Summary Mechanistic roles for many lncRNAs are poorly understood in part because their direct interactions with genomic loci and proteins are difficult to assess. Using a method to purify endogenous RNAs and their associated factors, we mapped the genomic binding sites for two highly expressed human lncRNAs, NEAT1 and MALAT1. We show that NEAT1 and MALAT1 localize to hundreds of genomic sites in human cells, primarily over active genes. NEAT1 and MALAT1 exhibit colocalization to many of these loci, but display distinct gene body binding patterns at these sites, suggesting independent but complementary functions for these RNAs. We also identified numerous proteins enriched by both lncRNAs, supporting complementary binding and function, in addition to unique associated proteins. Transcriptional inhibition or stimulation alters localization of NEAT1 on active chromatin sites, implying that underlying DNA sequence does not target NEAT1 to chromatin and that localization responds to cues involved in the transcription process.

Mammalian development requires effective mechanisms to repress genes whose expression would generate inappropriately specified cells. The Polycomb-repressive complex 1 (PRC1) family complexes are central to maintaining this repression. These include a set of canonical PRC1 complexes, each of which contains four core proteins, including one from the CBX family. These complexes have been shown previously to reside in membraneless organelles called Polycomb bodies, leading to speculation that canonical PRC1 might be found in a separate phase from the rest of the nucleus. We show here that reconstituted PRC1 readily phase-separates into droplets in vitro at low concentrations and physiological salt conditions. This behavior is driven by the CBX2 subunit. Point mutations in an internal domain of Cbx2 eliminate phase separation. These same point mutations eliminate the formation of puncta in cells and have been shown previously to eliminate nucleosome compaction in vitro and generate axial patterning defects in mice. Thus, the domain of CBX2 that is important for phase separation is the same domain shown previously to be important for chromatin compaction and proper development, raising the possibility of a mechanistic or evolutionary link between these activities.

Spt6 is a highly conserved histone chaperone that interacts directly with both RNA polymerase II and histones to regulate gene expression. To gain a comprehensive understanding of the roles of Spt6, we performed genome-wide analyses of transcription, chromatin structure, and histone modifications in a Schizosaccharomyces pombe spt6 mutant. Our results demonstrate dramatic changes to transcription and chromatin structure in the mutant, including elevated antisense transcripts at >70% of all genes and general loss of the ؉1 nucleosome. Furthermore, Spt6 is required for marks associated with active transcription, including trimethylation of histone H3 on lysine 4, previously observed in humans but not Saccharomyces cerevisiae, and lysine 36. Taken together, our results indicate that Spt6 is critical for the accuracy of transcription and the integrity of chromatin, likely via its direct interactions with RNA polymerase II and histones. Studies over the last few years have revealed that transcription across eukaryotic genomes is much more widespread and complex than previously believed (1). Although it was once thought that transcription occurs primarily across protein-coding regions, genome-wide studies have now shown that transcription is also prevalent in intergenic regions and on antisense strands, in organisms ranging from yeast to humans (2, 3). Although roles for a small amount of this transcription have been established, for most, we have little understanding of its biological functions. Furthermore, while some factors have been shown to control the level of noncoding and antisense transcripts, many questions remain regarding the regulation of their synthesis and stability.One factor that plays a prominent role in the genome-wide control of transcription is Spt6. Originally identified in Saccharomyces cerevisiae (4, 5), Spt6 is conserved throughout eukaryotes and also has homology to the prokaryotic activator Tex (6). Spt6 interacts directly with several important factors, including RNA polymerase II (RNAPII) (7-11), histones (12, 13), and the transcription factor Iws1/Spn1 (7,14,15), suggesting that it is multifunctional. Recent studies in mammalian cells show that Spt6 also interacts directly with other chromatin related factors, including H3K27 demethylases (16, 17). Several gene-specific studies have demonstrated roles for Spt6 in transcription initiation (18)(19)(20), elongation (21, 22), and termination (23, 24). In addition, Spt6 is required for H3K36 methylation (25-28) and regulates nucleosome positioning and occupancy, particularly over highly expressed genes (12,19,29). Finally, Spt6 can assemble nucleosomes in vitro in an ATP-independent fashion (12). These results suggest that Spt6 acts as a histone chaperone by restoring nucleosomes in the wake of RNAPII transcription (30,31).In vivo, Spt6 is critical for normal growth and transcription. It is either essential or nearly essential for viability in all organisms tested, and viable spt6 mutations cause severe defects. In S. cerevisiae spt6 mutants,...

The conserved eukaryotic Paf1 complex regulates RNA synthesis by RNA polymerase II at multiple levels, including transcript elongation, transcript termination, and chromatin modifications. To better understand the contributions of the Paf1 complex to transcriptional regulation, we generated mutations that alter conserved residues within the Rtf1 subunit of the Saccharomyces cerevisiae Paf1 complex. Importantly, single amino acid substitutions within a region of Rtf1 that is conserved from yeast to humans, which we termed the histone modification domain, resulted in the loss of histone H2B ubiquitylation and impaired histone H3 methylation. Phenotypic analysis of these mutations revealed additional defects in telomeric silencing, transcription elongation, and prevention of cryptic initiation. We also demonstrated that amino acid substitutions within the Rtf1 histone modification domain disrupt 39-end formation of snoRNA transcripts and identify a previously uncharacterized regulatory role for the histone H2B K123 ubiquitylation mark in this process. Cumulatively, our results reveal functionally important residues in Rtf1, better define the roles of Rtf1 in transcription and histone modification, and provide strong genetic support for the participation of histone modification marks in the termination of noncoding RNAs.

Histone modifications regulate transcription by RNA polymerase II and maintain a balance between active and repressed chromatin states. The conserved Paf1 complex (Paf1C) promotes specific histone modifications during transcription elongation, but the mechanisms by which it facilitates these marks are undefined. We previously identified a 90-amino acid region within the Rtf1 subunit of Paf1C that is necessary for Paf1C-dependent histone modifications in Saccharomyces cerevisiae. Here we show that this histone modification domain (HMD), when expressed as the only source of Rtf1, can promote H3 K4 and K79 methylation and H2B K123 ubiquitylation in yeast. The HMD can restore histone modifications in rtf1Δ cells whether or not it is directed to DNA by a fusion to a DNA binding domain. The HMD can facilitate histone modifications independently of other Paf1C subunits and does not bypass the requirement for Rad6-Bre1. The isolated HMD localizes to chromatin, and this interaction requires residues important for histone modification. When expressed outside the context of fulllength Rtf1, the HMD associates with and causes Paf1C-dependent histone modifications to appear at transcriptionally inactive loci, suggesting that its function has become deregulated. Finally, the Rtf1 HMDs from other species can function in yeast. Our findings suggest a direct and conserved role for Paf1C in coupling histone modifications to transcription elongation.transcription-coupled histone modifications | nucleosome I n eukaryotes, transcription occurs within the context of a restrictive, yet dynamic, chromatin environment. The posttranslational modification of histones represents a major mechanism by which cells control the structure of chromatin. Some modifications of histones include acetylation, methylation, and ubiquitylation. These modifications can alter the structural properties of nucleosomes and serve as specific effectors for the recruitment of proteins that further modify the chromatin template and regulate transcription (1).Monoubiquitylation of histone H2B on lysine (K) 123 in Saccharomyces cerevisiae is a conserved modification that is enriched on active genes but plays roles in both transcriptional repression and activation (2-4). Consistent with a repressive role, H2B monoubiquitylation stabilizes nucleosomes at yeast promoters (5), inhibits the association of the RNA polymerase (pol) II kinase Ctk1 with genes in yeast (6), and interferes with the recruitment of the elongation factor TFIIS to genes in human cells (7). In other studies, H2B monoubiquitylation has been shown to stimulate transcription of chromatin templates (8), promote nucleosome reassembly during transcription elongation (9), and inhibit chromatin compaction (10). H2B monoubiquitylation is also a prerequisite for other histone modifications that mark active genes. Ubiquitylation of H2B K123 by the Rad6-Bre1 ubiquitin conjugase-ligase proteins in yeast (11-13) is required for dimethylation and trimethylation of H3 K4 and K79 by the Set1/COMPASS and Dot1 methy...

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Compliance of the left atrial chamber was estimated with and without the appendage intact in six isolated canine left atria. Pressure-volume determinations were measured over a range of 5-30 mmHg for the whole left atrium and were repeated with the appendage excluded. The slope of the pressure vs. normalized volume data for the left atrium without the appendage (10.45 +/- 0.87) was significantly greater (P less than 0.01) than with the appendage intact (4.10 +/- 0.72). These data suggest that the left atrial appendage is more compliant than the remaining left atrium. Assuming that this relationship remains in vivo, the left atrial appendage may play an augmented role in maintaining hemodynamic function when filling pressures are elevated.

Behavioral experiences activate the Fos transcription factor (TF) in sparse populations of neurons that are critical for encoding and recalling specific events 1 – 3 . However, there is limited understanding of the mechanisms by which experience drives circuit reorganization to establish a network of Fos -activated cells. It is also unknown if Fos is required in this process beyond serving as a marker of recent neural activity and, if so, which of its many gene targets underlie circuit reorganization. Here we demonstrate that when mice engage in spatial exploration of novel environments, perisomatic inhibition of Fos -expressing hippocampal CA1 pyramidal neurons by parvalbumin (PV)-interneurons (INs) is enhanced, while perisomatic inhibition by cholecystokinin (CCK)-INs is weakened. This bidirectional modulation of inhibition is abolished when the function of the Fos TF complex is disrupted. Single-cell RNA-sequencing, ribosome-associated mRNA profiling, and chromatin analyses, combined with electrophysiology, reveal that Fos activates the transcription of Scg2 (secretogranin II), a gene that encodes multiple distinct neuropeptides, to coordinate these changes in inhibition. As PV- and CCK-INs mediate distinct features of pyramidal cell activity 4 – 6 , the Scg2-dependent reorganization of inhibitory synaptic input might be predicted to affect network function in vivo . Consistent with this prediction, hippocampal gamma rhythms and pyramidal cell coupling to CA1 theta are significantly altered with loss of Scg2 . These findings reveal an instructive role for Fos and Scg2 in establishing a network of Fos -activated neurons via the rewiring of local inhibition to form a selectively modulated state. The opposing plasticity mechanisms on distinct inhibitory pathways may support the consolidation of memories over time.