A Synthetic Approach to Reconstruct the Evolutionary and Functional Innovations of the Plant Histone Variant H2A.W.

Diversification of histone variants is marked by the acquisition of distinct motifs and functional properties through convergent evolution.1-4 H2A variants are distinguished by specific C-terminal motifs and tend to be segregated within defined domains of the genome.5,6 Whether evolution of these motifs pre-dated the evolution of segregation mechanisms or vice versa has remained unclear. A suitable model to address this question is the variant H2A.W, which evolved in plants through acquisition of a KSPK motif7 and is tightly associated with heterochromatin.4 We used fission yeast, where chromatin is naturally devoid of H2A.W, to study the impact of engineered chimeras combining yeast H2A with the KSPK motif. Biochemical assays showed that the KSPK motif conferred nucleosomes with specific properties. Despite uniform incorporation of the engineered H2A chimeras in the yeast genome, the KSPK motif specifically affected heterochromatin composition and function. We conclude that the KSPK motif promotes chromatin properties in yeast that are comparable to the properties and function of H2A.W in plant heterochromatin. We propose that the selection of functional motifs confer histone variants with properties that impact primarily a specific chromatin state. The association between a new histone variant and a preferred chromatin state can thus provide a setting for the evolution of mechanisms that segregate the new variant to this state, thereby enhancing the impact of the selected properties of the variant on genome activity.

Compartmentalization of DNA Damage Response between Heterochromatin and Euchromatin Is Mediated by Distinct H2A Histone Variants.

DNA double-strand break (DSB) repair depends on the ataxia telangiectasia mutated (ATM) kinase that phosphorylates the conserved C-terminal SQ motif present in the histone variant H2A.X [1-7]. In constitutive heterochromatin of mammals, DSB repair is delayed and relies on phosphorylation of the proteins HP1 and KAP1 by ATM [2, 8-14]. However, KAP1 is not conserved in plants and the HP1-related protein Like-HP1 (LHP1) is not localized at constitutive heterochromatin [15], suggesting that in plants, alternative mechanisms could be responsible for repair of DSBs in heterochromatin. In Arabidopsis, constitutive heterochromatin is marked by H3K9 methylation and the plant-specific histone variants H2A.W, which are distinguished by their C-terminal motif KSPKK and required for heterochromatin compaction [16-18]. We report that the Arabidopsis histone variant H2A.W.7 is confined to heterochromatin and carries a SQ motif that is phosphorylated by ATM. In response to DNA damage, phosphorylation of H2A.W.7 takes place in heterochromatin, while H2A.X phosphorylation takes place primarily in euchromatin. We propose that H2A.W.7 evolved in addition to H2A.X to facilitate DNA damage response in highly condensed heterochromatin, thus playing a role similar to KAP1 and HP1 phosphorylation in mammals. These data support the idea of the functional diversification of histone variants and their role in spatial compartmentalization of chromatin-related functions in eukaryotes.

H3S28 phosphorylation is a hallmark of the transcriptional response to cellular stress.

The selectivity of transcriptional responses to extracellular cues is reflected by the deposition of stimulus-specific chromatin marks. Although histone H3 phosphorylation is a target of numerous signaling pathways, its role in transcriptional regulation remains poorly understood. Here, for the first time, we report a genome-wide analysis of H3S28 phosphorylation in a mammalian system in the context of stress signaling. We found that this mark targets as many as 50% of all stress-induced genes, underlining its importance in signal-induced transcription. By combining ChIP-seq, RNA-seq, and mass spectrometry we identified the factors involved in the biological interpretation of this histone modification. We found that MSK1/2-mediated phosphorylation of H3S28 at stress-responsive promoters contributes to the dissociation of HDAC corepressor complexes and thereby to enhanced local histone acetylation and subsequent transcriptional activation of stress-induced genes. Our data reveal a novel function of the H3S28ph mark in the activation of mammalian genes in response to MAP kinase pathway activation.

An attempt at a molecular prediction of metastasis in patients with primary cutaneous melanoma.

BACKGROUND: Current prognostic clinical and morphological parameters are insufficient to accurately predict metastasis in individual melanoma patients. Several studies have described gene expression signatures to predict survival or metastasis of primary melanoma patients, however the reproducibility among these studies is disappointingly low. METHODOLOGY/PRINCIPAL FINDINGS: We followed extended REMARK/Gould Rothberg criteria to identify gene sets predictive for metastasis in patients with primary cutaneous melanoma. For class comparison, gene expression data from 116 patients with clinical stage I/II (no metastasis) and 72 with III/IV primary melanoma (with metastasis) at time of first diagnosis were used. Significance analysis of microarrays identified the top 50 differentially expressed genes. In an independent data set from a second cohort of 28 primary melanoma patients, these genes were analyzed by multivariate Cox regression analysis and leave-one-out cross validation for association with development of metastatic disease. In a multivariate Cox regression analysis, expression of the genes Ena/vasodilator-stimulated phosphoprotein-like (EVL) and CD24 antigen gave the best predictive value (p = 0.001; p = 0.017, respectively). A multivariate Cox proportional hazards model revealed these genes as a potential independent predictor, which may possibly add (both p = 0.01) to the predictive value of the most important morphological indicator, Breslow depth. CONCLUSION/SIGNIFICANCE: Combination of molecular with morphological information may potentially enable an improved prediction of metastasis in primary melanoma patients. A strength of the gene expression set is the small number of genes, which should allow easy reevaluation in independent data sets and adequately designed clinical trials.