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The conserved yeast E3 ubiquitin ligase Bre1 and its partner, the E2 ubiquitin-conjugating enzyme Rad6, monoubiquitinate histone H2B across gene bodies during the transcription cycle.sup.1. Although processive ubiquitination might--in principle--arise from Bre1 and Rad6 travelling with RNA polymerase II.sup.2, the mechanism of H2B ubiquitination across genic nucleosomes remains unclear. Here we implicate liquid-liquid phase separation.sup.3 as the underlying mechanism. Biochemical reconstitution shows that Bre1 binds the scaffold protein Lge1, which possesses an intrinsically disordered region that phase-separates via multivalent interactions. The resulting condensates comprise a core of Lge1 encapsulated by an outer catalytic shell of Bre1. This layered liquid recruits Rad6 and the nucleosomal substrate, which accelerates the ubiquitination of H2B. In vivo, the condensate-forming region of Lge1 is required to ubiquitinate H2B in gene bodies beyond the +1 nucleosome. Our data suggest that layered condensates of histone-modifying enzymes generate chromatin-associated 'reaction chambers', with augmented catalytic activity along gene bodies. Equivalent processes may occur in human cells, and cause neurological disease when impaired. The yeast E3 ligase Bre1 forms a core-shell condensate with the scaffold protein Lge1, implicating liquid-liquid phase separation as a mechanism in the ubiquitination of histone H2B along gene bodies., Author(s): Laura D. Gallego [sup.1] , Maren Schneider [sup.1] , Chitvan Mittal [sup.2] , Anete Romanauska [sup.1] , Ricardo M. Gudino Carrillo [sup.1] , Tobias Schubert [sup.1] , B. Franklin [...]

ATP-dependent chromatin remodellers allow access to DNA for transcription factors and the general transcription machinery, but whether mammalian chromatin remodellers (1-3) target specific nucleosomes to regulate transcription is unclear. Here [...]

The human genome is pervasively transcribed, yet only a small fraction is coding. Here we address whether this non-coding transcription arises at promoters, and detail the interactions of initiation factors TATA box binding protein (TBP), transcription factor IIB (TFIIB) and RNA polymerase (Pol) II. Using ChIP-exo (chromatin immunoprecipitation with lambda exonuclease digestion followed by high-throughput sequencing), we identify approximately 160,000 transcription initiation complexes across the human K562 genome, and more in other cancer genomes. Only about 5% associate with messenger RNA genes. The remainder associates with non-polyadenylated non-coding transcription. Regardless, Pol II moves into a transcriptionally paused state, and TBP and TFIIB remain at the promoter. Remarkably, the vast majority of locations contain the four core promoter elements-- upstream TFIIB recognition element (BRE.sub.u), TATA, downstream TFIIB recognition element (BRE.sub.d), and initiator element (INR)--in constrained positions. All but the INR also reside at Pol III promoters, where TBP makes similar contacts. This comprehensive and high-resolution genome-wide detection of the initiation machinery produces a consolidated view of transcription initiation events from yeast to humans at Pol II/III TATA-containing/TATA-less coding and non-coding genes. The ChIP-exo technique is used to map the organization of transcription initiation complexes across the human genome at near-base-pair resolution; most of the transcription initiation complexes give rise to non-coding, non-polyadenylated RNA, indicating that pervasive non-coding transcription arise from specific promoters and is regulated. Promoter recognition In this study Bryan Venters and Frank Pugh use the ChIP-exo technique to map the organization of transcription initiation complexes across the human genome at near-base-pair resolution in four ENCODE human cancer cell lines. The resulting data reveal the in vivo organization of more than 160,000 transcription initiation complexes and provide insights into the role of core promoter motifs such as TATA boxes, the production of divergent transcripts, and the transition of RNA polymerase II from transcription initiation to a paused state. Strikingly, most of the transcription initiation complexes give rise to non-coding, non-polyadenylated RNA, indicating that pervasive non-coding transcription arises from specific promoters and is regulated., Author(s): Bryan J. Venters [sup.1] [sup.2] , B. Franklin Pugh [sup.1] Author Affiliations: (1) Department of Biochemistry and Molecular Biology, Center for Eukaryotic Gene Regulation, The Pennsylvania State University, University [...]

Transcription and regulation of genes originate from transcription pre- initiation complexes (PICs). Their structural and positional organization across eukaryotic genomes is unknown. Here we applied lambda exonuclease to chromatin immunoprecipitates (termed ChIP-exo) to examine the precise location of 6,045 PICs in Saccharomyces. PICs, including RNA polymerase II and protein complexes TFIIA, TFIIB, TFIID (or TBP), TFIIE, TFIIF, TFIIH and TFIIK were positioned within promoters and excluded from coding regions. Exonuclease patterns were in agreement with crystallographic models of the PIC, and were sufficiently precise to identify TATA-like elements at so-called TATA-less promoters. These PICs and their transcription start sites were positionally constrained at TFIID-engaged downstream +1 nucleosomes. At TATA-box-containing promoters, which are depleted of TFIID, a +1 nucleosome was positioned to be in competition with the PIC, which may allow greater latitude in start-site selection. Our genomic localization of messenger RNA and non-coding RNA PICs reveals that two PICs, in inverted orientation, may occupy the flanking borders of nucleosome-free regions. Their unambiguous detection may help distinguish bona fide genes from transcriptional noise., Assembly of the PIC and its post-assembly control are critical early steps in the transcription of eukaryotic genes. TBP (TATA-binding protein) arrives at most promoters as part of the multi-subunit [...]

Comparative genomics of nucleosome positions provides a powerful means for understanding how the organization of chromatin and the transcription machinery co-evolve. Here we produce a high-resolution reference map of H2A.Z [...]

Author(s): Istvan Albert [1]; Travis N. Mavrich [1, 2]; Lynn P. Tomsho [1]; Ji Qi [1]; Sara J. Zanton [1, 2]; Stephan C. Schuster [1]; B. Franklin Pugh (corresponding author) [...]

Functional Domains and Upstream Activation Properties of Cloned Human TATA Binding Protein INITIATION OF TRANSCRIPTION OF EUKARYOTIC PROTEIN-coding genes is a complex process requiring RNA polymerase II (Pol II) as [...]