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.

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.

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.