Yamini Dalal

Structure, dynamics, and evolution of centromeric nucleosomes

Centromeres are defining features of eukaryotic chromosomes, providing sites of attachment for segregation during mitosis and meiosis. The fundamental unit of centromere structure is the centromeric nucleosome, which differs from the conventional nucleosome by the presence of a centromere-specific histone variant (CenH3) in place of canonical H3. We have shown that the CenH3 nucleosome core found in interphase Drosophila cells is a heterotypic tetramer, a “hemisome” consisting of one molecule each of CenH3, H4, H2A, and H2B, rather than the octamer of canonical histones that is found in bulk nucleosomes. The surprising discovery of hemisomes at centromeres calls for a reevaluation of evidence that has long been interpreted in terms of a more conventional nucleosome. We describe how the hemisome structure of centromeric nucleosomes can account for enigmatic properties of centromeres, including kinetochore accessibility, epigenetic inheritance, rapid turnover of misincorporated CenH3, and transcriptional quiescence of pericentric heterochromatin. Structural differences mediated by loop 1 are proposed to account for the formation of stable tetramers containing CenH3 rather than stable octamers containing H3. Asymmetric CenH3 hemisomes might interrupt the global condensation of octameric H3 arrays and present an asymmetric surface for kinetochore formation. We suggest that this simple mechanism for differentiation between centromeric and packaging nucleosomes evolved from an archaea-like ancestor at the dawn of eukaryotic evolution.

Tetrameric organization of vertebrate centromeric nucleosomes

Mitosis ensures equal genome segregation in the eukaryotic lineage. This process is facilitated by microtubule attachment to each chromosome via its centromere. In centromeres, canonical histone H3 is replaced in nucleosomes by a centromere-specific histone H3 variant (CENH3), providing the unique epigenetic signature required for microtubule binding. Due to recent findings of alternative CENH3 nucleosomal forms in invertebrate centromeres, it has been debated whether the classical octameric nucleosomal arrangement of two copies of CENH3, H4, H2A, and H2B forms the basis of the vertebrate centromere. To address this question directly, we examined CENH3 [centromere protein A (CENP-A)] nucleosomal organization in human cells, using a combination of nucleosome component analysis, atomic force microscopy (AFM), and immunoelectron microscopy (immuno-EM). We report that native CENP-A nucleosomes contain centromeric alpha satellite DNA, have equimolar amounts of H2A, H2B, CENP-A, and H4, and bind kinetochore proteins. These nucleosomes, when measured by AFM, yield one-half the dimensions of canonical octameric nucleosomes. Using immuno-EM, we find that one copy of CENP-A, H2A, H2B, and H4 coexist in CENP-A nucleosomes, in which internal C-terminal domains are accessible. Our observations indicate that CENP-A nucleosomes are organized as asymmetric heterotypic tetramers, rather than canonical octamers. Such altered nucleosomes form a chromatin fiber with distinct folding characteristics, which we utilize to discriminate tetramers directly within bulk chromatin. We discuss implications of our observations in the context of universal epigenetic and mechanical requirements for functional centromeres.

A long non-coding RNA is required for targeting centromeric protein A to the human centromere

The centromere is a specialized chromatin region marked by the histone H3 variant CENP-A. Although active centromeric transcription has been documented for over a decade, the role of centromeric transcription or transcripts has been elusive. Here, we report that centromeric α-satellite transcription is dependent on RNA Polymerase II and occurs at late mitosis into early G1, concurrent with the timing of new CENP-A assembly. Inhibition of RNA Polymerase II-dependent transcription abrogates the recruitment of CENP-A and its chaperone HJURP to native human centromeres. Biochemical characterization of CENP-A associated RNAs reveals a 1.3 kb molecule that originates from centromeres, which physically interacts with the soluble pre-assembly HJURP/CENP-A complex in vivo, and whose down-regulation leads to the loss of CENP-A and HJURP at centromeres. This study describes a novel function for human centromeric long non-coding RNAs in the recruitment of HJURP and CENP-A, implicating RNA-based chaperone targeting in histone variant assembly.

CENP-A nucleosomes localize to transcription factor hotspots and subtelomeric sites in human cancer cells

BackgroundThe histone H3 variant CENP-A is normally tightly regulated to ensure only one centromere exists per chromosome. Native CENP-A is often found overexpressed in human cancer cells and a range of human tumors. Consequently, CENP-A misregulation is thought to contribute to genome instability in human cancers. However, the consequences of such overexpression have not been directly elucidated in human cancer cells.ResultsTo investigate native CENP-A overexpression, we sought to uncover CENP-A-associated defects in human cells. We confirm that CENP-A is innately overexpressed in several colorectal cancer cell lines. In such cells, we report that a subset of structurally distinct CENP-A-containing nucleosomes associate with canonical histone H3, and with the transcription-coupled chaperones ATRX and DAXX. Furthermore, such hybrid CENP-A nucleosomes localize to DNase I hypersensitive and transcription factor binding sites, including at promoters of genes across the human genome. A distinct class of CENP-A hotspots also accumulates at subtelomeric chromosomal locations, including at the 8q24/Myc region long-associated with genomic instability. We show this 8q24 accumulation of CENP-A can also be seen in early stage primary colorectal tumors.ConclusionsOur data demonstrate that excess CENP-A accumulates at noncentromeric locations in the human cancer genome. These findings suggest that ectopic CENP-A nucleosomes could alter the state of the chromatin fiber, potentially impacting gene regulation and chromosome fragility.

Down the rabbit hole of centromere assembly and dynamics

The centromere is perhaps the most iconic feature on a eukaryotic chromosome. An amateur enthusiast equipped with a light microscope can easily identify the center of each metacentric chromosome, marking the spot responsible for accurate genome segregation. This review will highlight findings that provide novel insights into how centromeres are assembled and disassembled, the role centromeric proteins play in repair, epigenetic features uniquely found at the centromere, and the three dimensional organization of centromeres caught in the act of mitosis. These advances have unveiled a veritable wonderland of non-canonical features that drive centromere function.

Histone variants: the tricksters of the chromatin world☆

The eukaryotic genome exists in vivo at an equimolar ratio with histones, thus forming a polymer composed of DNA and histone proteins. Each nucleosomal unit in this polymer provides versatile capabilities and dynamic range. Substitutions of the individual components of the histone core with structurally distinct histone variants and covalent modifications alter the local fabric of the chromatin fiber, resulting in epigenetic changes that can be regulated by the cell. In this review, we highlight recent advances in the study of histone variant structure, assembly, and inheritance, their influence on nucleosome positioning, and their cumulative effect upon gene expression, DNA repair and the progression of disease. We also highlight fundamental questions that remain unanswered regarding the behavior of histone variants and their influence on cellular function in the normal and diseased states.

Single-epitope recognition imaging of native chromatin

BackgroundDirect visualization of chromatin has the potential to provide important insights into epigenetic processes. In particular, atomic force microscopy (AFM) can visualize single nucleosomes under physiological ionic conditions. However, AFM has mostly been applied to chromatin that has been reconstituted in vitro, and its potential as a tool for the dissection of native nucleosomes has not been explored. Recently we applied AFM to native Drosophila chromatin containing the centromere-specific histone 3 (CenH3), showing that it is greatly enriched in smaller particles. Taken together with biochemical analyses of CenH3 nucleosomes, we propose that centromeric nucleosomes are hemisomes, with one turn of DNA wrapped around a particle consisting of one molecule each of centromere-specific CenH3, H4, H2A and H2B.ResultsHere we apply a recognition mode of AFM imaging to directly identify CenH3 within histone core particles released from native centromeric chromatin. More than 90% of these particles were found to be tetrameric in height. The specificity of recognition was confirmed by blocking with a CenH3 peptide, and the strength of the interaction was quantified by force measurements. These results imply that the particles imaged by AFM are indeed mature CenH3-containing hemisomes.ConclusionEfficient and highly specific recognition of CenH3 in histone core particles isolated from native centromeric chromatin demonstrates that tetramers are the predominant form of centromeric nucleosomes in mature tetramers. Our findings provide proof of principle that this approach can yield insights into chromatin biology using direct and rapid detection of native nucleosomes in physiological salt concentrations.

The CENP-A nucleosome: a dynamic structure and role at the centromere.

The centromere is a specialized locus that directs the formation of the kinetochore protein complex for correct chromosome segregation. The specific centromere histone H3 variant CENP-A has been described as the epigenetic mark of this chromatin region. Several laboratories have explored its properties, its partners, and its role in centromere formation. Specifically, two types of CENP-A nucleosomes have been described, suggesting there may be more complexity involved in centromere structure than previously thought. Recent work adds to this paradox by questioning the role of CENP-A as a unique centromeric mark and highlighting the assembly of a functional kinetochore in the absence of CENP-A. In this review, we discuss recent literature on the CENP-A nucleosomes and the debate on its role in kinetochore formation and centromere identity.

Epigenetic specification of centromeres

Centromeres are the discrete sites of spindle microtubule attachment on chromosomes during mitosis and meiosis in all eukaryotes. These highly specialized chromatin structures typically occupy the same site for thousands of generations, yet the mechanism by which centromeres are established, maintained, and function remain a mystery. In metazoans, centromeric DNA sequence has proven not to be the key determinant of centromeric identity; therefore, centromeres are thought to be epigenetically specified by their specialized chromatin structure. In all eukaryotes, the centromere-specific histone H3 variant CenH3 replaces canonical H3 within nucleosomes at centric chromatin. This specialized nucleosome is the building block upon which all other centromere-associated proteins depend. This review highlights exciting new findings that have resulted in a paradigm shift in our understanding of CenH3 assembly into centromeric chromatin, CenH3 nucleosomal structure, CenH3 chromatin folding, the contribution of these factors to centromeric identity, and finally, the intimate role cell-cycle-regulated transcription and pericentric heterochromatin play in the maintenance and integrity of centromeres.

Long-range oscillation in a periodic DNA sequence motif may influence nucleosome array formation

We have experimentally examined the characteristics of nucleosome array formation in different regions of mouse liver chromatin, and have computationally analyzed the corresponding genomic DNA sequences. We have shown that the mouse adenosine deaminase (MADA) gene locus is packaged into an exceptionally regular nucleosome array with a shortened repeat, consistent with our computational prediction based on the DNA sequence. A survey of the mouse genome indicates that <10% of 70 kb windows possess a nucleosome-ordering signal, consisting of regular long-range oscillations in the period-10 triplet motif non-T, A/T, G (VWG), which is as strong as the signal in the MADA locus. A strong signal in the center of this locus, confirmed by in vitro chromatin assembly experiments, appears to cooperate with weaker, in-phase signals throughout the locus. In contrast, the mouse odorant receptor (MOR) locus, which lacks locus-wide signals, was representative of ∼40% of the mouse genomic DNA surveyed. Within this locus, nucleosome arrays were similar to those of bulk chromatin. Genomic DNA sequences which were computationally similar to MADA or MOR resulted in MADA- or MOR-like nucleosome ladders experimentally. Overall, we provide evidence that computationally predictable information in the DNA sequence may affect nucleosome array formation in animal tissue.