RESUMO
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. We report the development of a type of HAC that functions independently of these constraints. Formed by an initial CENP-A nucleosome seeding strategy, a construct lacking repetitive centromeric DNA formed several self-sufficient HACs that showed no uptake of genomic DNA. In contrast to traditional α-satellite HAC formation, the non-repetitive construct can form functional HACs without CENP-B or initial CENP-A nucleosome seeding, revealing distinct paths to centromere formation for different DNA sequence types. Our developments streamline the construction and characterization of HACs to facilitate mammalian synthetic genome efforts.
Assuntos
Centrômero/metabolismo , Cromossomos Artificiais Humanos/metabolismo , DNA Satélite/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Centrômero/genética , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Proteína B de Centrômero/deficiência , Proteína B de Centrômero/genética , Proteína B de Centrômero/metabolismo , Epigênese Genética , Humanos , Nucleossomos/química , Nucleossomos/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismoRESUMO
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. We will then discuss how several studies, which applied removal of endogenous centromeres or over-expression of the centromere-specific histone CENP-A, helped to investigate the contribution of chromatin context to centromere establishment. Finally, we will examine various biosynthetic approaches taking advantage of targeting specific proteins to ectopic sites in the genome to dissect the role of many centromere-associated proteins and chromatin modifiers for centromere inheritance and function. Together, these studies showed that chromatin context matters, particularly proximity to heterochromatin or repetitive DNA sequences. Moreover, despite the important contribution of centromeric DNA, the centromere-specific histone H3-variant CENP-A emerges as a key epigenetic mark to establish and maintain functional centromeres on artificial chromosomes or at ectopic sites of the genome.
Assuntos
Proteína Centromérica A/metabolismo , Centrômero/genética , Centrômero/metabolismo , Cromatina/genética , Cromatina/metabolismo , Cromossomos Artificiais/genética , Epigênese GenéticaRESUMO
Modern drug discovery relies on combinatorial screening campaigns to find drug molecules targeting specific disease-associated proteins. The success of such campaigns often relies on functional and structural information of the selected therapeutic target, only achievable once its purification is mastered. With the aim of bypassing the protein purification process to gain insights on the druggability, ligand binding, and/or characterization of protein-protein interactions, herein, we describe the Extract2Chip method. This approach builds on the immobilization of site-specific biotinylated proteins of interest, directly from cellular extracts, on avidin-coated sensor chips to allow for the characterization of molecular interactions via surface plasmon resonance (SPR). The developed method was initially validated using Cyclophilin D (CypD) and subsequently applied to other drug discovery projects in which the targets of interest were difficult to express, purify, and crystallize. Extract2Chip was successfully applied to the characterization of Yes-associated protein (YAP): Transcriptional enhancer factor TEF (TEAD1) protein-protein interaction inhibitors, in the validation of a ternary complex assembly composed of Dyskerin pseudouridine synthase 1 (DKC1) and RuvBL1/RuvBL2, and in the establishment of a fast-screening platform to select the most suitable NUAK family SNF1-like kinase 2 (NUAK2) surrogate for binding and structural studies. The described method paves the way for a potential revival of the many drug discovery campaigns that have failed to deliver due to the lack of suitable and sufficient protein supply.
Assuntos
Descoberta de Drogas , Ressonância de Plasmônio de Superfície , Ressonância de Plasmônio de Superfície/métodos , Descoberta de Drogas/métodos , Proteínas , Cromatografia de Afinidade , Ligação ProteicaRESUMO
The class 4 P-type ATPases ("flippases") maintain membrane asymmetry by translocating phosphatidylethanolamine and phosphatidylserine from the outer leaflet to the cytosolic leaflet of the plasma membrane. In Saccharomyces cerevisiae, five related gene products (Dnf1, Dnf2, Dnf3, Drs2, and Neo1) are implicated in flipping of phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine. In MAT A: cells responding to α-factor, we found that Dnf1, Dnf2, and Dnf3, as well as the flippase-activating protein kinase Fpk1, localize at the projection ("shmoo") tip where polarized growth is occurring and where Ste5 (the central scaffold protein of the pheromone-initiated MAPK cascade) is recruited. Although viable, a MAT A: dnf1∆ dnf2∆ dnf3∆ triple mutant exhibited a marked decrease in its ability to respond to α-factor, which we could attribute to pronounced reduction in Ste5 stability resulting from an elevated rate of its Cln2â Cdc28-initiated degradation. Similarly, a MAT A: dnf1∆ dnf3∆ drs2∆ triple mutant also displayed marked reduction in its ability to respond to α-factor, which we could attribute to inefficient recruitment of Ste5 to the plasma membrane due to severe mislocalization of the cellular phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate pools. Thus proper remodeling of plasma membrane aminoglycerolipids and phosphoinositides is necessary for efficient recruitment, stability, and function of the pheromone signaling apparatus.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Membrana Celular/química , Feromônios/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transdução de Sinais , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/genética , ATPases Transportadoras de Cálcio/genética , Fosfatidilinositóis/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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. We targeted histone H3 chimeras to chromosomally integrated Lac operator sequences by fusing each of the chimeras to the Lac repressor. Using this approach, we found surprising contributions from a small portion of the N-terminal tail and the CENP-A targeting domain in the initial recruitment of two essential constitutive centromere proteins, CENP-C and CENP-T. Our results indicate that the regions of CENP-A required for early events in centromere establishment differ from those that are required for maintaining centromere identity.