Dephosphorylating RNA polymerase II: Transcription in metazoans requires coordination of multiple factors to control the progression of polymerases and the integrity of their RNA products. Zheng et al. identified a new dual-enzyme complex called INTAC, which is composed of protein phosphatase 2A (PP2A) core enzyme and the multisubunit RNA endonuclease Integrator. Structural and functional studies show that INTAC functions as a noncanonical PP2A holoenzyme that dephosphorylates the C-terminal domain of RNA polymerase II to attenuate transcription. This study provides a direct connection between PP2A-mediated dephosphorylation and transcriptional regulation, two fundamental cellular processes. Science , this issue p. eabb5872

UHRF1 (Ubiquitin-like, with PHD and RING finger domains 1) plays an important role in DNA CpG methylation, heterochromatin function and gene expression. Overexpression of UHRF1 has been suggested to contribute to tumorigenesis. However, regulation of UHRF1 is largely unknown. Here we show that the deubiquitylase USP7 interacts with UHRF1. Using interaction-defective and catalytic mutants of USP7 for complementation experiments, we demonstrate that both physical interaction and catalytic activity of USP7 are necessary for UHRF1 ubiquitylation and stability regulation. Mass spectrometry analysis identified phosphorylation of serine (S) 652 within the USP7-interacting domain of UHRF1, which was further confirmed by a UHRF1 S652 phosphor (S652ph)-specific antibody. Importantly, the S652ph antibody identifies phosphorylated UHRF1 in mitotic cells and consistently S652 can be phosphorylated by the M phase-specific kinase CDK1-cyclin B in vitro. UHRF1 S652 phosphorylation significantly reduces UHRF1 interaction with USP7 in vitro and in vivo, which is correlated with a decreased UHRF1 stability in the M phase of the cell cycle. In contrast, UHRF1 carrying the S652A mutation, which renders UHRF1 resistant to phosphorylation at S652, is more stable. Importantly, cells carrying the S652A mutant grow more slowly suggesting that maintaining an appropriate level of UHRF1 is important for cell proliferation regulation. Taken together, our findings uncovered a cell cycle-specific signaling event that relieves UHRF1 from its interaction with USP7, thus exposing UHRF1 to proteasome-mediated degradation. These findings identify a molecular mechanism by which cellular UHRF1 level is regulated, which may impact cell proliferation.

Nuclei of eukaryotes contain various higher-order chromatin architectures and nuclear bodies (NBs), which are critical for proper nuclear functions. Recent studies showed that active chromatin regions are associated with nuclear speckles (NSs), a type of NBs involved in RNA processing. However, the functional roles of NSs in 3D genome organization remain unclear. Using mouse hepatocytes as the model, we knocked down SRRM2, a core protein component scaffolding NSs, and performed Hi-C experiments to examine genome-wide chromatin interactions. We found that Srrm2 depletion disrupted the NSs and changed the expression of 1282 genes. The intra-chromosomal interactions were decreased in type A (active) compartments and increased in type B (repressive) compartments. Furthermore, upon Srrm2 knockdown, the insulation of TADs was decreased specifically in active compartments, and the most significant reduction occurred in A1 sub-compartments. Interestingly, the change of intra-TAD chromatin interactions upon Srrm2 depletion was not associated with the alteration of gene expression. We show that disruption of NSs by Srrm2 knockdown causes a global decrease in chromatin interactions in active compartments, indicating critical functions of NSs in the organization of the 3D genome.

Pleiotropic transcription regulator RNA polymerase II (Pol II)–associated factor 1 (PAF1) governs multiple transcriptional steps and the deposition of several epigenetic marks. However, it remains unclear how ultimate transcriptional outcome is determined by PAF1 and whether it relates to PAF1-controlled epigenetic marks. We use rapid degradation systems and reveal direct PAF1 functions in governing pausing partially by recruiting Integrator-PP2A (INTAC), in addition to ensuring elongation. Following acute PAF1 degradation, most destabilized polymerase undergoes effective release, which presumably relies on skewed balance between INTAC and P-TEFb, resulting in hyperphosphorylated substrates including SPT5. Impaired Pol II progression during elongation, along with altered pause release frequency, determines the final transcriptional outputs. Moreover, PAF1 degradation causes a cumulative decline in histone modifications. These epigenetic alterations in chromatin likely further influence the production of transcripts from PAF1 target genes.

While several inhibitors targeting RNA polymerase II (Pol II) kinases have been applied for inhibiting RNA Pol II phosphorylation, there are few approaches for inducing RNA Pol II hyperphosphorylation. Here, we present a protocol for constructing the INTS8 degradation tag (dTAG) system combined with ectopic expression of N-terminally truncated INTS8 (INTS8-ΔN) in DLD-1 cells. We describe steps for INTS8-dTAG cell line construction, validation of knockin and degradation, and INTS8-ΔN rescue. We then detail validation of RNA Pol II phosphorylation upregulation. For complete details on the use and execution of this protocol, please refer to Hu et al. (2023).¹.