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...

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.

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We provide a protocol for precision nuclear run-on sequencing (PRO-seq) and its variant, PRO-cap, which map the location of active RNA polymerases (PRO-seq) or transcription start sites (TSSs) (PRO-cap) genome-wide at high resolution. The density of RNA polymerases at a particular genomic locus directly reflects the level of nascent transcription at that region. Nuclei are isolated from cells and, under nuclear run-on conditions, transcriptionally engaged RNA polymerases incorporate one or, at most, a few biotin-labeled nucleotide triphosphates (biotin-NTPs) into the 3′ end of nascent RNA. The biotin-labeled nascent RNA is used to prepare sequencing libraries, which are sequenced from the 3′ end to provide high-resolution positional information for the RNA polymerases. PRO-seq provides much higher sensitivity than ChIP-seq, and it generates a much larger fraction of usable sequence reads than ChIP-seq or NET-seq (native elongating transcript sequencing). Similarly to NET-seq, PRO-seq maps the RNA polymerase at up to base-pair resolution with strand specificity, but unlike NET-seq it does not require immunoprecipitation. With the protocol provided here, PRO-seq (or PRO-cap) libraries for high-throughput sequencing can be generated in 4–5 working days. The method has been applied to human, mouse, Drosophila melanogaster and Caenorhabditis elegans cells and, with slight modifications, to yeast.

Precise spatio-temporal control of gene activity is essential for organismal development, growth, and survival in a changing environment. Decisive steps in gene regulation involve the pausing of RNA polymerase II (Pol II) in early elongation, and the controlled release of paused polymerase into productive RNA synthesis. Here we describe the factors that enable pausing and the events that trigger Pol II release into the gene. We also discuss open questions in the field concerning the stability of paused Pol II, nucleosomes as obstacles to elongation, and potential roles of pausing in defining the precision and dynamics of gene expression.Cell type-and condition-specific patterns of gene expression involve tight control of multiple steps in the transcription cycle. Early regulatory events govern the association of DNA-binding transcription factors (TFs) with their target motifs and the recruitment of RNA polymerase II (Pol II) and general TFs (GTFs) to the promoter region of a gene. These events enable transcription initiation and the synthesis of a short, nascent RNA. However, Pol II then pauses promoter-proximally, awaiting further signals before entering the gene body. The fate of the paused early elongation complex is absolutely decisive for gene output. If paused Pol II successfully transitions into productive elongation, a functional full-length mRNA can be made. However, a failure in the maturation of paused Pol II toward productive RNA synthesis short circuits the process of gene expression. Thus, understanding the factors that govern stable Pol II pausing and release are of paramount importance toward an appreciation of gene regulation in human health and disease.

Summary We report the immediate effects of estrogen signaling on the transcriptome of breast cancer cells using Global Run-On and sequencing (GRO-seq). The data were analyzed using a new bioinformatic approach that allowed us to identify transcripts directly from the GRO-seq data. We found that estrogen signaling directly regulates a strikingly large fraction of the transcriptome in a rapid, robust, and unexpectedly transient manner. In addition to protein coding genes, estrogen regulates the distribution and activity of all three RNA polymerases, and virtually every class of non-coding RNA that has been described to date. We also identified a large number of previously undetected estrogen-regulated intergenic transcripts, many of which are found proximal to estrogen receptor binding sites. Collectively, our results provide the most comprehensive measurement of the primary and immediate estrogen effects to date and a resource for understanding rapid signal-dependent transcription in other systems.

Summary RNA polymerase II (Pol II) transcribes hundreds of kilobases of DNA, limiting the production of mRNAs and lncRNAs. We used Global Run-on Sequencing (GRO-seq) to measure the rates of transcription by Pol II following gene activation. Elongation rates vary as much as 4-fold at different genomic loci and in response to two distinct cellular signaling pathways [i.e., 17β-estradiol (E2) and TNFα]. The rates are slowest near the promoter and increase during the first ~15 kb transcribed. Gene body elongation rates correlate with Pol II density, resulting in systematically higher rates of transcript production at genes with higher Pol II density. Pol II dynamics following short inductions indicate that E2 stimulates gene expression by increasing Pol II initiation, whereas TNFα reduces Pol II residence time at pause sites. Collectively, our results identify previously uncharacterized variation in the rate of transcription and highlight elongation as an important, variable, and regulated rate-limiting step during transcription.