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- Analysis of Protein Turnover by Quantitative SNAP-Based Pulse-Chase ImagingPublication . Bodor, Dani L.; Rodríguez, Mariluz Gómez; Moreno, Nuno; Jansen, Lars E.T.Assessment of protein dynamics in living cells is crucial for understanding their biological properties and functions. The SNAP-tag, a self labeling suicide enzyme, presents a tool with unique features that can be adopted for determining protein dynamics in living cells. Here we present detailed protocols for the use of SNAP in fluorescent pulse-chase and quench-chase-pulse experiments. These time-slicing methods provide powerful tools to assay and quantify the fate and turnover rate of proteins of different ages. We cover advantages and pitfalls of SNAP-tagging in fixed- and live-cell studies and evaluate the recently developed fast-acting SNAPf variant. In addition, to facilitate the analysis of protein turnover datasets, we present an automated algorithm for spot recognition and quantification.
- Assembly in G1 phase and long-term stability are unique intrinsic features of CENP-A nucleosomesPublication . Bodor, D. L.; Valente, L. P.; Mata, J. F.; Black, B. E.; Jansen, L. E. T.Centromeres are the site of kinetochore formation during mitosis. Centromere protein A (CENP-A), the centromere-specific histone H3 variant, is essential for the epigenetic maintenance of centromere position. Previously we showed that newly synthesized CENP-A is targeted to centromeres exclusively during early G1 phase and is subsequently maintained across mitotic divisions. Using SNAP-based fluorescent pulse labeling, we now demonstrate that cell cycle-restricted chromatin assembly at centromeres is unique to CENP-A nucleosomes and does not involve assembly of other H3 variants. Strikingly, stable retention is restricted to the CENP-A/H4 core of the nucleosome, which we find to outlast general chromatin across several cell divisions. We further show that cell cycle timing of CENP-A assembly is independent of centromeric DNA sequences and instead is mediated by the CENP-A targeting domain. Unexpectedly, this domain also induces stable transmission of centromeric nucleosomes, independent of the CENP-A deposition factor HJURP. This demonstrates that intrinsic properties of the CENP-A protein direct its cell cycle-restricted assembly and induces quantitative mitotic transmission of the CENP-A/H4 nucleosome core, ensuring long-term stability and epigenetic maintenance of centromere position.
- Basic properties of epigenetic systems: lessons from the centromerePublication . Gómez-Rodríguez, Mariluz; Jansen, Lars ETChromatin-based epigenetic inheritance cooperates with cis-acting DNA sequence information to propagate gene expression states and chromosome architecture across cell division cycles. Histone proteins and their modifications are central components of epigenetic systems but how, and to what extent, they are propagated is a matter of continued debate. Centromeric nucleosomes, marked by the histone H3 variant CENP-A, are stable across mitotic divisions and are assembled in a locus specific and cell cycle controlled manner. The mechanism of inheritance of this unique chromatin domain has important implications for how general nucleosome transmission is controlled in space and time.
- Cdk Activity Couples Epigenetic Centromere Inheritance to Cell Cycle ProgressionPublication . Silva, Mariana C.C.; Bodor, Dani L.; Stellfox, Madison E.; Martins, Nuno M.C.; Hochegger, Helfrid; Foltz, Daniel R.; Jansen, Lars E.T.Centromeres form the site of chromosome attachment to microtubules during mitosis. Identity of these loci is maintained epigenetically by nucleosomes containing the histone H3 variant CENP-A. Propagation of CENP-A chromatin is uncoupled from DNA replication initiating only during mitotic exit. We now demonstrate that inhibition of Cdk1 and Cdk2 activities is sufficient to trigger CENP-A assembly throughout the cell cycle in a manner dependent on the canonical CENP-A assembly machinery. We further show that the key CENP-A assembly factor Mis18BP1(HsKNL2) is phosphorylated in a cell cycle-dependent manner that controls its centromere localization during mitotic exit. These results strongly support a model in which the CENP-A assembly machinery is poised for activation throughout the cell cycle but kept in an inactive noncentromeric state by Cdk activity during S, G2, and M phases. Alleviation of this inhibition in G1 phase ensures tight coupling between DNA replication, cell division, and subsequent centromere maturation.
- Chromosomes. CENP-C reshapes and stabilizes CENP-A nucleosomes at the centromerePublication . Falk, S. J.; Guo, L. Y.; Sekulic, N.; Smoak, E. M.; Mani, T.; Logsdon, G. A.; Gupta, K.; Jansen, L. E. T.; Van Duyne, G. D.; Vinogradov, S. A.; Lampson, M. A.; Black, B. E.Inheritance of each chromosome depends upon its centromere. A histone H3 variant, centromere protein A (CENP-A), is essential for epigenetically marking centromere location. We find that CENP-A is quantitatively retained at the centromere upon which it is initially assembled. CENP-C binds to CENP-A nucleosomes and is a prime candidate to stabilize centromeric chromatin. Using purified components, we find that CENP-C reshapes the octameric histone core of CENP-A nucleosomes, rigidifies both surface and internal nucleosome structure, and modulates terminal DNA to match the loose wrap that is found on native CENP-A nucleosomes at functional human centromeres. Thus, CENP-C affects nucleosome shape and dynamics in a manner analogous to allosteric regulation of enzymes. CENP-C depletion leads to rapid removal of CENP-A from centromeres, indicating their collaboration in maintaining centromere identity.
- A Dual Inhibitory Mechanism Sufficient to Maintain Cell-Cycle-Restricted CENP-A AssemblyPublication . Stankovic, Ana; Guo, Lucie Y.; Mata, João F.; Bodor, Dani L.; Cao, Xing-Jun; Bailey, Aaron O.; Shabanowitz, Jeffrey; Hunt, Donald F.; Garcia, Benjamin A.; Black, Ben E.; Jansen, Lars E.T.Chromatin featuring the H3 variant CENP-A at the centromere is critical for its mitotic function and epigenetic maintenance. Assembly of centromeric chromatin is restricted to G1 phase through inhibitory action of Cdk1/2 kinases in other phases of the cell cycle. Here, we identify the two key targets sufficient to maintain cell-cycle control of CENP-A assembly. We uncovered a single phosphorylation site in the licensing factor M18BP1 and a cyclin A binding site in the CENP-A chaperone, HJURP, that mediated specific inhibitory phosphorylation. Simultaneous expression of mutant proteins lacking these residues results in complete uncoupling from the cell cycle. Consequently, CENP-A assembly is fully recapitulated under high Cdk activities, indistinguishable from G1 assembly. We find that Cdk-mediated inhibition is exerted by sequestering active factors away from the centromere. Finally, we show that displacement of M18BP1 from the centromere is critical for the assembly mechanism of CENP-A.
- Dynamics of Histone H3 Deposition In Vivo Reveal a Nucleosome Gap-Filling Mechanism for H3.3 to Maintain Chromatin IntegrityPublication . Ray-Gallet, Dominique; Woolfe, Adam; Vassias, Isabelle; Pellentz, Céline; Lacoste, Nicolas; Puri, Aastha; Schultz, David C.; Pchelintsev, Nikolay A.; Adams, Peter D.; Jansen, Lars E.T.; Almouzni, GenevièveEstablishment of a proper chromatin landscape is central to genome function. Here, we explain H3 variant distribution by specific targeting and dynamics of deposition involving the CAF-1 and HIRA histone chaperones. Impairing replicative H3.1 incorporation via CAF-1 enables an alternative H3.3 deposition at replication sites via HIRA. Conversely, the H3.3 incorporation throughout the cell cycle via HIRA cannot be replaced by H3.1. ChIP-seq analyses reveal correlation between HIRA-dependent H3.3 accumulation and RNA pol II at transcription sites and specific regulatory elements, further supported by their biochemical association. The HIRA complex shows unique DNA binding properties, and depletion of HIRA increases DNA sensitivity to nucleases. We propose that protective nucleosome gap filling of naked DNA by HIRA leads to a broad distribution of H3.3, and HIRA association with Pol II ensures local H3.3 enrichment at specific sites. We discuss the importance of this H3.3 deposition as a salvage pathway to maintain chromatin integrity.
- Enhancer regions show high histone H3.3 turnover that changes during differentiationPublication . Deaton, Aimee M; Gómez-Rodríguez, Mariluz; Mieczkowski, Jakub; Tolstorukov, Michael Y; Kundu, Sharmistha; Sadreyev, Ruslan I; Jansen, Lars ET; Kingston, Robert EThe organization of DNA into chromatin is dynamic; nucleosomes are frequently displaced to facilitate the ability of regulatory proteins to access specific DNA elements. To gain insight into nucleosome dynamics, and to follow how dynamics change during differentiation, we used a technique called time-ChIP to quantitatively assess histone H3.3 turnover genome-wide during differentiation of mouse ESCs. We found that, without prior assumptions, high turnover could be used to identify regions involved in gene regulation. High turnover was seen at enhancers, as observed previously, with particularly high turnover at super-enhancers. In contrast, regions associated with the repressive Polycomb-Group showed low turnover in ESCs. Turnover correlated with DNA accessibility. Upon differentiation, numerous changes in H3.3 turnover rates were observed, the majority of which occurred at enhancers. Thus, time-ChIP measurement of histone turnover shows that active enhancers are unusually dynamic in ESCs and changes in highly dynamic nucleosomes predominate at enhancers during differentiation.
- Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochorePublication . Bergmann, Jan H; Rodríguez, Mariluz Gómez; Martins, Nuno M C; Kimura, Hiroshi; Kelly, David A; Masumoto, Hiroshi; Larionov, Vladimir; Jansen, Lars E T; Earnshaw, William CKinetochores assemble on distinct 'centrochromatin' containing the histone H3 variant CENP-A and interspersed nucleosomes dimethylated on H3K4 (H3K4me2). Little is known about how the chromatin environment at active centromeres governs centromeric structure and function. Here, we report that centrochromatin resembles K4-K36 domains found in the body of some actively transcribed housekeeping genes. By tethering the lysine-specific demethylase 1 (LSD1), we specifically depleted H3K4me2, a modification thought to have a role in transcriptional memory, from the kinetochore of a synthetic human artificial chromosome (HAC). H3K4me2 depletion caused kinetochores to suffer a rapid loss of transcription of the underlying α-satellite DNA and to no longer efficiently recruit HJURP, the CENP-A chaperone. Kinetochores depleted of H3K4me2 remained functional in the short term, but were defective in incorporation of CENP-A, and were gradually inactivated. Our data provide a functional link between the centromeric chromatin, α-satellite transcription, maintenance of CENP-A levels and kinetochore stability.
- Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variantPublication . Earnshaw, W. C.; Allshire, R. C.; Black, B. E.; Bloom, K.; Brinkley, B. R.; Brown, W.; Cheeseman, I. M.; Choo, K. H. A.; Copenhaver, G. P.; DeLuca, J. G.; Desai, A.; Diekmann, S.; Erhardt, S.; Fitzgerald-Hayes, M.; Foltz, D.; Fukagawa, T.; Gassmann, R.; Gerlich, D. W.; Glover, D. M.; Gorbsky, G. J.; Harrison, S. C.; Heun, P.; Hirota, T.; Jansen, L. E. T.; Karpen, G.; Kops, G. J. P. L.; Lampson, M. A.; Lens, S. M.; Losada, A.; Luger, K.; Maiato, H.; Maddox, P. S.; Margolis, R. L.; Masumoto, H.; McAinsh, A. D.; Mellone, B. G.; Meraldi, P.; Musacchio, A.; Oegema, K.; O’Neill, R. J.; Salmon, E. D.; Scott, K. C.; Straight, A. F.; Stukenberg, P. T.; Sullivan, B. A.; Sullivan, K. F.; Sunkel, C. E.; Swedlow, J. R.; Walczak, C. E.; Warburton, P. E.; Westermann, S.; Willard, H. F.; Wordeman, L.; Yanagida, M.; Yen, T. J.; Yoda, K.; Cleveland, D. W.The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.