Explore & actuate: the future of personalized medicine in oncology through emerging technologies.

PURPOSE OF REVIEW: The future of medicine is aimed to equip the physician with tools to assess the individual health of the patient for the uniqueness of the disease that separates it from the rest. The integration of omics technologies into clinical practice, reviewed here, would open new avenues for addressing the spatial and temporal heterogeneity of cancer. The rising cancer burden patiently awaits the advent of such an approach to personalized medicine for routine clinical settings. RECENT FINDINGS: To weigh the translational potential, multiple technologies were categorized based on the extractable information from the different types of samples used, to the various omic-levels of molecular information that each technology has been able to advance over the last 2 years. This review uses a multifaceted classification that helps to assess translational potential in a meaningful way toward clinical adaptation. SUMMARY: The importance of distinguishing technologies based on the flow of information from exploration to actuation puts forth a framework that allows the clinicians to better adapt a chosen technology or use them in combination to enhance their goals toward personalized medicine.

A potential screening method for epigenetic drugs: uncovering stress-induced gene silencing in Chlamydomonas.

Histone modifications and DNA methylation together govern promoter availability, thereby influencing gene expression. This study queries the unicellular chlorophyte, Chlamydomonas reinhardtii using a three step "epigenetic assay" design to phenotypically track the variegation of a randomly integrated Paromomycin resistance transgene(s) (PmR). Based on its position of integration, the PmR gene expression hinged on two epigenetic hallmarks: the spreading of heterochromatin, and the transmissible memory of epigenetic states across generations. Using a spot-dilution analysis, the loss of antibiotic resistance phenotype was scored from 0 to 4, four being maximally silenced. Appropriate construct designs were used to demonstrate that the cis-spread of heterochromatin could be interfered with a stronger euchromatic barrier (TUB2 promoter). When assayed for metal ion stress, a combination of Mn deficiency with excess Cu or Zn stress was shown to induce gene silencing in Chlamydomonas. Cu stress resulted in the accumulation of intracellular ROS, while Zn stress elevated the sensitivity to ROS. As proof of functional conservation, mammalian epigenetic drugs demonstrably interfered with stress-induced gene silencing. Finally, a selected group of transgenic clones responsive to HDACi sodium butyrate, when tested in a gradient plate format showed similarity in phenotype to the plant-derived compound cinnamic acid. This indicated a possible commonality in their mode of action, unlike curcumin which might have a different mechanism. Thus, using binned libraries, based on a common set of responses to known drugs, a cost-effective high-throughput screening strategy for epigenetically active compounds from plants or other sources is described.

A Novel Method to Generate MNase Ladders Reveal Rapid Chromatin Remodeling upon Gametogenesis and Mating in Chlamydomonas.

To circumvent nuclei isolation for nucleosomal mapping of wild-type (cell walled) algal cells, we developed a quick and versatile methodology, by abrasion of whole cells (Chlamydomonas, Scenedesmus and yeast), allowing Micrococcal Nuclease (MNase) direct access to nuclear chromatin, in situ. Varying parameters such as bead abrasion, vortex and incubation conditions, we optimized capture of an 'early digest' which may probe chromatin differentially, based on nucleosome accessibility. A comparison of such ladders across vegetative cells, gametes and zygotes revealed an increase in the average nucleosomal repeat length (+17-34nt) upon gametogenesis, indicating a trend of chromatin compaction. Using PCR, we compared promoter enrichment in increasing orders of fractionated nucleosomal repeats (mono-, di-, up to penta-), each differing in cleavability based on chromatin accessibility. Concordant with higher gene expression (mating locus), promoters revealed an enrichment in mono-nucleosomal fractions. Interestingly, the zygote specific gene, MT0828 displayed rapid remodelling from penta-nucleosomal enrichment when completely repressed (vegetative), to intermediate states during gametogenesis (24hrs), which finally shifted to being largely mono-nucleosomal, when induced (1h zygotes). Summarizing three candidate genes from the mating locus, we conclude that the MNase based 'Chromatin Accessibility Assay' can track a range of large-scale rapid chromatin remodelling transitions within the binaries of gene expression.

DNA Methylation Patterns Separate Senescence from Transformation Potential and Indicate Cancer Risk.

Overall shared DNA methylation patterns between senescence (Sen) and cancers have led to the model that tumor-promoting epigenetic patterns arise through senescence. We show that transformation-associated methylation changes arise stochastically and independently of programmatic changes during senescence. Promoter hypermethylation events in transformation involve primarily pro-survival and developmental genes, similarly modified in primary tumors. Senescence-associated hypermethylation mainly involves metabolic regulators and appears early in proliferating "near-senescent" cells, which can be immortalized but are refractory to transformation. Importantly, a subset of transformation-associated hypermethylated developmental genes exhibits highest methylation gains at all age-associated cancer risk states across tissue types. These epigenetic changes favoring cell self-renewal and survival, arising during tissue aging, are fundamentally important for stratifying cancer risk and concepts for cancer prevention.

Genome-wide positioning of bivalent mononucleosomes.

BACKGROUND: Bivalent chromatin refers to overlapping regions containing activating histone H3 Lys4 trimethylation (H3K4me3) and inactivating H3K27me3 marks. Existence of such bivalent marks on the same nucleosome has only recently been suggested. Previous genome-wide efforts to characterize bivalent chromatin have focused primarily on individual marks to define overlapping zones of bivalency rather than mapping positions of truly bivalent mononucleosomes. RESULTS: Here, we developed an efficacious sequential ChIP technique for examining global positioning of individual bivalent nucleosomes. Using next generation sequencing approaches we show that although individual H3K4me3 and H3K27me3 marks overlap in broad zones, bivalent nucleosomes are focally enriched in the vicinity of the transcription start site (TSS). These seem to occupy the H2A.Z nucleosome positions previously described as salt-labile nucleosomes, and are correlated with low gene expression. Although the enrichment profiles of bivalent nucleosomes show a clear dependency on CpG island content, they demonstrate a stark anti-correlation with methylation status. CONCLUSIONS: We show that regional overlap of H3K4me3 and H3K27me3 chromatin tend to be upstream to the TSS, while bivalent nucleosomes with both marks are mainly promoter proximal near the TSS of CpG island-containing genes with poised/low expression. We discuss the implications of the focal enrichment of bivalent nucleosomes around the TSS on the poised chromatin state of promoters in stem cells.

Oxidative damage targets complexes containing DNA methyltransferases, SIRT1, and polycomb members to promoter CpG Islands.

Cancer cells simultaneously harbor global losses and gains in DNA methylation. We demonstrate that inducing cellular oxidative stress by hydrogen peroxide treatment recruits DNA methyltransferase 1 (DNMT1) to damaged chromatin. DNMT1 becomes part of a complex(es) containing DNMT3B and members of the polycomb repressive complex 4. Hydrogen peroxide treatment causes relocalization of these proteins from non-GC-rich to GC-rich areas. Key components are similarly enriched at gene promoters in an in vivo colitis model. Although high-expression genes enriched for members of the complex have histone mark and nascent transcription changes, CpG island-containing low-expression genes gain promoter DNA methylation. Thus, oxidative damage induces formation and relocalization of a silencing complex that may explain cancer-specific aberrant DNA methylation and transcriptional silencing.

Aberrant silencing of cancer-related genes by CpG hypermethylation occurs independently of their spatial organization in the nucleus.

Aberrant promoter DNA-hypermethylation and repressive chromatin constitutes a frequent mechanism of gene inactivation in cancer. There is great interest in dissecting the mechanisms underlying this abnormal silencing. Studies have shown changes in the nuclear organization of chromatin in tumor cells as well as the association of aberrant methylation with long-range silencing of neighboring genes. Furthermore, certain tumors show a high incidence of promoter methylation termed as the CpG island methylator phenotype. Here, we have analyzed the role of nuclear chromatin architecture for genes in hypermethylated inactive versus nonmethylated active states and its relation with long-range silencing and CpG island methylator phenotype. Using combined immunostaining for active/repressive chromatin marks and fluorescence in situ hybridization in colorectal cancer cell lines, we show that aberrant silencing of these genes occurs without requirement for their being positioned at heterochromatic domains. Importantly, hypermethylation, even when associated with long-range epigenetic silencing of neighboring genes, occurs independent of their euchromatic or heterochromatic location. Together, these results indicate that, in cancer, extensive changes around promoter chromatin of individual genes or gene clusters could potentially occur locally without preference for nuclear position and/or causing repositioning. These findings have important implications for understanding relationships between nuclear organization and gene expression patterns in cancer.

Chz1, a nuclear chaperone for histone H2AZ.

The histone variant H2AZ marks nucleosomes flanking the promoters of most genes of budding yeast. The incorporation of H2AZ into chromatin is dependent on the SWR1 complex, which catalyses the replacement of conventional histone H2A with H2AZ. In cells, the pool of unincorporated histone H2AZ has previously been found in association with Nap1, a chaperone for conventional histone H2A-H2B. Here, we report the discovery of Chz1, a histone chaperone that has preference for H2AZ and can also deliver a source of the histone variant for SWR1-dependent histone replacement. Bacterially expressed Chz1 forms a heterotrimer with H2AZ-H2B, stabilizing the association of the histone dimer. We have identified a conserved motif important for histone variant recognition within the H2AZ-interacting domain of Chz1. The presence of this motif in other metazoan proteins suggests that H2AZ-specific chaperones may be widely conserved.

Swc2 is a widely conserved H2AZ-binding module essential for ATP-dependent histone exchange.

The histone variant H2AZ is incorporated preferentially at specific locations in chromatin to modulate chromosome functions. In Saccharomyces cerevisiae, deposition of histone H2AZ is mediated by the multiprotein SWR1 complex, which catalyzes ATP-dependent exchange of nucleosomal histone H2A for H2AZ. Here, we define interactions between SWR1 components and H2AZ, revealing a link between the ATPase domain of Swr1 and three subunits required for the binding of H2AZ. We discovered that Swc2 binds directly to and is essential for transfer of H2AZ. Swc6 and Arp6 are necessary for the association of Swc2 and for nucleosome binding, whereas other subunits, Swc5 and Yaf9, are required for H2AZ transfer but neither H2AZ nor nucleosome binding. Finally, the C-terminal alpha-helix of H2AZ is crucial for its recognition by SWR1. These findings provide insight on the initial events of histone exchange.

ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex.

The conserved histone variant H2AZ has an important role in the regulation of gene expression and the establishment of a buffer to the spread of silent heterochromatin. How histone variants such as H2AZ are incorporated into nucleosomes has been obscure. We have found that Swr1, a Swi2/Snf2-related adenosine triphosphatase, is the catalytic core of a multisubunit, histone-variant exchanger that efficiently replaces conventional histone H2A with histone H2AZ in nucleosome arrays. Swr1 is required for the deposition of histone H2AZ at specific chromosome locations in vivo, and Swr1 and H2AZ commonly regulate a subset of yeast genes. These findings define a previously unknown role for the adenosine triphosphate-dependent chromatin remodeling machinery.

DNA dynamics in RecA-DNA filaments: ATP hydrolysis-related flexibility in DNA.

RecA-catalyzed DNA recombination is initiated by a mandatory, high-energy form of DNA in RecA-nucleoprotein filaments, where bases are highly unstacked and the backbone is highly unwound. Interestingly, only the energetics consequent to adenosine triphosphate (ATP) binding, rather than its hydrolysis, seems sufficient to mediate such a high-energy structural hallmark of a recombination filament. The structural consequence of ATP hydrolysis on the DNA part of the filament thus remains largely unknown. We report time-resolved fluorescence dynamics of bases in RecA-DNA complexes and demonstrate that DNA bases in the same exhibit novel, motional dynamics with a rotational correlation time of 7-10 ns, specifically in the presence of ATP hydrolysis. When the ongoing ATP hydrolysis of RecA-DNA filament is "poisoned" by a nonhydrolyzable form of ATP (ATPgammaS), the motional dynamics cease and reveal a global motion with a rotational correlation time of >20 ns. Such ATP hydrolysis-induced flexibility ensues in single-stranded as well as double-stranded bases of RecA-DNA filaments. These results suggest that the role of ATP hydrolysis is to induce a high level of backbone flexibility in RecA-DNA filament, a dynamic property that is likely to be important for efficient strand exchanges in ATP hydrolysis specific RecA reactions. It is the absence of these motions that may cause high rigidity in RecA-DNA filaments in ATPgammaS. Dynamic light scattering measurement comparisons of RecA-ss-DNA filaments formed in ATPgammaS vs that of ATP confirmed such an interpretation, where the former showed a complex of larger (30 nm) hydrodynamic radius than that of latter (12-15 nm). Taken together, these results reveal a more dynamic state of DNA in RecA-DNA filament that is hydrolyzing ATP, which encourage us to model the role of ATP hydrolysis in RecA-mediated DNA transactions.

RecA-promoted sliding of base pairs within DNA repeats: quantitative analysis by a slippage assay.

RecA that catalyses efficient homology search and exchange of DNA bases has to effect major transitions in the structure as well as the dynamics of bases within RecA-DNA filament. RecA induces slippage of paired strands in poly(dA)-poly(dT) duplex using the energy of ATP hydrolysis. Here, we have adopted the targeted ligation assay and quantified the strand slippage within a short central cassette of (dA)(4)-(dT)(4) duplex. The design offers a stringent test case for scoring a cross-talk between A residues with those of T that are diagonally placed on the opposite strand at either -3, -2, -1, +1, +2, or +3 pairing frames. As expected, the cross-talk levels in RecA mediated as well as thermally annealed duplexes were maximal in non-diagonal pairing frame (i.e., 0-frame), the levels of which fell off gradually as the frames became more diagonal, i.e., -3<-2<-1 or +3<+2<+1. Interestingly, the level of cross-talk in naked duplexes was intrinsically less efficient in minus frames than their plus frame counterparts. The asymmetry created in naked duplexes by such a disparity between minus versus plus frames was partially obviated by RecA. Moreover, RecA promoted a significantly higher level of cross-talk selectively in -2 and -1 frames, as compared to that in naked DNA, which suggests a model that the elevated cross-talk in RecA filament may be limited to base pairs housed within the same rather than adjacent RecA monomers.