The chromatin-binding domain of Ki-67 together with p53 protects human chromosomes from mitotic damage.

Vertebrate mammals express a protein called Ki-67 which is most widely known as a clinically useful marker of highly proliferative cells. Previous studies of human cells indicated that acute depletion of Ki-67 can elicit a delay at the G1/S boundary of the cell cycle, dependent on induction of the checkpoint protein p21. Consistent with those observations, we show here that acute Ki-67 depletion causes hallmarks of DNA damage, and the damage occurs even in the absence of checkpoint signaling. This damage is not observed in cells traversing S phase but is instead robustly detected in mitotic cells. The C-terminal chromatin-binding domain of Ki-67 is necessary and sufficient to protect cells from this damage. We also observe synergistic effects when Ki-67 and p53 are simultaneously depleted, resulting in increased levels of chromosome bridges at anaphase, followed by the appearance of micronuclei. Therefore, these studies identify the C terminus of Ki-67 as an important module for genome stability.

The nuclear bodies conference: Hubs of genomic activity, June 24-28, Western Shore, Nova Scotia, Canada.

This conference brought together leaders in the investigation of various bodies that populate the nucleus, a field that complements research on chromosome biology. These nuclear bodies had been receiving increasing attention as hubs of genome activity and the new findings reported at the conference strongly confirmed and significantly expanded this principle.

Two contrasting classes of nucleolus-associated domains in mouse fibroblast heterochromatin.

In interphase eukaryotic cells, almost all heterochromatin is located adjacent to the nucleolus or to the nuclear lamina, thus defining nucleolus-associated domains (NADs) and lamina-associated domains (LADs), respectively. Here, we determined the first genome-scale map of murine NADs in mouse embryonic fibroblasts (MEFs) via deep sequencing of chromatin associated with purified nucleoli. We developed a Bioconductor package called NADfinder and demonstrated that it identifies NADs more accurately than other peak-calling tools, owing to its critical feature of chromosome-level local baseline correction. We detected two distinct classes of NADs. Type I NADs associate frequently with both the nucleolar periphery and the nuclear lamina, and generally display characteristics of constitutive heterochromatin, including late DNA replication, enrichment of H3K9me3, and little gene expression. In contrast, Type II NADs associate with nucleoli but do not overlap with LADs. Type II NADs tend to replicate earlier, display greater gene expression, and are more often enriched in H3K27me3 than Type I NADs. The nucleolar associations of both classes of NADs were confirmed via DNA-FISH, which also detected Type I but not Type II probes enriched at the nuclear lamina. Type II NADs are enriched in distinct gene classes, including factors important for differentiation and development. In keeping with this, we observed that a Type II NAD is developmentally regulated, and present in MEFs but not in undifferentiated embryonic stem (ES) cells.

Distinct features of nucleolus-associated domains in mouse embryonic stem cells.

Heterochromatin in eukaryotic interphase cells frequently localizes to the nucleolar periphery (nucleolus-associated domains (NADs)) and the nuclear lamina (lamina-associated domains (LADs)). Gene expression in somatic cell NADs is generally low, but NADs have not been characterized in mammalian stem cells. Here, we generated the first genome-wide map of NADs in mouse embryonic stem cells (mESCs) via deep sequencing of chromatin associated with biochemically purified nucleoli. As we had observed in mouse embryonic fibroblasts (MEFs), the large type I subset of NADs overlaps with constitutive LADs and is enriched for features of constitutive heterochromatin, including late replication timing and low gene density and expression levels. Conversely, the type II NAD subset overlaps with loci that are not lamina-associated, but in mESCs, type II NADs are much less abundant than in MEFs. mESC NADs are also much less enriched in H3K27me3 modified regions than are NADs in MEFs. Additionally, comparision of MEF and mESC NADs revealed enrichment of developmentally regulated genes in cell-type-specific NADs. Together, these data indicate that NADs are a developmentally dynamic component of heterochromatin. These studies implicate association with the nucleolar periphery as a mechanism for developmentally regulated gene expression and will facilitate future studies of NADs during mESC differentiation.

Ki-67: more than a proliferation marker.

Ki-67 protein has been widely used as a proliferation marker for human tumor cells for decades. In recent studies, multiple molecular functions of this large protein have become better understood. Ki-67 has roles in both interphase and mitotic cells, and its cellular distribution dramatically changes during cell cycle progression. These localizations correlate with distinct functions. For example, during interphase, Ki-67 is required for normal cellular distribution of heterochromatin antigens and for the nucleolar association of heterochromatin. During mitosis, Ki-67 is essential for formation of the perichromosomal layer (PCL), a ribonucleoprotein sheath coating the condensed chromosomes. In this structure, Ki-67 acts to prevent aggregation of mitotic chromosomes. Here, we present an overview of functional roles of Ki-67 across the cell cycle and also describe recent experiments that clarify its role in regulating cell cycle progression in human cells.

Correction to: Novel genetic tools for probing individual H3 molecules in each nucleosome.

In the original publication, Fig. 1 was incorrectly published. The amino acid sequence was shifted to the left relative to the rest of the diagram in the published version and the corrected figure is given here.

Novel genetic tools for probing individual H3 molecules in each nucleosome.

In eukaryotes, genomic DNA is packaged into the nucleus together with histone proteins, forming chromatin. The fundamental repeating unit of chromatin is the nucleosome, a naturally symmetric structure that wraps DNA and is the substrate for numerous regulatory post-translational modifications. However, the biological significance of nucleosomal symmetry until recently had been unexplored. To investigate this issue, we developed an obligate pair of histone H3 heterodimers, a novel genetic tool that allowed us to modulate modification sites on individual H3 molecules within nucleosomes in vivo. We used these constructs for molecular genetic studies, for example demonstrating that H3K36 methylation on a single H3 molecule per nucleosome in vivo is sufficient to restrain cryptic transcription. We also used asymmetric nucleosomes for mass spectrometric analysis of dependency relationships among histone modifications. Furthermore, we extended this system to the centromeric H3 isoform (Cse4/CENP-A), gaining insights into centromeric nucleosomal symmetry and structure. In this review, we summarize our findings and discuss the utility of this novel approach.

New partners for HP1 in transcriptional gene silencing.

A new study in this issue of Molecular Cell (Yamane et al., 2010) demonstrates how chromatin assembly proteins HIRA/Asf1 help enforce transcriptional gene silencing in heterochromatin by bridging interactions between HP1 and histone deacetylase complexes.

Grabbing the genome by the NADs.

The regions of the genome that interact frequently with the nucleolus have been termed nucleolar-associated domains (NADs). Deep sequencing and DNA-fluorescence in situ hybridization (FISH) experiments have revealed that these domains are enriched for repetitive elements, regions of the inactive X chromosome (Xi), and several RNA polymerase III-transcribed genes. NADs are often marked by chromatin modifications characteristic of heterochromatin, including H3K27me3, H3K9me3, and H4K20me3, and artificial targeting of genes to this area is correlated with reduced expression. It has therefore been hypothesized that NAD localization to the nucleolar periphery contributes to the establishment and/or maintenance of heterochromatic silencing. Recently published studies from several multicellular eukaryotes have begun to reveal the trans-acting factors involved in NAD localization, including the insulator protein CCCTC-binding factor (CTCF), chromatin assembly factor (CAF)-1 subunit p150, several nucleolar proteins, and two long non-coding RNAs (lncRNAs). The mechanisms by which these factors coordinate with one another in regulating NAD localization and/or silencing are still unknown. This review will summarize recently published studies, discuss where additional research is required, and speculate about the mechanistic and functional implications of genome organization around the nucleolus.

Chemical screening identifies filastatin, a small molecule inhibitor of Candida albicans adhesion, morphogenesis, and pathogenesis.

Infection by pathogenic fungi, such as Candida albicans, begins with adhesion to host cells or implanted medical devices followed by biofilm formation. By high-throughput phenotypic screening of small molecules, we identified compounds that inhibit adhesion of C. albicans to polystyrene. Our lead candidate compound also inhibits binding of C. albicans to cultured human epithelial cells, the yeast-to-hyphal morphological transition, induction of the hyphal-specific HWP1 promoter, biofilm formation on silicone elastomers, and pathogenesis in a nematode infection model as well as alters fungal morphology in a mouse mucosal infection assay. We term this compound filastatin based on its strong inhibition of filamentation, and we use chemical genetic experiments to show that it acts downstream of multiple signaling pathways. These studies show that high-throughput functional assays targeting fungal adhesion can provide chemical probes for study of multiple aspects of fungal pathogenesis.

Chromatin-mediated Candida albicans virulence.

Candida albicans is the most prevalent human fungal pathogen. To successfully propagate an infection, this organism relies on the ability to change morphology, express virulence-associated genes and resist DNA damage caused by the host immune system. Many of these events involve chromatin alterations that are crucial for virulence. This review will focus on the studies that have been conducted on how chromatin function affects pathogenicity of C. albicans and other fungi. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly.

Chromatin-mediated Candida albicans virulence.

Candida albicans is the most prevalent human fungal pathogen. To successfully propagate an infection, this organism relies on the ability to change morphology, express virulence-associated genes and resist DNA damage caused by the host immune system. Many of these events involve chromatin alterations that are crucial for virulence. This review will focus on the studies that have been conducted on how chromatin function affects pathogenicity of C. albicans and other fungi. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly.

A small molecule inhibitor of fungal histone acetyltransferase Rtt109.

The histone acetyltransferase Rtt109 is the sole enzyme responsible for acetylation of histone H3 lysine 56 (H3K56) in fungal organisms. Loss of Rtt109 renders fungal cells extremely sensitive to genotoxic agents, and prevents pathogenesis in several clinically important species. Here, via a high throughput chemical screen of >300,000 compounds, we discovered a chemical inhibitor of Rtt109 that does not inhibit other acetyltransferase enzymes. This compound inhibits Rtt109 regardless of which histone chaperone cofactor protein (Asf1 or Vps75) is present, and appears to inhibit Rtt109 via a tight-binding, uncompetitive mechanism.

Molecular functions of the histone acetyltransferase chaperone complex Rtt109-Vps75.

Histone acetylation and nucleosome remodeling regulate DNA damage repair, replication and transcription. Rtt109, a recently discovered histone acetyltransferase (HAT) from Saccharomyces cerevisiae, functions with the histone chaperone Asf1 to acetylate lysine K56 on histone H3 (H3K56), a modification associated with newly synthesized histones. In vitro analysis of Rtt109 revealed that Vps75, a Nap1 family histone chaperone, could also stimulate Rtt109-dependent acetylation of H3K56. However, the molecular function of the Rtt109-Vps75 complex remains elusive. Here we have probed the molecular functions of Vps75 and the Rtt109-Vps75 complex through biochemical, structural and genetic means. We find that Vps75 stimulates the kcat of histone acetylation by approximately 100-fold relative to Rtt109 alone and enhances acetylation of K9 in the H3 histone tail. Consistent with the in vitro evidence, cells lacking Vps75 showed a substantial reduction (60%) in H3K9 acetylation during S phase. X-ray structural, biochemical and genetic analyses of Vps75 indicate a unique, structurally dynamic Nap1-like fold that suggests a potential mechanism of Vps75-dependent activation of Rttl09. Together, these data provide evidence for a multifunctional HAT-chaperone complex that acetylates histone H3 and deposits H3-H4 onto DNA, linking histone modification and nucleosome assembly.

Histone acetyltransferase Rtt109 is required for Candida albicans pathogenesis.

Candida albicans is a ubiquitous opportunistic pathogen that is the most prevalent cause of hospital-acquired fungal infections. In mammalian hosts, C. albicans is engulfed by phagocytes that attack the pathogen with DNA-damaging reactive oxygen species (ROS). Acetylation of histone H3 lysine 56 (H3K56) by the fungal-specific histone acetyltransferase Rtt109 is important for yeast model organisms to survive DNA damage and maintain genome integrity. To assess the importance of Rtt109 for C. albicans pathogenicity, we deleted the predicted homolog of Rtt109 in the clinical C. albicans isolate, SC5314. C. albicans rtt109(-/-) mutant cells lack acetylated H3K56 (H3K56ac) and are hypersensitive to genotoxic agents. Additionally, rtt109(-/-) mutant cells constitutively display increased H2A S129 phosphorylation and elevated DNA repair gene expression, consistent with endogenous DNA damage. Importantly, C. albicans rtt109(-/-) cells are significantly less pathogenic in mice and more susceptible to killing by macrophages in vitro than are wild-type cells. Via pharmacological inhibition of the host NADPH oxidase enzyme, we show that the increased sensitivity of rtt109(-/-) cells to macrophages depends on the host's ability to generate ROS, providing a mechanistic link between the drug sensitivity, gene expression, and pathogenesis phenotypes. We conclude that Rtt109 is particularly important for fungal pathogenicity, suggesting a unique target for therapeutic antifungal compounds.

Catalytic activation of histone acetyltransferase Rtt109 by a histone chaperone.

Most histone acetyltransferases (HATs) function as multisubunit complexes in which accessory proteins regulate substrate specificity and catalytic efficiency. Rtt109 is a particularly interesting example of a HAT whose specificity and catalytic activity require association with either of two histone chaperones, Vps75 or Asf1. Here, we utilize biochemical, structural, and genetic analyses to provide the detailed molecular mechanism for activation of a HAT (Rtt109) by its activating subunit Vps75. The rate-determining step of the activated complex is the transfer of the acetyl group from acetyl CoA to the acceptor lysine residue. Vps75 stimulates catalysis (> 250-fold), not by contributing a catalytic base, but by stabilizing the catalytically active conformation of Rtt109. To provide structural insight into the functional complex, we produced a molecular model of Rtt109-Vps75 based on X-ray diffraction of crystals of the complex. This model reveals distinct negative electrostatic surfaces on an Rtt109 molecule that interface with complementary electropositive ends of a symmetrical Vps75 dimer. Rtt109 variants with interface point substitutions lack the ability to be fully activated by Vps75, and one such variant displayed impaired Vps75-dependent histone acetylation functions in yeast, yet these variants showed no adverse effect on Asf1-dependent Rtt109 activities in vitro and in vivo. Finally, we provide evidence for a molecular model in which a 12 complex of Rtt109-Vps75 acetylates a heterodimer of H3-H4. The activation mechanism of Rtt109-Vps75 provides a valuable framework for understanding the molecular regulation of HATs within multisubunit complexes.

Nucleolus and centromere TSA-Seq reveals variable localization of heterochromatin in different cell types.

Genome differential positioning within interphase nuclei remains poorly explored. We extended and validated TSA-seq to map genomic regions near nucleoli and pericentric heterochromatin in four human cell lines. Our study confirmed that smaller chromosomes localize closer to nucleoli but further deconvolved this by revealing a preference for chromosome arms below 36-46 Mbp in length. We identified two lamina associated domain subsets through their differential nuclear lamina versus nucleolar positioning in different cell lines which showed distinctive patterns of DNA replication timing and gene expression across all cell lines. Unexpectedly, active, nuclear speckle-associated genomic regions were found near typically repressive nuclear compartments, which is attributable to the close proximity of nuclear speckles and nucleoli in some cell types, and association of centromeres with nuclear speckles in hESCs. Our study points to a more complex and variable nuclear genome organization than suggested by current models, as revealed by our TSA-seq methodology.

Proliferating cell nuclear antigen (PCNA) is required for cell cycle-regulated silent chromatin on replicated and nonreplicated genes.

In Saccharomyces cerevisiae, silent chromatin is formed at HMR upon the passage through S phase, yet neither the initiation of DNA replication at silencers nor the passage of a replication fork through HMR is required for silencing. Paradoxically, mutations in the DNA replication processivity factor, POL30, disrupt silencing despite this lack of requirement for DNA replication in the establishment of silencing. We tested whether pol30 mutants could establish silencing at either replicated or non-replicated HMR loci during S phase and found that pol30 mutants were defective in establishing silencing at HMR regardless of its replication status. Although previous studies tie the silencing defect of pol30 mutants to the chromatin assembly factors Asf1p and CAF-1, we found pol30 mutants did not exhibit a gross defect in packaging HMR into chromatin. Rather, the pol30 mutants exhibited defects in histone modifications linked to ASF1 and CAF-1-dependent pathways, including SAS-I- and Rtt109p-dependent acetylation events at H4-K16 and H3-K9 (plus H3-K56; Miller, A., Yang, B., Foster, T., and Kirchmaier, A. L. (2008) Genetics 179, 793-809). Additional experiments using FLIM-FRET revealed that Pol30p interacted with SAS-I and Rtt109p in the nuclei of living cells. However, these interactions were disrupted in pol30 mutants with defects linked to ASF1- and CAF-1-dependent pathways. Together, these results imply that Pol30p affects epigenetic processes by influencing the composition of chromosomal histone modifications.

Cell cycle- and chaperone-mediated regulation of H3K56ac incorporation in yeast.

Acetylation of histone H3 lysine 56 is a covalent modification best known as a mark of newly replicated chromatin, but it has also been linked to replication-independent histone replacement. Here, we measured H3K56ac levels at single-nucleosome resolution in asynchronously growing yeast cultures, as well as in yeast proceeding synchronously through the cell cycle. We developed a quantitative model of H3K56ac kinetics, which shows that H3K56ac is largely explained by the genomic replication timing and the turnover rate of each nucleosome, suggesting that cell cycle profiles of H3K56ac should reveal most first-time nucleosome incorporation events. However, since the deacetylases Hst3/4 prevent use of H3K56ac as a marker for histone deposition during M phase, we also directly measured M phase histone replacement rates. We report a global decrease in turnover rates during M phase and a further specific decrease in turnover at several early origins of replication, which switch from rapidly replaced in G1 phase to stably bound during M phase. Finally, by measuring H3 replacement in yeast deleted for the H3K56 acetyltransferase Rtt109 and its two co-chaperones Asf1 and Vps75, we find evidence that Rtt109 and Asf1 preferentially enhance histone replacement at rapidly replaced nucleosomes, whereas Vps75 appears to inhibit histone turnover at those loci. These results provide a broad perspective on histone replacement/incorporation throughout the cell cycle and suggest that H3K56 acetylation provides a positive-feedback loop by which replacement of a nucleosome enhances subsequent replacement at the same location.

Close to the edge: Heterochromatin at the nucleolar and nuclear peripheries.

Chromatin is a dynamic structure composed of DNA, RNA, and proteins, regulating storage and expression of the genetic material in the nucleus. Heterochromatin plays a crucial role in driving the three-dimensional arrangement of the interphase genome, and in preserving genome stability by maintaining a subset of the genome in a silent state. Spatial genome organization contributes to normal patterns of gene function and expression, and is therefore of broad interest. Mammalian heterochromatin, the focus of this review, mainly localizes at the nuclear periphery, forming Lamina-associated domains (LADs), and at the nucleolar periphery, forming Nucleolus-associated domains (NADs). Together, these regions comprise approximately one-half of mammalian genomes, and most but not all loci within these domains are stochastically placed at either of these two locations after exit from mitosis at each cell cycle. Excitement about the role of these heterochromatic domains in early development has recently been heightened by the discovery that LADs appear at some loci in the preimplantation mouse embryo prior to other chromosomal features like compartmental identity and topologically-associated domains (TADs). While LADs have been extensively studied and mapped during cellular differentiation and early embryonic development, NADs have been less thoroughly studied. Here, we summarize pioneering studies of NADs and LADs, more recent advances in our understanding of cis/trans-acting factors that mediate these localizations, and discuss the functional significance of these associations.