PRO-seq: Precise Mapping of Engaged RNA Pol II at Single-Nucleotide Resolution.

Gene regulation is dependent on the production of mRNAs and a repertoire of non-coding RNAs by RNA polymerase II (RNAPII). Precision run-on sequencing (PRO-seq) maps the position of engaged RNAPII complexes at single-nucleotide resolution and can reveal direct targets of regulation, locations of enhancers, and transcription mechanisms that are difficult or impossible to measure by analysis of total cellular RNA. Briefly, this method first involves permeabilizing cells with mild detergents to remove intracellular NTPs and halt transcription. Transcription is then resumed in the presence of biotin-NTPs and sarkosyl to allow transcriptional incorporation of a single biotinylated NTP by RNAPII. The biotin moiety is then bound to streptavidin beads to stringently enrich for nascent RNAs. Sequencing libraries are then generated such that the first base read corresponds to the 3' end of the nascent transcript. Here, we describe our current protocol for generating PRO-seq libraries from metazoan cells, including adaptations of previously published protocols to incorporate unique molecular identifiers, reduce ligation bias, and improve library yields. Additional commentary describes quality control and processing of PRO-seq data and references for more advanced downstream analysis such as gene and enhancer identification. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Cell permeabilization for PRO-seq Basic Protocol 2: Construction of PRO-seq libraries Support Protocol: Adenylation of 3' adapter.

Global identification of SWI/SNF targets reveals compensation by EP400.

Mammalian SWI/SNF chromatin remodeling complexes move and evict nucleosomes at gene promoters and enhancers to modulate DNA access. Although SWI/SNF subunits are commonly mutated in disease, therapeutic options are limited by our inability to predict SWI/SNF gene targets and conflicting studies on functional significance. Here, we leverage a fast-acting inhibitor of SWI/SNF remodeling to elucidate direct targets and effects of SWI/SNF. Blocking SWI/SNF activity causes a rapid and global loss of chromatin accessibility and transcription. Whereas repression persists at most enhancers, we uncover a compensatory role for the EP400/TIP60 remodeler, which reestablishes accessibility at most promoters during prolonged loss of SWI/SNF. Indeed, we observe synthetic lethality between EP400 and SWI/SNF in cancer cell lines and human cancer patient data. Our data define a set of molecular genomic features that accurately predict gene sensitivity to SWI/SNF inhibition in diverse cancer cell lines, thereby improving the therapeutic potential of SWI/SNF inhibitors.

U1 snRNP increases RNA Pol II elongation rate to enable synthesis of long genes.

The expansion of introns within mammalian genomes poses a challenge for the production of full-length messenger RNAs (mRNAs), with increasing evidence that these long AT-rich sequences present obstacles to transcription. Here, we investigate RNA polymerase II (RNAPII) elongation at high resolution in mammalian cells and demonstrate that RNAPII transcribes faster across introns. Moreover, we find that this acceleration requires the association of U1 snRNP (U1) with the elongation complex at 5' splice sites. The role of U1 to stimulate elongation rate through introns reduces the frequency of both premature termination and transcriptional arrest, thereby dramatically increasing RNA production. We further show that changes in RNAPII elongation rate due to AT content and U1 binding explain previous reports of pausing or termination at splice junctions and the edge of CpG islands. We propose that U1-mediated acceleration of elongation has evolved to mitigate the risks that long AT-rich introns pose to transcript completion.

Integrator endonuclease drives promoter-proximal termination at all RNA polymerase II-transcribed loci.

RNA polymerase II (RNAPII) pausing in early elongation is critical for gene regulation. Paused RNAPII can be released into productive elongation by the kinase P-TEFb or targeted for premature termination by the Integrator complex. Integrator comprises endonuclease and phosphatase activities, driving termination by cleavage of nascent RNA and removal of stimulatory phosphorylation. We generated a degron system for rapid Integrator endonuclease (INTS11) depletion to probe the direct consequences of Integrator-mediated RNA cleavage. Degradation of INTS11 elicits nearly universal increases in active early elongation complexes. However, these RNAPII complexes fail to achieve optimal elongation rates and exhibit persistent Integrator phosphatase activity. Thus, only short transcripts are significantly upregulated following INTS11 loss, including transcription factors, signaling regulators, and non-coding RNAs. We propose a uniform molecular function for INTS11 across all RNAPII-transcribed loci, with differential effects on particular genes, pathways, or RNA biotypes reflective of transcript lengths rather than specificity of Integrator activity.

Screening thousands of transcribed coding and non-coding regions reveals sequence determinants of RNA polymerase II elongation potential.

Precise regulation of transcription by RNA polymerase II (RNAPII) is critical for organismal growth and development. However, what determines whether an engaged RNAPII will synthesize a full-length transcript or terminate prematurely is poorly understood. Notably, RNAPII is far more susceptible to termination when transcribing non-coding RNAs than when synthesizing protein-coding mRNAs, but the mechanisms underlying this are unclear. To investigate the impact of transcribed sequence on elongation potential, we developed a method to screen the effects of thousands of INtegrated Sequences on Expression of RNA and Translation using high-throughput sequencing (INSERT-seq). We found that higher AT content in non-coding RNAs, rather than specific sequence motifs, drives RNAPII termination. Further, we demonstrate that 5' splice sites autonomously stimulate processive transcription, even in the absence of polyadenylation signals. Our results reveal a potent role for the transcribed sequence in dictating gene output and demonstrate the power of INSERT-seq toward illuminating these contributions.

Co-transcriptional splicing regulates 3' end cleavage during mammalian erythropoiesis.

Pre-mRNA processing steps are tightly coordinated with transcription in many organisms. To determine how co-transcriptional splicing is integrated with transcription elongation and 3' end formation in mammalian cells, we performed long-read sequencing of individual nascent RNAs and precision run-on sequencing (PRO-seq) during mouse erythropoiesis. Splicing was not accompanied by transcriptional pausing and was detected when RNA polymerase II (Pol II) was within 75-300 nucleotides of 3' splice sites (3'SSs), often during transcription of the downstream exon. Interestingly, several hundred introns displayed abundant splicing intermediates, suggesting that splicing delays can take place between the two catalytic steps. Overall, splicing efficiencies were correlated among introns within the same transcript, and intron retention was associated with inefficient 3' end cleavage. Remarkably, a thalassemia patient-derived mutation introducing a cryptic 3'SS improved both splicing and 3' end cleavage of individual ß-globin transcripts, demonstrating functional coupling between the two co-transcriptional processes as a determinant of productive gene output.

methyl-ATAC-seq measures DNA methylation at accessible chromatin.

Chromatin features are characterized by genome-wide assays for nucleosome location, protein binding sites, three-dimensional interactions, and modifications to histones and DNA. For example, assay for transposase accessible chromatin sequencing (ATAC-seq) identifies nucleosome-depleted (open) chromatin, which harbors potentially active gene regulatory sequences; and bisulfite sequencing (BS-seq) quantifies DNA methylation. When two distinct chromatin features like these are assayed separately in populations of cells, it is impossible to determine, with certainty, where the features are coincident in the genome by simply overlaying data sets. Here, we describe methyl-ATAC-seq (mATAC-seq), which implements modifications to ATAC-seq, including subjecting the output to BS-seq. Merging these assays into a single protocol identifies the locations of open chromatin and reveals, unambiguously, the DNA methylation state of the underlying DNA. Such combinatorial methods eliminate the need to perform assays independently and infer where features are coincident.

TRAIP is a master regulator of DNA interstrand crosslink repair.

Cells often use multiple pathways to repair the same DNA lesion, and the choice of pathway has substantial implications for the fidelity of genome maintenance. DNA interstrand crosslinks covalently link the two strands of DNA, and thereby block replication and transcription; the cytotoxicity of these crosslinks is exploited for chemotherapy. In Xenopus egg extracts, the collision of replication forks with interstrand crosslinks initiates two distinct repair pathways. NEIL3 glycosylase can cleave the crosslink1; however, if this fails, Fanconi anaemia proteins incise the phosphodiester backbone that surrounds the interstrand crosslink, generating a double-strand-break intermediate that is repaired by homologous recombination2. It is not known how the simpler NEIL3 pathway is prioritized over the Fanconi anaemia pathway, which can cause genomic rearrangements. Here we show that the E3 ubiquitin ligase TRAIP is required for both pathways. When two replisomes converge at an interstrand crosslink, TRAIP ubiquitylates the replicative DNA helicase CMG (the complex of CDC45, MCM2-7 and GINS). Short ubiquitin chains recruit NEIL3 through direct binding, whereas longer chains are required for the unloading of CMG by the p97 ATPase, which enables the Fanconi anaemia pathway. Thus, TRAIP controls the choice between the two known pathways of replication-coupled interstrand-crosslink repair. These results, together with our other recent findings3,4 establish TRAIP as a master regulator of CMG unloading and the response of the replisome to obstacles.