is a chromatin-binding protein implicated in cancer and autoimmune diseases that functions as a scaffold for transcription factors at promoters and super-enhancers. Although chromatin decompaction and transcriptional activation of target genes are associated with BRD4 binding, the mechanisms involved are unknown. We report that BRD4 is a histone acetyltransferase (HAT) that acetylates histones H3 and H4 with a pattern distinct from those of other HATs. Both mouse and human BRD4 have intrinsic HAT activity. Importantly, BRD4 acetylates H3 K122, a residue critical for nucleosome stability, thus resulting in nucleosome eviction and chromatin decompaction. Nucleosome clearance by BRD4 occurs genome wide, including at its targets MYC, FOS and AURKB (Aurora B kinase), resulting in increased transcription. These findings suggest a model wherein BRD4 actively links chromatin structure and transcription: it mediates chromatin decompaction by acetylating and evicting nucleosomes at target genes, thereby activating transcription.

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BRD4 is a histone acetyltransferase that evicts nucleosomes from chromatin

Down-regulation and mutations of the nuclear-architecture proteins lamin A and C cause misshapen nuclei and altered chromatin organization associated with cancer and laminopathies, including the premature-aging disease Hutchinson-Gilford progeria syndrome (HGPS). Here, we identified the small molecule "Remodelin" that improved nuclear architecture, chromatin organization, and fitness of both human lamin A/C-depleted cells and HGPS-derived patient cells and decreased markers of DNA damage in these cells. Using a combination of chemical, cellular, and genetic approaches, we identified the acetyl-transferase protein NAT10 as the target of Remodelin that mediated nuclear shape rescue in laminopathic cells via microtubule reorganization. These findings provide insights into how NAT10 affects nuclear architecture and suggest alternative strategies for treating laminopathies and aging.

/pdf/chemical-inhibition-of-nat10-corrects-defects-of-44ivi4umm4.pdf

Chemical Inhibition of NAT10 Corrects Defects of Laminopathic Cells

Acetylation is a key modulator of genome accessibility through decondensation of the chromatin structure. The balance between acetylation and opposite deacetylation is, in fact, a prerequisite for several cell functions and differentiation. To find modulators of the histone acetyltransferase Gcn5p, we performed a phenotypic screening on a set of newly synthesized molecules derived from thiazole in budding yeast Saccharomyces cerevisiae. We selected compounds that induce growth inhibition in yeast strains deleted in genes encoding known histone acetyltransferases. A novel molecule CPTH2, cyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl)hydrazone, was selected based on its inhibitory effect on the growth of a gcn5Delta strain. We demonstrated a specific chemical-genetic interaction between CPTH2 and HAT Gcn5p, indicating that CPTH2 inhibits the Gcn5p dependent functional network. CPTH2 inhibited an in vitro HAT reaction, which is reverted by increasing concentration of histone H3. In vivo, it decreased acetylation of bulk histone H3 at the specific H3-AcK14 site. On the whole, our results demonstrate that CPTH2 is a novel HAT inhibitor modulating Gcn5p network in vitro and in vivo.

A novel histone acetyltransferase inhibitor modulating Gcn5 network: cyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl)hydrazone.

Synthesis of a novel series of thiazole-based histone acetyltransferase inhibitors.

Epigenetic changes and chromatin signaling regulate gene expression by modifying chromatin structure. Multisubunit chromatin complexes reversibly remodel nucleosome organization and act in the catalytic modification of histone N-terminal tails, producing a combinatorial code of posttranslational modifications (25, 48, 50). Gcn5p (2, 14, 30), the ancestor of the histone acetyltransferase (HAT) family, marks histone H3 and H4 tails with an ɛ-acetyl group on specific lysines (41, 57). Gcn5 does not act on its own but rather as the catalytic subunit of two separate and conserved HAT complexes named ADA and SAGA (Spt-Ada-Gcn5-acetyltransferase). Specifically, Spt proteins are exclusively present in SAGA, while Spt20p is necessary for maintaining the integrity and function of the whole complex (58). Histone acetylation by Gcn5p is implicated in the displacement of nucleosomes from promoters during transcriptional activation and also in aiding the recruitment of TATA binding protein, RNA polymerase II, and coactivators (17). Acetylation therefore facilitates the formation of an accessible “open” chromatin structure corresponding to the transcribing genome (9, 31, 51). During cell division, chromatin remodeling expands to wide chromosomal regions, producing long waves of compaction and decondensation over the whole genome at each cell division (5, 55). Disturbing the HAT/histone deacetylase balance therefore alters protein activities on a cellular scale, which leads to various diseases, including cancer.

Several reports have highlighted the involvement of Gcn5p alone or in combination with other HATs such as Sas3 in the cell cycle (19, 22) and in the transcriptional regulation of a set of genes required at the end of telophase (28). In mammalian cells, loss of the homologue Gcn512 is lethal during embryogenesis, induces a high level of apoptosis (60), and affects G2/M transition in null mouse embryonic stem cells (32). In yeast, the specialized centromeric nucleosome, which contains the histone H3 variant Cse4p (37), is necessary for the assembly and interactions of inner kinetochore components at the centromere (7) and for the correct attachment of the chromosomes to the spindle in metaphase. Mutations in the centromeric/kinetochore components or epigentic modifications of this structure may lead to chromosome missegregation and G2/M delay. Since kinetochore assembly depends on the structure of the underlying centromere, an aberrant acetylation at this site may lead to a defective kinetochore, resulting in mitotic defects and cell death (56, 63). In fission yeast, the acetylation of histone H4 by Alp5 is also required for the correct attachment of the chromosome to the mitotic spindle (39, 46). Hyperacetylation following treatment with histone deacetylase inhibitors alters centromeric chromatin and induces high chromosome loss (10, 40, 59). Taken together, this evidence demonstrates that epigenetic regulation extends far behind histones. A growing number of nonhistone protein substrates (e.g., transcription factors) have been found to be modified at the posttranslational level and have a direct impact on key signal transduction pathways (27). Structural proteins such as the outer kinetochore component Dam1 were reported to be methylated by the histone methyltransferase Set1 (64).

These findings suggest that HATs, their substrates, and the cellular pathways that they fine-tune are still poorly understood, (65) despite the growing number of chromatin modifiers known to be involved in different cellular processes. Here we report that Gcn5p controls the metaphase-to-anaphase transition in budding yeast and is required for correct chromosome segregation and centromere/kinetochore function in mitosis. Genetic interactions between Gcn5p and DNA-bound kinetochore components together with the effect of Gcn5p on the variant centromeric nucleosome and its physical association at the centromere demonstrate that this HAT is directly implicated in the control of metaphase-to-anaphase transition.

Gcn5p Plays an Important Role in Centromere Kinetochore Function in Budding Yeast

Aneuploidy, the unbalanced segregation of chromosomes during cell division, is recurrent in many tumors and the cause of birth defects and genetic diseases. Centromeric chromatin represents the chromosome attachment site to the mitotic spindle, marked by specialized nucleosomes containing a specific histone variant, CEN-H3/Cse4, in yeast. Mislocalization of Cse4 outside the centromere is deleterious and may cause aberrant chromosome behavior and mitotic loss. For this reason, ubiquitylation by the E3-ubiquitin ligase Psh1 and subsequent proteolysis tightly regulates its restricted localization. Among multiproteic machineries, the SAGA complex is not merely engaged in acetylation but also directly involved in deubiquitylation. In this study, we investigated the role of SAGA-DUB’s Ubp8-driven deubiquitylation of the centromeric histone variant Cse4 in budding yeast. We found that Ubp8 works in concert with the E3-ubiquitin ligase Psh1, and that its loss causes defective deubiquitylation and the accumulation of a short ubiquitin oligomer on Cse4. We also show that lack of Ubp8 and defective deubiquitylation increase mitotic instability, cause faster Cse4 proteolysis and induce mislocalization of the centromeric histone outside the centromere. Our data provide evidence for a fundamental role of DUB-Ubp8 in deubiquitylation and the stability of the centromeric histone in budding yeast.

SAGA DUB-Ubp8 Deubiquitylates Centromeric Histone Variant Cse4.

Epigenetics, the heritable changes that do not involve DNA sequences, give dynamic propulsion to a static genome and modulate genome accessibility by acting through multiple layers of regulation, ultimately ending with a variable organization of chromatin. The main player in chromatin reprograming is the nucleosome, which modifies its association to DNA depending on a variety of post-translational modifications (PTMs) on histone tails. PTMs act directly on higher order chromatin structures and affect the degree of DNA wrap around the histone octamer and the interaction and/or sequential recruitment of chromatin-associated proteins and transcription factors at defined regions.1 Recently genomic approaches revealed a far more complex epigenome involving key proteins of cell signaling.

Stefano Vernarecci

Papers

A novel histone acetyltransferase inhibitor modulating Gcn5 network: cyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl)hydrazone.

Synthesis of a novel series of thiazole-based histone acetyltransferase inhibitors.

Gcn5p Plays an Important Role in Centromere Kinetochore Function in Budding Yeast

SAGA DUB-Ubp8 Deubiquitylates Centromeric Histone Variant Cse4.

Tuning acetylated chromatin with HAT inhibitors: a novel tool for therapy.