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- 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.
- 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.
- 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.
- Genetics. Sowing the seeds of centromeres.Publication . Jansen, L. E. T.The centromere is a chromatin-based platform that accumulates microtubule-binding proteins that drive chromosome segregation during cell division. Despite their size (on the order of megabases of DNA in mammals) and conserved role, centromeres have the remarkable capacity to leave their usual comfort zone and to reform at a new chromosomal site (1). Although found rarely, these so-called neocentromeres are by most measures bona fide and segregate chromosomes with high fidelity. What accounts for this nomadic behavior?
- A Rapid FACS-Based Strategy to Isolate Human Gene Knockin and Knockout ClonesPublication . Mata, João F.; Lopes, Telma; Gardner, Rui; Jansen, Lars E. T.Gene targeting protocols for mammalian cells remain inefficient and labor intensive. Here we describe FASTarget, a rapid, fluorescent cell sorting based strategy to isolate rare gene targeting events in human somatic cells. A fluorescent protein is used as a means for direct selection of targeted clones obviating the need for selection and outgrowth of drug resistant clones. Importantly, the use of a promoter-less, ATG-less construct greatly facilitates the recovery of correctly targeted cells. Using this method we report successful gene targeting in up to 94% of recovered human somatic cell clones. We create functional EYFP-tagged knockin clones in both transformed and non-transformed human somatic cell lines providing a valuable tool for mammalian cell biology. We further demonstrate the use of this technology to create gene knockouts. Using this generally applicable strategy we can recover gene targeted clones within approximately one month from DNA construct delivery to obtaining targeted monoclonal cell lines.
- 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.
- Temporal control of epigenetic centromere specificationPublication . Valente, Luis P; Silva, Mariana C C; Jansen, Lars E TAll living organisms require accurate mechanisms to faithfully inherit their genetic material during cell division. The centromere is a unique locus on each chromosome that supports a multiprotein structure called the kinetochore. During mitosis, the kinetochore is responsible for connecting chromosomes to spindle microtubules, allowing faithful segregation of the duplicated genome. In most organisms, centromere position and function is not defined by the local DNA sequence context but rather by an epigenetic chromatin-based mechanism. Centromere protein A (CENP-A) is central to this process, as chromatin assembled from this histone H3 variant is essential for assembly of the centromere complex, as well as for its epigenetic maintenance. As a major determinant of centromere function, CENP-A assembly requires tight control, both in its specificity for the centromere and in timing of assembly. In the last few years, there have been several new insights into the molecular mechanism that allow this process to occur. We will review these here and discuss the general implications of the mechanism of cell cycle coupling of centromere inheritance.
- Reductionism at the vertebrate kinetochorePublication . Stankovic, A.; Jansen, L. E. T.The kinetochore forms the site of attachment for mitotic spindle microtubules driving chromosome segregation. The interdependent protein interactions in this large structure have made it difficult to dissect the function of its components. In this issue, Hori et al. (2013. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201210106) present a novel and powerful methodology to address the sufficiency of individual proteins for the creation of a functional de novo centromere.
- 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.
- 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.