Efficient formation of single-copy human artificial chromosomes.

Large DNA assembly methodologies underlie milestone achievements in synthetic prokaryotic and budding yeast chromosomes. While budding yeast control chromosome inheritance through ~125-base pair DNA sequence-defined centromeres, mammals and many other eukaryotes use large, epigenetic centromeres. Harnessing centromere epigenetics permits human artificial chromosome (HAC) formation but is not sufficient to avoid rampant multimerization of the initial DNA molecule upon introduction to cells.

Centromere structure and function: lessons from Drosophila.

The fruit fly Drosophila melanogaster serves as a powerful model organism for advancing our understanding of biological processes, not just by studying its similarities with other organisms including ourselves but also by investigating its differences to unravel the underlying strategies that evolved to achieve a common goal. This is particularly true for centromeres, specialized genomic regions present on all eukaryotic chromosomes that function as the platform for the assembly of kinetochores. These multiprotein structures play an essential role during cell division by connecting chromosomes to spindle microtubules in mitosis and meiosis to mediate accurate chromosome segregation.

Efficient Formation of Single-copy Human Artificial Chromosomes.

Large DNA assembly methodologies underlie milestone achievements in synthetic prokaryotic and budding yeast chromosomes. While budding yeast control chromosome inheritance through ~125 bp DNA sequence-defined centromeres, mammals and many other eukaryotes use large, epigenetic centromeres. Harnessing centromere epigenetics permits human artificial chromosome (HAC) formation but is not sufficient to avoid rampant multimerization of the initial DNA molecule upon introduction to cells.

The ins and outs of CENP-A: Chromatin dynamics of the centromere-specific histone.

Centromeres are highly specialised chromosome domains defined by the presence of an epigenetic mark, the specific histone H3 variant called CENP-A (centromere protein A). They constitute the genomic regions on which kinetochores form and when defective cause segregation defects that can lead to aneuploidy and cancer. Here, we discuss how CENP-A is established and maintained to propagate centromere identity while subjected to dynamic chromatin remodelling during essential cellular processes like DNA repair, replication, and transcription.

Spt6 is a maintenance factor for centromeric CENP-A.

Replication and transcription of genomic DNA requires partial disassembly of nucleosomes to allow progression of polymerases. This presents both an opportunity to remodel the underlying chromatin and a danger of losing epigenetic information. Centromeric transcription is required for stable incorporation of the centromere-specific histone dCENP-A in M/G1 phase, which depends on the eviction of previously deposited H3/H3.

SWR1 and NuA4 complexes are defined by DOMINO isoforms.

Histone acetylation and deposition of H2A.Z variant are integral aspects of active transcription. In , the single DOMINO chromatin regulator complex is thought to combine both activities an unknown mechanism.

Structural basis for centromere maintenance by Drosophila CENP-A chaperone CAL1.

Centromeres are microtubule attachment sites on chromosomes defined by the enrichment of histone variant CENP-A-containing nucleosomes. To preserve centromere identity, CENP-A must be escorted to centromeres by a CENP-A-specific chaperone for deposition. Despite this essential requirement, many eukaryotes differ in the composition of players involved in centromere maintenance, highlighting the plasticity of this process.

Reconstituting Drosophila Centromere Identity in Human Cells.

The centromere is an essential chromosomal region required for accurate chromosome segregation. Most eukaryotic centromeres are defined epigenetically by the histone H3 variant, centromere protein (CENP)-A, yet how its self-propagation is achieved remains poorly understood. Here, we develop a heterologous system to reconstitute epigenetic inheritance of centromeric chromatin by ectopically targeting the Drosophila centromere proteins dCENP-A, dCENP-C, and CAL1 to LacO arrays in human cells.

Human Artificial Chromosomes that Bypass Centromeric DNA.

Recent breakthroughs with synthetic budding yeast chromosomes expedite the creation of synthetic mammalian chromosomes and genomes. Mammals, unlike budding yeast, depend on the histone H3 variant, CENP-A, to epigenetically specify the location of the centromere-the locus essential for chromosome segregation. Prior human artificial chromosomes (HACs) required large arrays of centromeric α-satellite repeats harboring binding sites for the DNA sequence-specific binding protein, CENP-B.

High-resolution mapping of centromeric protein association using APEX-chromatin fibers.

Background: The centromere is a specialized chromosomal locus that forms the basis for the assembly of a multi-protein complex, the kinetochore and ensures faithful chromosome segregation during every cell division. The repetitive nature of the underlying centromeric sequence represents a major obstacle for high-resolution mapping of protein binding using methods that rely on annotated genomes. Here, we present a novel microscopy-based approach called "APEX-chromatin fibers" for localizing protein binding over the repetitive centromeric sequences at kilobase resolution.

Oligomerization of Drosophila Nucleoplasmin-Like Protein is required for its centromere localization.

The evolutionarily conserved nucleoplasmin family of histone chaperones has two paralogues in Drosophila, named Nucleoplasmin-Like Protein (NLP) and Nucleophosmin (NPH). NLP localizes to the centromere, yet molecular underpinnings of this localization are unknown. Moreover, similar to homologues in other organisms, NLP forms a pentamer in vitro, but the biological significance of its oligomerization has not been explored.

Centromere transcription allows CENP-A to transit from chromatin association to stable incorporation.

Centromeres are essential for chromosome segregation and are specified epigenetically by the presence of the histone H3 variant CENP-A. In flies and humans, replenishment of the centromeric mark is uncoupled from DNA replication and requires the removal of H3 "placeholder" nucleosomes. Although transcription at centromeres has been previously linked to the loading of new CENP-A, the underlying molecular mechanism remains poorly understood.

Artificial Chromosomes and Strategies to Initiate Epigenetic Centromere Establishment.

In recent years, various synthetic approaches have been developed to address the question of what directs centromere establishment and maintenance. In this chapter, we will discuss how approaches aimed at constructing synthetic centromeres have co-evolved with and contributed to shape the theory describing the determinants of centromere identity. We will first review lessons learned from artificial chromosomes created from "naked" centromeric sequences to investigate the role of the underlying DNA for centromere formation.

Identification of Drosophila centromere associated proteins by quantitative affinity purification-mass spectrometry.

Centromeres of higher eukaryotes are epigenetically defined by the centromere specific histone H3 variant CENP-A(CID). CENP-A(CID) builds the foundation for the assembly of a large network of proteins. In contrast to mammalian systems, the protein composition of Drosophila centromeres has not been comprehensively investigated.

Both tails and the centromere targeting domain of CENP-A are required for centromere establishment.

The centromere-defined by the presence of nucleosomes containing the histone H3 variant, CENP-A-is the chromosomal locus required for the accurate segregation of chromosomes during cell division. Although the sequence determinants of human CENP-A required to maintain a centromere were reported, those that are required for early steps in establishing a new centromere are unknown. In this paper, we used gain-of-function histone H3 chimeras containing various regions unique to CENP-A to investigate early events in centromere establishment.

Fly versus man: evolutionary impairment of nucleolar targeting affects the degradome of Drosophila's Taspase1.

Human Taspase1 is essential for development and cancer by processing critical regulators, such as the mixed-lineage leukemia protein. Likewise, its ortholog, trithorax, is cleaved by Drosophila Taspase1 (dTaspase1), implementing a functional coevolution. To uncover novel mechanism regulating protease function, we performed a functional analysis of dTaspase1 and its comparison to the human ortholog.

Identification of novel Drosophila centromere-associated proteins.

Centromeres are chromosomal regions crucial for correct chromosome segregation during mitosis and meiosis. They are epigenetically defined by centromeric proteins such as the centromere-specific histone H3-variant centromere protein A (CENP-A). In humans, 16 additional proteins have been described to be constitutively associated with centromeres throughout the cell cycle, known as the constitutive centromere-associated network (CCAN).

Nucleolus and nuclear periphery: velcro for heterochromatin.

Heterochromatin was first defined by Emil Heitz in 1928 by light microscopy. In the 1950s electron microscopy studies revealed that heterochromatin preferentially localizes to the nuclear periphery and around the nucleolus. While the use of genomic approaches led to the genome wide identification of lamina-associated and nucleolus-associated chromatin domains (LADs, NADs), recent studies now shed light on the processes mediating this topology and its dynamics.

CAL1 is the Drosophila CENP-A assembly factor.

Centromeres are specified epigenetically by the incorporation of the histone H3 variant CENP-A. In humans, amphibians, and fungi, CENP-A is deposited at centromeres by the HJURP/Scm3 family of assembly factors, but homologues of these chaperones are absent from a number of major eukaryotic lineages such as insects, fish, nematodes, and plants. In Drosophila, centromeric deposition of CENP-A requires the fly-specific protein CAL1.

A pair of centromeric proteins mediates reproductive isolation in Drosophila species.

Speciation involves the reproductive isolation of natural populations due to the sterility or lethality of their hybrids. However, the molecular basis of hybrid lethality and the evolutionary driving forces that provoke it remain largely elusive. The hybrid male rescue (Hmr) and the lethal hybrid rescue (Lhr) genes serve as a model to study speciation in Drosophilids because their interaction causes lethality in male hybrid offspring.

The nucleoplasmin homolog NLP mediates centromere clustering and anchoring to the nucleolus.

Centromere clustering during interphase is a phenomenon known to occur in many different organisms and cell types, yet neither the factors involved nor their physiological relevance is well understood. Using Drosophila tissue culture cells and flies, we identified a network of proteins, including the nucleoplasmin-like protein (NLP), the insulator protein CTCF, and the nucleolus protein Modulo, to be essential for the positioning of centromeres. Artificial targeting further demonstrated that NLP and CTCF are sufficient for clustering, while Modulo serves as the anchor to the nucleolus.

Drosophila CENH3 is sufficient for centromere formation.

CENH3 is a centromere-specific histone H3 variant essential for kinetochore assembly. Despite its central role in centromere function, there has been no conclusive evidence supporting CENH3 as sufficient to determine centromere identity. To address this question, we artificially targeted Drosophila CENH3 (CENP-A/CID) as a CID-GFP-LacI fusion protein to stably integrated lac operator (lacO) arrays.

Heterochromatin boundaries are hotspots for de novo kinetochore formation.

The centromere-specific histone H3 variant CENH3 (also known as CENP-A) is considered to be an epigenetic mark for establishment and propagation of centromere identity. Pulse induction of CENH3 (Drosophila CID) in Schneider S2 cells leads to its incorporation into non-centromeric regions and generates CID islands that resist clearing from chromosome arms for multiple cell generations. We demonstrate that CID islands represent functional ectopic kinetochores, which are non-randomly distributed on the chromosome and show a preferential localization near telomeres and pericentric heterochromatin in transcriptionally silent, intergenic chromatin domains.