SummaryTranscription initiation entails chromatin opening followed by pre-initiation complex formation and RNA polymerase II recruitment. Subsequent polymerase elongation requires additional signals, resulting in increased residence time downstream of the start site, a phenomenon referred to as pausing. Here, we harnessed single-molecule footprinting to quantify distinct steps of initiation in vivo throughout the Drosophila genome. This identifies the impact of promoter structure on initiation dynamics in relation to nucleosomal occupancy. Additionally, perturbation of transcriptional initiation reveals an unexpectedly high turnover of polymerases at paused promoters—an observation confirmed at the level of nascent RNAs. These observations argue that absence of elongation is largely caused by premature termination rather than by stable polymerase stalling. In support of this non-processive model, we observe that induction of the paused heat shock promoter depends on continuous initiation. Our study provides a framework to quantify protein binding at single-molecule resolution and refines concepts of transcriptional pausing.

Topoisomerases are essential for DNA replication in dividing cells, but their genomic targets and function in postmitotic cells remain poorly understood. Here we show that a switch in the expression from Topoisomerases IIα (Top2α) to IIβ (Top2β) occurs during neuronal differentiation in vitro and in vivo. Genome-scale location analysis in stem cell-derived postmitotic neurons reveals Top2β binding to chromosomal sites that are methylated at lysine 4 of histone H3, a feature of regulatory regions. Indeed Top2β-bound sites are preferentially promoters and become targets during the transition from neuronal progenitors to neurons, at a time when cells exit the cell cycle. Absence of Top2β protein or its activity leads to changes in transcription and chromatin accessibility at many target genes. Top2β deficiency does not impair stem cell properties and early steps of neuronal differentiation but causes premature death of postmitotic neurons. This neuronal degeneration is caused by up-regulation of Ngfr p75, a gene bound and repressed by Top2β. These findings suggest a chromatin-based targeting of Top2β to regulatory regions in the genome to govern the transcriptional program associated with neuronal differentiation and longevity.epigenetic regulation | neurogenesis | gene expression | genomewide assays T opoisomerases are essential for solving topological problems arising from DNA-templated processes such as replication, transcription, recombination, chromatin remodeling, chromosome condensation, and segregation (1-5). The type I subfamily of topoisomerases achieves this task by passing one strand of the DNA through a break in the opposing strand; proteins in the type II subfamily pass a region of duplex strands from the same or a different molecule through a double-stranded gap generated in DNA (1-5). Mammalian cells encode two isozymes of type II enzymes that have highly homologous N-terminal ATPase and central core domains but differ at their C-termini (6). These two isozymes, Topoisomerases IIα (Top2α) and IIβ (Top2β), have almost identical enzymatic properties in vitro (7, 8); however, their expression patterns are dissimilar. Top2α is the main isoform expressed in proliferating cells, shows high expression in S/G2/M phases of the cell cycle, and plays important roles in DNA replication and chromosome condensation/segregation during the cell cycle (9-12).The cellular functions of Top2β are much less well understood. It is expressed in all mammalian cells throughout the cell cycle but is up-regulated robustly when cells reach a postmitotic state of terminal differentiation (13-15). For example, the postmitotic granule cells in the external germinal layer of the developing rat cerebellum show a transition from Top2α to Top2β (14), and blocking Top2β catalytic activity affects the expression of about one third of genes induced during differentiation of rat cerebellar granule neurons (16). Genetic deletion of Top2b in mice causes neural defects including aberrant axonal elongation and branching and perinatal death e...

Significance Polycomb repressive complex 2 functions in gene repression and acts by methylating histone H3 at lysine 27 (H3K27me3). Despite its relevance, it remains elusive how this complex is recruited to its target sites in the genome. Here, we used repeated genomic targeting in embryonic stem cells to identify DNA sequence determinants that autonomously confer H3K27me3 recruitment. We show that surprisingly small CG-rich DNA sequences are sufficient to recruit H3K27me3, but only if they are devoid of DNA methylation and transcriptional activity. This study provides new insights into the mechanisms recruiting H3K27me3 and the cross-talk between diverse chromatin modifications.

Post-translational modifications of proteins have emerged as a major mechanism for regulating gene expression. However, our understanding of how histone modifications directly affect chromatin function remains limited. In this study, we investigate acetylation of histone H3 at lysine 64 (H3K64ac), a previously uncharacterized acetylation on the lateral surface of the histone octamer. We show that H3K64ac regulates nucleosome stability and facilitates nucleosome eviction and hence gene expression in vivo. In line with this, we demonstrate that H3K64ac is enriched in vivo at the transcriptional start sites of active genes and it defines transcriptionally active chromatin. Moreover, we find that the p300 co-activator acetylates H3K64, and consistent with a transcriptional activation function, H3K64ac opposes its repressive counterpart H3K64me3. Our findings reveal an important role for a histone modification within the nucleosome core as a regulator of chromatin function and they demonstrate that lateral surface modifications can define functionally opposing chromatin states.DOI: http://dx.doi.org/10.7554/eLife.01632.001

DNA methylation is considered a stable epigenetic mark, yet methylation patterns can vary during differentiation and in diseases such as cancer. Local levels of DNA methylation result from opposing enzymatic activities, the rates of which remain largely unknown. Here we developed a theoretical and experimental framework enabling us to infer methylation and demethylation rates at 860,404 CpGs in mouse embryonic stem cells. We find that enzymatic rates can vary as much as two orders of magnitude between CpGs with identical steady-state DNA methylation. Unexpectedly, de novo and maintenance methylation activity is reduced at transcription factor binding sites, while methylation turnover is elevated in transcribed gene bodies. Furthermore, we show that TET activity contributes substantially more than passive demethylation to establishing low methylation levels at distal enhancers. Taken together, our work unveils a genome-scale map of methylation kinetics, revealing highly variable and context-specific activity for the DNA methylation machinery.

Histone modification, polymerase binding, mRNA half-life, and miRNA abundance measurements in mouse cells are used to dissect the relative contribution of each to mRNA levels, revealing control primarily at the level of transcription, with minor contributions from post-transcriptional processes.

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