Summary ATP-dependent chromatin remodelers regulate chromatin structure during multiple stages of transcription. We report that RSC, an essential chromatin remodeler, is recruited to the open reading frames (ORFs) of actively transcribed genes genome-wide, suggesting a role for RSC in regulating transcription elongation. Consistent with such a role, Pol II occupancy in the ORFs of weakly transcribed genes is drastically reduced upon depletion of the RSC catalytic subunit Sth1. RSC inactivation also reduced histone H3 occupancy across transcribed regions. Remarkably, the strongest effects on Pol II and H3 occupancy were confined to the genes displaying the greatest RSC ORF enrichment. Additionally, RSC recruitment to the ORF requires the activities of the SAGA and NuA4 HAT complexes and is aided by the activities of the Pol II CTD Ser2 kinases Bur1 and Ctk1. Overall, our findings strongly implicate ORF-associated RSC in governing Pol II function and in maintaining chromatin structure over transcribed regions.

BackgroundGenomic imprinting is essential for mammalian development and provides a unique paradigm to explore intra-cellular differences in chromatin configuration. So far, the detailed allele-specific chromatin organization of imprinted gene domains has mostly been lacking. Here, we explored the chromatin structure of the two conserved imprinted domains controlled by paternal DNA methylation imprints—the Igf2-H19 and Dlk1-Dio3 domains—and assessed the involvement of the insulator protein CTCF in mouse cells.ResultsBoth imprinted domains are located within overarching topologically associating domains (TADs) that are similar on both parental chromosomes. At each domain, a single differentially methylated region is bound by CTCF on the maternal chromosome only, in addition to multiple instances of bi-allelic CTCF binding. Combinations of allelic 4C-seq and DNA-FISH revealed that bi-allelic CTCF binding alone, on the paternal chromosome, correlates with a first level of sub-TAD structure. On the maternal chromosome, additional CTCF binding at the differentially methylated region adds a further layer of sub-TAD organization, which essentially hijacks the existing paternal-specific sub-TAD organization. Perturbation of maternal-specific CTCF binding site at the Dlk1-Dio3 locus, using genome editing, results in perturbed sub-TAD organization and bi-allelic Dlk1 activation during differentiation.ConclusionsMaternal allele-specific CTCF binding at the imprinted Igf2-H19 and the Dlk1-Dio3 domains adds an additional layer of sub-TAD organization, on top of an existing three-dimensional configuration and prior to imprinted activation of protein-coding genes. We speculate that this allele-specific sub-TAD organization provides an instructive or permissive context for imprinted gene activation during development.

Spt6 is a multifunctional histone chaperone involved in the maintenance of chromatin structure during elongation by RNA polymerase II (Pol II). Spt6 has a tandem SH2 (tSH2) domain within its C terminus that recognizes Pol II C-terminal domain (CTD) peptides phosphorylated on Ser2, Ser5, or Try1 in vitro. Deleting the tSH2 domain, however, only has a partial effect on Spt6 occupancy in vivo, suggesting that more complex mechanisms are involved in the Spt6 recruitment. Our results show that the Ser2 kinases Bur1 and Ctk1, but not the Ser5 kinase Kin28, cooperate in recruiting Spt6, genomewide. Interestingly, the Ser2 kinases promote the association of Spt6 in early transcribed regions and not toward the 3= ends of genes, where phosphorylated Ser2 reaches its maximum level. In addition, our results uncover an unexpected role for histone deacetylases (Rpd3 and Hos2) in promoting Spt6 interaction with elongating Pol II. Finally, our data suggest that phosphorylation of the Pol II CTD on Tyr1 promotes the association of Spt6 with the 3= ends of transcribed genes, independently of Ser2 phosphorylation. Collectively, our results show that a complex network of interactions, involving the Spt6 tSH2 domain, CTD phosphorylation, and histone deacetylases, coordinate the recruitment of Spt6 to transcribed genes in vivo. D ynamic reorganization of chromatin structure is important for the regulation of transcription by RNA polymerase II (Pol II). Several ATP-dependent chromatin remodelers, histone-modifying enzymes, and histone chaperones promote the disruption of chromatin structure to allow transcription by Pol II and to restore chromatin structure in the wake of transcription (1, 2). Spt6 is a highly conserved and multifunctional protein shown to regulate multiple steps of transcription, including initiation (3, 4) and elongation (5, 6). In addition, it is important for other DNAdependent processes such as recombination (7,8), mRNA export (9), and viral replication (10).Spt6 interacts with histones H3 and H4 (11) and functions as a histone chaperone to regulate cotranscriptional nucleosome reassembly and to modulate chromatin structure, including histone modifications (11-15). Depletion of Spt6 in yeast (Saccharomyces cerevisiae) causes a widespread reduction in histone H3 primarily from the 5= ends of transcribed genes, indicating the importance of Spt6 in maintaining histone occupancy (16). One of the effects of losing Spt6 function is altered gene expression and activation of intragenic cryptic transcription (17-19). Aberrant transcription is also associated with histone deacetylase (HDAC) mutants. Impairing the function of the HDAC Rpd3-small (Rpd3S) and Hos2-Set3 complexes leads to cryptic and antisense transcription genome-wide (20, 21). It is not clear whether Spt6 and HDACs collaborate to suppress spurious transcription.Spt6 localization to transcribed regions strongly correlates with Pol II occupancy (15,22). It possesses a tandem Src homology 2 domain (tSH2) that interacts with phosphorylated Pol II C-terminal doma...

Graphical Abstract Highlights d Dense transposon integration identified genes important for forming heterochromatin d This transposon method identified many candidates that are essential genes d Four candidates are canonical mRNA cleavage and polyadenylation factors d Iss1 is required for heterochromatin assembly by recruiting Mmi1 to the exosome SUMMARY Heterochromatin functions as a scaffold for factors responsible for gene silencing and chromosome segregation. Heterochromatin can be assembled by multiple pathways, including RNAi and RNA surveillance. We identified factors that form heterochromatin using dense profiles of transposable element integration in Schizosaccharomyces pombe. The candidates include a large number of essential proteins such as four canonical mRNA cleavage and polyadenylation factors. We find that Iss1, a subunit of the poly(A) polymerase module, plays a role in forming heterochromatin in centromere repeats that is independent of RNAi. Genome-wide maps reveal that Iss1 accumulates at genes regulated by RNA surveillance. Iss1 interacts with RNA surveillance factors Mmi1 and Rrp6, and importantly, Iss1 contributes to RNA elimination that forms heterochromatin at meiosis genes. Our profile of transposable element integration supports the model that a network of mRNA cleavage and polyadenylation factors coordinates RNA surveillance, including the mechanism that forms heterochromatin at meiotic genes.