SUMMARY The essential helicase-like protein Sen1 mediates termination of RNA Polymerase II (Pol II) transcription at snoRNAs and other non-coding RNAs in yeast. A mutation in the Pol II subunit Rpb1 that increases the elongation rate increases readthrough transcription at Sen1-mediated terminators. Termination and growth defects in sen1 mutant cells are partially suppressed by a slowly transcribing Pol II mutant and are exacerbated by a faster transcribing Pol II mutant. Deletion of the nuclear exosome subunit Rrp6 allows visualization of non-coding RNA intermediates that are terminated but not yet processed. Sen1 mutants or faster transcribing Pol II increase the average lengths of pre-processed snoRNA, CUT, and SUT transcripts, while slowed Pol II transcription produces shorter transcripts. These connections between transcription rate and Sen1 activity support a model whereby kinetic competition between elongating Pol II and Sen1 helicase establishes the temporal and spatial window for early Pol II termination.

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CRISPR-Cas9-mediated gene interference (CRISPRi) and activation (CRISPRa) approaches hold promise for functional gene studies and genome-wide screens in human pluripotent stem cells (hPSCs). However, in contrast to CRISPR-Cas9 nuclease approaches, the efficiency of CRISPRi/a depends on continued expression of the dead Cas9 (dCas9) effector and guide RNA (gRNA), which can vary substantially depending on transgene design and delivery. Here, we design and generate new fluorescently labeled piggyBac (PB) vectors to deliver uniform and sustained expression of multiplexed gRNAs. In addition, we generate hPSC lines harboring AAVS1-integrated, inducible and fluorescent dCas9-KRAB and dCas9-VPR transgenes to allow for accurate quantification and tracking of cells that express both the dCas9 effectors and gRNAs. We then employ these systems to target the TCF4 gene in hPSCs and assess expression levels of the dCas9 effectors, individual gRNAs and targeted gene. We also assess the performance of our PB system for single gRNA delivery, confirming its utility for library format applications. collectively, our results provide proof-of-principle application of a stable, multiplexed pB gRnA delivery system that can be widely exploited to further enable genome engineering studies in hPSCs. Paired with diverse CRISPR tools including our dual fluorescence CRISPRi/a cell lines, this system can facilitate functional dissection of individual genes and pathways as well as larger-scale screens for studies of development and disease.

Summary Human pluripotent stem cells (hPSCs) have proven to be valuable tools for both drug discovery and the development of cell-based therapies. However, the long non-coding RNA XIST , which is essential for the establishment and maintenance of X chromosome inactivation, is repressed during culture, thereby causing erosion of dosage compensation in female hPSCs. Here, we report that the de novo DNA methyltransferases DNMT3A/3B are necessary for XIST repression in female hPSCs. We found that the deletion of both genes, but not the individual genes, inhibited XIST silencing, maintained the heterochromatin mark of H3K27me3, and did not cause global overdosage in X-linked genes. Meanwhile, DNMT3A/3B deletion after XIST repression failed to restore X chromosome inactivation. Our findings revealed that de novo DNA methyltransferases are primary factors responsible for initiating erosion of dosage compensation in female hPSCs, and XIST silencing is stably maintained in a de novo DNA-methylation-independent manner.

SUMMARY The C-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II (RNApII), coordinates recruitment of RNA- and chromatin-modifying factors to transcription complexes. It is unclear whether the CTD communicates with the catalytic core region of Rpb1 and thus must be physically connected, or instead can function as an independent domain. To address this question, CTD was transferred to other RNApII subunits. Fusions to Rpb4 or Rpb6, two RNApII subunits located near the original position of CTD, support viability in a strain carrying a truncated Rpb1. In contrast, CTD fusion to Rpb9 on the other side of RNApII does not. Rpb4-CTD and Rpb6-CTD proteins are functional for phosphorylation and recruitment of various factors, albeit with some restrictions and minor defects. Normal CTD functions are not transferred to RNApI or RNApIII by Rbp6-CTD. These results show that, with some spatial constraints, CTD can function even when disconnected from Rpb1.