Graham Dellaire

Changes in chromatin structure and mobility in living cells at sites of DNA double-strand breaks

The repair of DNA double-strand breaks (DSBs) is facilitated by the phosphorylation of H2AX, which organizes DNA damage signaling and chromatin remodeling complexes in the vicinity of the lesion (Pilch, D.R., O.A. Sedelnikova, C. Redon, A. Celeste, A. Nussenzweig, and W.M. Bonner. 2003. Biochem. Cell Biol. 81:123–129; Morrison, A.J., and X. Shen. 2005. Cell Cycle. 4:568–571; van Attikum, H., and S.M. Gasser. 2005. Nat. Rev. Mol. Cell. Biol. 6:757–765). The disruption of DNA integrity induces an alteration of chromatin architecture that has been proposed to activate the DNA damage transducing kinase ataxia telangiectasia mutated (ATM; Bakkenist, C.J., and M.B. Kastan. 2003. Nature. 421:499–506). However, little is known about the physical properties of damaged chromatin. In this study, we use a photoactivatable version of GFP-tagged histone H2B to examine the mobility and structure of chromatin containing DSBs in living cells. We find that chromatin containing DSBs exhibits limited mobility but undergoes an energy-dependent local expansion immediately after DNA damage. The localized expansion observed in real time corresponds to a 30–40% reduction in the density of chromatin fibers in the vicinity of DSBs, as measured by energy-filtering transmission electron microscopy. The observed opening of chromatin occurs independently of H2AX and ATM. We propose that localized adenosine triphosphate–dependent decondensation of chromatin at DSBs establishes an accessible subnuclear environment that facilitates DNA damage signaling and repair.

Expression Patterns and Post-translational Modifications Associated with Mammalian Histone H3 Variants

Covalent histone modifications and the incorporation of histone variants bring about changes in chromatin structure that in turn alter gene expression. Interest in non-allelic histone variants has been renewed, in part because of recent work on H3 (and other) histone variants. However, only in mammals do three non-centromeric H3 variants (H3.1, H3.2, and H3.3) exist. Here, we show that mammalian cell lines can be separated into two different groups based on their expression of H3.1, H3.2, and H3.3 at both mRNA and protein levels. Additionally, the ratio of these variants changes slightly during neuronal differentiation of murine ES cells. This difference in H3 variant expression between cell lines could not be explained by changes in growth rate, cell cycle stages, or chromosomal ploidy, but rather suggests other possibilities, such as changes in H3 variant incorporation during differentiation and tissue- or species-specific H3 variant expression. Moreover, quantitative mass spectrometry analysis of human H3.1, H3.2, and H3.3 showed modification differences between these three H3 variants, suggesting that they may have different biological functions. Specifically, H3.3 contains marks associated with transcriptionally active chromatin, whereas H3.2, in contrast, contains mostly silencing modifications that have been associated with facultative heterochromatin. Interestingly, H3.1 is enriched in both active and repressive marks, although the latter marks are different from those observed in H3.2. Although the biological significance as to why mammalian cells differentially employ three highly similar H3 variants remains unclear, our results underscore potential functional differences between them and reinforce the general view that H3.1 and H3.2 in mammalian cells should not be treated as equivalent proteins.

HuCHRAC, a human ISWI chromatin remodelling complex contains hACF1 and two novel histone-fold proteins

Chromatin remodelling complexes containing the nucleosome‐dependent ATPase ISWI were first isolated from Drosophila embryos (NURF, CHRAC and ACF). ISWI was the only common component reported in these complexes. Our purification of human CHRAC (HuCHRAC) shows that ISWI chromatin remodelling complexes can have a conserved subunit composition in completely different cell types, suggesting a conserved function of ISWI. We show that the human homologues of two novel putative histone‐fold proteins in Drosophila CHRAC are present in HuCHRAC. The two human histone‐fold proteins form a stable complex that binds naked DNA but not nucleosomes. HuCHRAC also contains human ACF1 (hACF1), the homologue of Acf1, a subunit of Drosophila ACF. The N‐terminus of mouse ACF1 was reported as a heterochromatin‐targeting domain. hACF1 is a member of a family of proteins with a related domain structure that all may target heterochromatin. We discuss a possible function for HuCHRAC in heterochromatin dynamics. HuCHRAC does not contain topoisomerase II, which was reported earlier as a subunit of Drosophila CHRAC.

Application of Quantum Dots as Probes for Correlative Fluorescence, Conventional, and Energy-filtered Transmission Electron Microscopy

Luminescent semiconductor quantum dots (QDs) are a new class of fluorescent label with wide-ranging applications for cell imaging. The electron density and elemental composition of these materials permit the extension of their use as probes in conventional electron microscopy (TEM) and energy-filtered TEM (EFTEM). Here we illustrate the feasibility of using streptavidin-conjugated QDs as TEM tags by labeling a nuclear protein on cell sections and obtaining correlative fluorescence and TEM data. We also show that QD probes can be employed in conjunction with immunogold for co-localization of proteins at the ultrastructural level. Furthermore, by obtaining cadmium elemental maps of CdSe/ZnS QDs distributed on a nuclear structure, we demonstrate the potential of QDs for co-localization of multiple proteins when used in combination with EFTEM.

A mechanism for the suppression of homologous recombination in G1 cells

DNA repair by homologous recombination is highly suppressed in G1 cells to ensure that mitotic recombination occurs solely between sister chromatids. Although many homologous recombination factors are cell-cycle regulated, the identity of the events that are both necessary and sufficient to suppress recombination in G1 cells is unknown. Here we report that the cell cycle controls the interaction of BRCA1 with PALB2–BRCA2 to constrain BRCA2 function to the S/G2 phases in human cells. We found that the BRCA1-interaction site on PALB2 is targeted by an E3 ubiquitin ligase composed of KEAP1, a PALB2-interacting protein, in complex with cullin-3 (CUL3)–RBX1 (ref. 6). PALB2 ubiquitylation suppresses its interaction with BRCA1 and is counteracted by the deubiquitylase USP11, which is itself under cell cycle control. Restoration of the BRCA1–PALB2 interaction combined with the activation of DNA-end resection is sufficient to induce homologous recombination in G1, as measured by RAD51 recruitment, unscheduled DNA synthesis and a CRISPR–Cas9-based gene-targeting assay. We conclude that the mechanism prohibiting homologous recombination in G1 minimally consists of the suppression of DNA-end resection coupled with a multi-step block of the recruitment of BRCA2 to DNA damage sites that involves the inhibition of BRCA1–PALB2–BRCA2 complex assembly. We speculate that the ability to induce homologous recombination in G1 cells with defined factors could spur the development of gene-targeting applications in non-dividing cells.

PML bodies: a meeting place for genomic loci?

Promyelocytic leukemia (PML) bodies have been implicated in a variety of cellular processes, such as cell-cycle regulation, apoptosis, proteolysis, tumor suppression, DNA repair and transcription. Despite this, the function of PML bodies is still unknown. Direct and indirect evidence supports the hypothesis that PML bodies interact with specific genes or genomic loci. This includes the finding that the stability of PML bodies is affected by cell stress and changes in chromatin structure. PML bodies also facilitate the transcription and replication of double-stranded DNA viral genomes. Moreover, PML bodies associate with specific regions of high transcriptional activity in the cellular genome. We propose that PML bodies functionally interact with chromatin and are important for the regulation of gene expression.

Zebrafish xenografts as a tool for in vivo studies on human cancer

The zebrafish has become a powerful vertebrate model for genetic studies of embryonic development and organogenesis and increasingly for studies in cancer biology. Zebrafish facilitate the performance of reverse and forward genetic approaches, including mutagenesis and small molecule screens. Moreover, several studies report the feasibility of xenotransplanting human cells into zebrafish embryos and adult fish. This model provides a unique opportunity to monitor tumor‐induced angiogenesis, invasiveness, and response to a range of treatments in vivo and in real time. Despite the high conservation of gene function between fish and humans, concern remains that potential differences in zebrafish tissue niches and/or missing microenvironmental cues could limit the relevance and translational utility of data obtained from zebrafish human cancer cell xenograft models. Here, we summarize current data on xenotransplantation of human cells into zebrafish, highlighting the advantages and limitations of this model in comparison to classical murine models of xenotransplantation.

Heterochromatin and the DNA damage response: the need to relax 1

Higher order chromatin structure has an impact on all nuclear functions, including the DNA damage response. Over the past several years, it has become increasingly clear that heterochromatin and euchromatin represent separate entities with respect to both damage sensitivity and repair. The chromatin compaction present in heterochromatin helps to protect this DNA from damage; however, when lesions do occur, the compaction restricts the ability of DNA damage response proteins to access the site, as evidenced by its ability to block the expansion of H2AX phosphorylation. As such, DNA damage in heterochromatin is refractory to repair, which requires the surrounding chromatin structure to be decondensed. In the case of DNA double-strand breaks, this relaxation is at least partially mediated by the ATM kinase phosphorylating and inhibiting the function of the transcriptional repressor KAP1. This review will focus on the functions of KAP1 and other proteins involved in the maintenance or restriction of heterochromatin, including HP1 and TIP60, in the DNA damage response. As heterochromatin is important for maintaining genomic stability, cells must maintain a delicate balance between allowing repair factors access to these regions and ensuring that these regions retain their organization to prevent increased DNA damage and chromosomal mutations.

Chromatin Contributes to Structural Integrity of Promyelocytic Leukemia Bodies through a SUMO-1-independent Mechanism

Promyelocytic leukemia (PML) protein is implicated in transcriptional regulation, apoptosis, DNA repair, and tumor suppression. It is not known, however, whether PML and other components of PML bodies function within the vicinity of the bodies or elsewhere in the nucleoplasm. In this study, we demonstrate that chromatin organization around PML bodies influences their morphology, dynamics, and structural integrity by a SUMO-1-independent mechanism. Following transcriptional inhibition and during early apoptosis, chromatin retracts from the periphery of PML bodies, coinciding with the formation of new PML-containing structures through fission of supramolecular PML-containing microbodies. Both fission and fusion of microbodies with parental PML bodies indicate a loss of structural integrity of the bodies, dependent on the state of the surrounding chromatin. This is supported by the observation that treatment of live cells with DNase I could reproduce the structural instability of PML bodies. In addition, PML bodies, which are normally surrounded by chromatin and are positionally stable, become more dynamic following these treatments, presumably due to the loss of chromatin contacts. Overexpression of SUMO-1, a modification required for PML body formation, did not prevent PML body fission, indicating that chromatin-based integrity of PML body structure occurs through a SUMO-1-independent mechanism.

The number of PML nuclear bodies increases in early S phase by a fission mechanism

Promyelocytic leukemia (PML) nuclear bodies have been implicated in a variety of cellular processes including apoptosis, tumour suppression, anti-viral response, DNA repair and transcriptional regulation. PML nuclear bodies are both positionally and structurally stable over extended periods of interphase. As demonstrated in this study, the structural stability is lost as cells enter S phase, evidenced both by distortions in shape and by fission and fusion events. At the end of this period of structural instability, the number of PML nuclear bodies has increased by a factor of twofold. Association of the fission products with chromatin implies that the PML nuclear bodies respond to changes in chromatin organisation or topology, and thus could play a role in monitoring genome integrity during DNA synthesis or in the continued maintenance of functional chromosomal domains prior to mitosis.