CRISPR activity against phage and conjugative plasmid DNA molecules suggests that CRISPR systems may also prevent plasmid DNA transformation. We therefore introduced pG0(wt) and pG0(mut) nes-target and -flanking sequences (200 base pairs) in either orientation into the staphylococcal plasmid pC194 (23), generating pNes(wt) and pNes(mut), respectively (Fig. 3A). Flanking DNA was included in the inserts to ensure the presence of any sequences outside of the target that may contribute to CRISPR interference (24). Plasmids were transformed by electroporation into wildtype RP62a and isogenic Dcrispr LAM104 strains. pC194 and both pNes(mut) plasmids were transformed into both strains, whereas the pNes(wt) plasmids were transformed only into the Dcrispr mutant (Fig. 3B). We also performed pNes(wt)/ pNes(mut) mixed transformations of RP62a or LAM104 strains to test interference in an internally controlled fashion. Again, only pNes(mut) plasmids were recovered from RP62a transformants, whereas pNes(wt) and pNes(mut) plasmids were found in LAM104 transformant colonies ( fig. S4). It remains to be established whether natural transformation, which involves the uptake of a single DNA strand (25), is subject to CRISPR interference. Nonetheless, our experiments suggest that CRISPR systems can counteract multiple routes of plasmid transfer.These transformation data provide additional evidence that crRNAs target DNA molecules. First, interference occurred regardless of the insert orientation in pNes(wt); this, combined with the lack of compelling evidence for CRISPRderived double-stranded RNA ( fig. S2) (4, 6, 7), is consistent with spc1 targeting either DNA strand rather than a unidirectional transcript. Second, the target sites in the pNes(wt) and pNes(mut) plasmids are located between the transcriptional terminators of the rep and cat genes (Fig. 3A) (23,26,27). This minimizes the likelihood that this region of the plasmid is even transcribed, which is consistent with its dispensability for plasmid maintenance (23,28).Altogether, these data provide strong functional evidence that CRISPR interference acts at the DNA level and therefore differs fundamentally from the RNA interference (RNAi) phenomenon observed in eukaryotes and with which CRISPR activity was originally compared (29). A DNA targeting mechanism for CRISPR interference implies a means to prevent its action at the encoding CRISPR locus itself, as well as other potential chromosomal loci, such as prophage sequences. Little information exists to suggest how crRNAs would avoid targeting "self " DNA, although the role of flanking sequences during CRISPR interference (24) could contribute to target specificity. From a practical standpoint, the ability to direct the specific addressable destruction of DNA that contains any given 24-to 48-nucleotide target sequence could have considerable functional utility, especially if the system can function outside of its native bacterial or archaeal context. Furthermore, our results demonstrate that CRISPR function is not limited to...

Recent years have witnessed a sea change in our understanding of transcription regulation: whereas traditional models focused solely on the events that brought RNA polymerase II (Pol II) to a gene promoter to initiate RNA synthesis, emerging evidence points to the pausing of Pol II during early elongation as a widespread regulatory mechanism in higher eukaryotes. Current data indicate that pausing is particularly enriched at genes in signal-responsive pathways. Here the evidence for pausing of Pol II from recent high-throughput studies will be discussed, as well as the potential interconnected functions of promoter-proximally paused Pol II.

Despite the conventional distinction between them, promoters and enhancers share many features in mammals, including divergent transcription and similar modes of transcription factor binding. Here, we examine the architecture of transcription initiation through comprehensive mapping of transcription start sites (TSSs) in human lymphoblastoid B-cell (GM12878) and chronic myelogenous leukemic (K562) tier 1, ENCODE cell lines. Using a nuclear run-on protocol called GRO-cap, which captures TSSs for both stable and unstable transcripts, we conduct detailed comparisons of thousands of promoters and enhancers in human cells. These analyses reveal a common architecture of initiation, including tightly spaced (110 bp) divergent initiation, similar frequencies of core-promoter sequence elements, highly positioned flanking nucleosomes, and two modes of transcription factor binding. Post-initiation transcript stability provides a more fundamental distinction between promoters and enhancers than patterns of histone modifications, transcription factors or co-activators. These results support a unified model of transcription initiation at promoters and enhancers.

Recent advances in sequencing techniques that measure nascent transcripts and that reveal the positioning of RNA polymerase II (Pol II) have shown that the pausing of Pol II in promoter-proximal regions and its release to initiate a phase of productive elongation are key steps in transcription regulation. Moreover, after the release of Pol II from the promoter-proximal region, elongation rates are highly dynamic throughout the transcription of a gene, and vary on a gene-by-gene basis. Interestingly, Pol II elongation rates affect co-transcriptional processes such as splicing, termination and genome stability. Increasing numbers of factors and regulatory mechanisms have been associated with the steps of transcription elongation by Pol II, revealing that elongation is a highly complex process. Elongation is thus now recognized as a key phase in the regulation of transcription by Pol II.

Transcription regulation occurs frequently through promoter-associated pausing of RNA polymerase II (Pol II). We developed a Precision nuclear Run-On and sequencing assay (PRO-seq) to map the genome-wide distribution of transcriptionally-engaged Pol II at base-pair resolution. Pol II accumulates immediately downstream of promoters, at intron-exon junctions that are efficiently used for splicing, and over 3' poly-adenylation sites. Focused analyses of promoters reveal that pausing is not fixed relative to initiation sites nor is it specified directly by the position of a particular core promoter element or the first nucleosome. Core promoter elements function beyond initiation, and when optimally positioned they act collectively to dictate the position and strength of pausing. We test this ‘Complex Interaction’ model with insertional mutagenesis of the Drosophila Hsp70 core promoter.

Production of mRNA depends critically on the rate of RNA polymerase II (Pol II) elongation. To dissect Pol II dynamics in mouse ES cells, we inhibited Pol II transcription at either initiation or promoter-proximal pause escape with Triptolide or Flavopiridol, and tracked Pol II kinetically using GRO-seq. Both inhibitors block transcription of more than 95% of genes, showing that pause escape, like initiation, is a ubiquitous and crucial step within the transcription cycle. Moreover, paused Pol II is relatively stable, as evidenced from half-life measurements at ∼3200 genes. Finally, tracking the progression of Pol II after drug treatment establishes Pol II elongation rates at over 1000 genes. Notably, Pol II accelerates dramatically while transcribing through genes, but slows at exons. Furthermore, intergenic variance in elongation rates is substantial, and is influenced by a positive effect of H3K79me2 and negative effects of exon density and CG content within genes.DOI: http://dx.doi.org/10.7554/eLife.02407.001

PARP-1 is the most abundantly expressed member of a family of proteins that catalyze the transfer of ADP-ribose units from NAD+ to target proteins. Herein, we describe previously uncharacterized nucleosome binding properties of PARP-1 that promote the formation of compact, transcriptionally repressed chromatin structures. PARP-1 binds in a specific manner to nucleosomes and modulates chromatin structure through NAD+-dependent automodification, without modifying core histones or promoting the disassembly of nucleosomes. The automodification activity of PARP-1 is potently stimulated by nucleosomes, causing the release of PARP-1 from chromatin. The NAD+-dependent activities of PARP-1 are reversed by PARG, a poly(ADP-ribose) glycohydrolase, and are inhibited by ATP. In vivo, PARP-1 incorporation is associated with transcriptionally repressed chromatin domains that are spatially distinct from both histone H1-repressed domains and actively transcribed regions. Thus, PARP-1 functions both as a structural component of chromatin and a modulator of chromatin structure through its intrinsic enzymatic activity.

We have purified a human histone H4 lysine 20 methyltransferase and cloned the encoding gene, PR/SET07. A mutation in Drosophila pr-set7 is lethal: second instar larval death coincides with the loss of H4 lysine 20 methylation, indicating a fundamental role for PR-Set7 in development. Transcriptionally competent regions lack H4 lysine 20 methylation, but the modification coincided with condensed chromosomal regions on polytene chromosomes, including chromocenter and euchromatic arms. The Drosophila male X chromosome, which is hyperacetylated at H4 lysine 16, has significantly decreased levels of lysine 20 methylation compared to that of females. In vitro, methylation of lysine 20 and acetylation of lysine 16 on the H4 tail are competitive. Taken together, these results support the hypothesis that methylation of H4 lysine 20 maintains silent chromatin, in part, by precluding neighboring acetylation on the H4 tail.