DNA:RNA hybrid formation is emerging as a significant cause of genome instability in biological systems ranging from bacteria to mammals. Here we describe the genome-wide distribution of DNA:RNA hybrid prone loci in Saccharomyces cerevisiae by DNA:RNA immunoprecipitation (DRIP) followed by hybridization on tiling microarray. These profiles show that DNA:RNA hybrids preferentially accumulated at rDNA, Ty1 and Ty2 transposons, telomeric repeat regions and a subset of open reading frames (ORFs). The latter are generally highly transcribed and have high GC content. Interestingly, significant DNA:RNA hybrid enrichment was also detected at genes associated with antisense transcripts. The expression of antisense-associated genes was also significantly altered upon overexpression of RNase H, which degrades the RNA in hybrids. Finally, we uncover mutant-specific differences in the DRIP profiles of a Sen1 helicase mutant, RNase H deletion mutant and Hpr1 THO complex mutant compared to wild type, suggesting different roles for these proteins in DNA:RNA hybrid biology. Our profiles of DNA:RNA hybrid prone loci provide a resource for understanding the properties of hybrid-forming regions in vivo, extend our knowledge of hybrid-mitigating enzymes, and contribute to models of antisense-mediated gene regulation. A summary of this paper was presented at the 26th International Conference on Yeast Genetics and Molecular Biology, August 2013.

Genome instability via RNA:DNA hybrid-mediated R loops has been observed in mutants involved in various aspects of transcription and RNA processing. The prevalence of this mechanism among essential chromosome instability (CIN) genes remains unclear. In a secondary screen for increased Rad52 foci in CIN mutants, representing~25% of essential genes, we identified seven essential subunits of the mRNA cleavage and polyadenylation (mCP) machinery. Genome-wide analysis of fragile sites by chromatin immunoprecipitation (ChIP) and microarray (ChIP-chip) of phosphorylated H2A in these mutants supported a transcription-dependent mechanism of DNA damage characteristic of R loops. In parallel, we directly detected increased RNA:DNA hybrid formation in mCP mutants and demonstrated that CIN is suppressed by expression of the R-loop-degrading enzyme RNaseH. To investigate the conservation of CIN in mCP mutants, we focused on FIP1L1, the human ortholog of yeast FIP1, a conserved mCP component that is part of an oncogenic fusion in eosinophilic leukemia. We found that truncation fusions of yeast FIP1 analogous to those in cancer cause loss of function and that siRNA knockdown of FIP1L1 in human cells increases DNA damage and chromosome breakage. Our findings illuminate how mCP maintains genome integrity by suppressing R-loop formation and suggest that this function may be relevant to certain human cancers.

Biological embedding occurs when life experience alters biological processes to affect later life health and well-being. Although extensive correlative data exist supporting the notion that epigenetic mechanisms such as DNA methylation underlie biological embedding, causal data are lacking. We describe specific epigenetic mechanisms and their potential roles in the biological embedding of experience. We also consider the nuanced relationships between the genome, the epigenome, and gene expression. Our ability to connect biological embedding to the epigenetic landscape in its complexity is challenging and complicated by the influence of multiple factors. These include cell type, age, the timing of experience, sex, and DNA sequence. Recent advances in molecular profiling and epigenome editing, combined with the use of comparative animal and human longitudinal studies, should enable this field to transition from correlative to causal analyses.

Chang et al. link the RECQ-like helicase BLM and its yeast orthologue Sgs1 to preventing DNA damage caused by the accumulation of DNA:RNA hybrid structures called R-loops. This adds to a growing family of helicases implicated in R-loop resolution.

Ectopic R-loop accumulation causes DNA replication stress and genome instability. To avoid these outcomes, cells possess a range of anti-R-loop mechanisms, including RNaseH that degrades the RNA moiety in R-loops. To comprehensively identify anti-R-loop mechanisms, we performed a genome-wide trigenic interaction screen in yeast lacking RNH1 and RNH201. We identified >100 genes critical for fitness in the absence of RNaseH, which were enriched for DNA replication fork maintenance factors including the MRE11-RAD50-NBS1 (MRN) complex. While MRN has been shown to promote R-loops at DNA double-strand breaks, we show that it suppresses R-loops and associated DNA damage at transcription–replication conflicts. This occurs through a non-nucleolytic function of MRE11 that is important for R-loop suppression by the Fanconi Anemia pathway. This work establishes a novel role for MRE11-RAD50-NBS1 in directing tolerance mechanisms at transcription–replication conflicts.

Our past experiences shape our current and future behavior. These experiences must leave some enduring imprint on our brains, altering neural circuits that mediate behavior and contributing to our individual differences. As a framework for understanding how experiences might produce lasting changes in neural circuits, Clayton [D. F. Clayton, Neurobiol. Learn. Mem. 74, 185–216 (2000)] introduced the concept of the genomic action potential (gAP)—a structured genomic response in the brain to acute experience. Similar to the familiar electrophysiological action potential (eAP), the gAP also provides a means for integrating afferent patterns of activity but on a slower timescale and with longer-lasting effects. We revisit this concept in light of contemporary work on experience-dependent modification of neural circuits. We review the “Immediate Early Gene” (IEG) response, the starting point for understanding the gAP. We discuss evidence for its involvement in the encoding of experience to long-term memory across time and biological levels of organization ranging from individual cells to cell ensembles and whole organisms. We explore distinctions between memory encoding and homeostatic functions and consider the potential for perpetuation of the imprint of experience through epigenetic mechanisms. We describe a specific example of a gAP in humans linked to individual differences in the response to stress. Finally, we identify key objectives and new tools for continuing research in this area.

The incorporation of histone variants into nucleosomes represents one way of altering the chromatin structure to accommodate diverse functions. Histone variant H2A.Z has specific roles in gene regulation, heterochromatin boundary formation, and genomic integrity. The precise features required for H2A.Z to function and specify an identity different from canonical H2A remain to be fully explored. Analysis of the C-terminal docking domain of H2A.Z in Saccharomyces cerevisiae using epistatic miniarray profile (E-MAP) uncovered nuanced requirements of the H2A.Z C-terminal region for cell growth when additional genes were compromised. Moreover, the H2A.Z(1-114) truncation, lacking the last 20 amino acids of the protein, did not support regular H2A.Z functions, such as resistance to genotoxic stress, restriction of heterochromatin in its native context, GAL1 gene activation, and chromatin anchoring. The corresponding region of H2A could fully rescue the strong defects caused by loss of this functionally essential region in the C terminus of H2A.Z. Despite the dramatic reduction in function, the H2A.Z(1-114) truncation still bound the H2A.Z deposition complex SWR1-C, the histone chaperone Chz1, and histone H2B. These data are consistent with a model in which retaining the variant in chromatin after its deposition by SWR1-C is a crucial determinant of its function.

e Recognition of histone marks by reader modules is thought to be at the heart of epigenetic mechanisms. These protein domains are considered to function by targeting regulators to chromosomal loci carrying specific histone modifications. This is important for proper gene regulation as well as propagation of epigenetic information. The NuA4 acetyltransferase complex contains two of these reader modules, an H3K4me3-specific plant homeodomain (PHD) within the Yng2 subunit and an H3K36me2/3-specific chromodomain in the Eaf3 subunit. While each domain showed a close functional interaction with the respective histone mark that it recognizes, at the biochemical level, genetic level (as assessed with epistatic miniarray profile screens), and phenotypic level, cells with the combined loss of both readers showed greatly enhanced phenotypes. Chromatin immunoprecipitation coupled with next-generation sequencing experiments demonstrated that the Yng2 PHD specifically directs H4 acetylation near the transcription start site of highly expressed genes, while Eaf3 is important downstream on the body of the genes. Strikingly, the recruitment of the NuA4 complex to these loci was not significantly affected. Furthermore, RNA polymerase II occupancy was decreased only under conditions where both PHD and chromodomains were lost, generally in the second half of the gene coding regions. Altogether, these results argue that methylated histone reader modules in NuA4 are not responsible for its recruitment to the promoter or coding regions but, rather, are required to orient its acetyltransferase catalytic site to the methylated histone 3-bearing nucleosomes in the surrounding chromatin, cooperating to allow proper transition from transcription initiation to elongation. Chromatin is a dynamic nucleoprotein structure that compacts the long DNA molecule into the small nuclear space, simultaneously creating a mechanism to regulate DNA access to machineries implicated in essential nuclear processes, namely, transcription, DNA replication, DNA repair, and meiotic recombination. Modulation of the chromatin structure is highly regulated by four broad classes of nuclear factors: chromatin remodelers, histone chaperones, histone variants, and histone modifiers. These players essentially target nucleosomes, basic units of chromatin consisting of 146 bp of DNA wrapped around an octamer of histone proteins. Histone modifiers catalyze posttranslational modifications (PTM), including acetylation, phosphorylation, and methylation, that can directly affect DNA-histone contacts (e.g., acetylation) and/or create a binding platform for PTM readers (1, 2). These readers correspond to proteins/complexes containing distinct domains/modules that can bind specific modified histone residues. Such motifs include bromodomains that recognize acetylated residues; chromodomains (CHD); PWWP, Tudor, and MBT domains for methylated residues; BRCT, BIR, and 14-3-3 domains for phosphorylated histones; and plant homeodomain (PHD) fingers that can bind different modification...