Transcriptional Memory: Mothers SET the Table for Daughters

SummaryEukaryotic gene transcription within individual cells of a population is often associated with heterogeneous pulses of gene activity. A recent study, however, shows that mothers and their daughters share similar transcriptional frequencies, and inheritance of mother's transcriptional tendencies requires methylation of histone H3 by a Set1 methyltransferase

Chromatin Remodeling: Nucleosomes Bulging at the Seams

ATP-dependent chromatin remodeling enzymes, such as SWI/SNF, hydrolyze thousands of ATPs to regulate gene expression on chromatin fibers. Recent mechanistic studies suggest that these enzymes generate localized changes in DNA topology that drive formation of multiple, remodeled nucleosomal states

Yeast Sirtuin Family Members Maintain Transcription Homeostasis to Ensure Genome Stability

The mammalian sirtuin, SIRT6, is a key tumor suppressor that maintains genome stability and regulates transcription, though how SIRT6 family members control genome stability is unclear. Here, we use multiple genome-wide approaches to demonstrate that the yeast SIRT6 homologs, Hst3 and Hst4, prevent genome instability by tuning levels of both coding and noncoding transcription. While nascent RNAs are elevated in the absence of Hst3 and Hst4, a global impact on steady-state mRNAs is masked by the nuclear exosome, indicating that sirtuins and the exosome provide two levels of regulation to maintain transcription homeostasis. We find that, in the absence of Hst3 and Hst4, increased transcription is associated with excessive DNA-RNA hybrids (R-loops) that appear to lead to new DNA double-strand breaks. Importantly, dissolution of R-loops suppresses the genome instability phenotypes of hst3 hst4 mutants, suggesting that the sirtuins maintain genome stability by acting as a rheostat to prevent promiscuous transcription

Direct interactions promote eviction of the Sir3 heterochromatin protein by the SWI/SNF chromatin remodeling enzyme

Heterochromatin is a specialized chromatin structure that is central to eukaryotic transcriptional regulation and genome stability. Despite its globally repressive role, heterochromatin must also be dynamic, allowing for its repair and replication. In budding yeast, heterochromatin formation requires silent information regulators (Sirs) Sir2p, Sir3p, and Sir4p, and these Sir proteins create specialized chromatin structures at telomeres and silent mating-type loci. Previously, we found that the SWI/SNF chromatin remodeling enzyme can catalyze the ATP-dependent eviction of Sir3p from recombinant nucleosomal arrays, and this activity enhances early steps of recombinational repair in vitro. Here, we show that the ATPase subunit of SWI/SNF, Swi2p/Snf2p, interacts with the heterochromatin structural protein Sir3p. Two interaction surfaces are defined, including an interaction between the ATPase domain of Swi2p and the nucleosome binding, Bromo-Adjacent-Homology domain of Sir3p. A SWI/SNF complex harboring a Swi2p subunit that lacks this Sir3p interaction surface is unable to evict Sir3p from nucleosomes, even though its ATPase and remodeling activities are intact. In addition, we find that the interaction between Swi2p and Sir3p is key for SWI/SNF to promote resistance to replication stress in vivo and for establishment of heterochromatin at telomeres

Distinct transcriptional roles for Histone H3-K56 acetylation during the cell cycle in Yeast

Dynamic disruption and reassembly of promoter-proximal nucleosomes is a conserved hallmark of transcriptionally active chromatin. Histone H3-K56 acetylation (H3K56Ac) enhances these turnover events and promotes nucleosome assembly during S phase. Here we sequence nascent transcripts to investigate the impact of H3K56Ac on transcription throughout the yeast cell cycle. We find that H3K56Ac is a genome-wide activator of transcription. While H3K56Ac has a major impact on transcription initiation, it also appears to promote elongation and/or termination. In contrast, H3K56Ac represses promiscuous transcription that occurs immediately following replication fork passage, in this case by promoting efficient nucleosome assembly. We also detect a stepwise increase in transcription as cells transit S phase and enter G2, but this response to increased gene dosage does not require H3K56Ac. Thus, a single histone mark can exert both positive and negative impacts on transcription that are coupled to different cell cycle events

RNA polymerase II depletion promotes transcription of alternative mRNA species

BACKGROUND: Cells respond to numerous internal and external stresses, such as heat, cold, oxidative stress, DNA damage, and osmotic pressure changes. In most cases, the primary response to stress is transcriptional induction of genes that assist the cells in tolerating the stress and facilitate the repair of the cellular damage. However, when the transcription machinery itself is stressed, responding by such standard mechanisms may not be possible. RESULTS: In this study, we demonstrate that depletion or inactivation of RNA polymerase II (RNAPII) changes the preferred polyadenylation site usage for several transcripts, and leads to increased transcription of a specific subset of genes. Surprisingly, depletion of RNA polymerase I (RNAPI) also promotes altered polyadenylation site usage, while depletion of RNA polymerase III (RNAPIII) does not appear to have an impact. CONCLUSIONS: Our results demonstrate that stressing the transcription machinery by depleting either RNAPI or RNAPII leads to a novel transcriptional response that results in induction of specific mRNAs and altered polyadenylation of many of the induced transcripts

INO80C Remodeler Maintains Genomic Stability by Preventing Promiscuous Transcription at Replication Origins

The proper coordination of transcription with DNA replication and repair is central for genomic stability. We investigate how the INO80C chromatin remodeling enzyme might coordinate these genomic processes. We find that INO80C co-localizes with the origin recognition complex (ORC) at yeast replication origins and is bound to replication initiation sites in mouse embryonic stem cells (mESCs). In yeast, INO80C recruitment requires origin sequences but does not require ORC, suggesting that recruitment is independent of pre-replication complex assembly. In both yeast and ESCs, INO80C co-localizes at origins with Mot1 and NC2 transcription factors, and genetic studies suggest that they function together to promote genome stability. Interestingly, nascent transcript sequencing demonstrates that INO80C and Mot1 prevent pervasive transcription through origin sequences, and absence of these factors leads to formation of new DNA double-strand breaks. We propose that INO80C and Mot1/NC2 function through distinct pathways to limit origin transcription, maintaining genomic stability

A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 +/- 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of +/- 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies

Comparison of bacterial maxillary sinus cultures between odontogenic sinusitis and chronic rhinosinusitis

BACKGROUND: Bacterial odontogenic sinusitis (ODS) is distinct from other forms of rhinosinusitis. Diagnosing ODS can be challenging because of nonspecific clinical presentations and underrepresentation in the literature. The purpose of this study was to compare maxillary sinus bacterial cultures between patients with ODS and chronic rhinosinusitis (CRS), to determine whether certain bacteria are associated with ODS. METHODS: This was a retrospective case-control study of 276 consecutive patients from August 2015 to August 2019 who underwent endoscopic sinus surgery (ESS) for bacterial ODS, CRS without nasal polyps (CRSsNP), or CRS with nasal polyps (CRSwNP). When present, pus was sterilely cultured from maxillary sinuses after maxillary antrostomy, and aerobic and anaerobic cultures were immediately sent for processing. Demographics and culture results were compared between ODS and CRS patients, and then separately between ODS and CRSsNP, and ODS and CRSwNP. ODS culture results were also compared between different dental pathologies (endodontic vs oroantral fistula). RESULTS: The following bacteria were significantly more likely in ODS compared to CRS: mixed anaerobes, Fusobacterium spp., Eikenella corrodens, Streptococcus intermedius, Streptococcus anginosus, and Streptococcus constellatus. Staphylococcus aureus and Pseudomonas aeruginosa were inversely related to ODS. There were no significant differences in cultures between the different dental pathologies. CONCLUSION: Certain bacteria were more likely to be associated with ODS compared to CRS when purulence was cultured from the maxillary sinus. Physicians should evaluate for an odontogenic source of sinusitis when these ODS-associated bacteria are identified in maxillary sinus cultures

Multivalent interactions drive nucleosome binding and efficient chromatin deacetylation by SIRT6

The protein deacetylase SIRT6 maintains cellular homeostasis through multiple pathways that include the deacetylation of histone H3 and repression of transcription. Prior work suggests that SIRT6 is associated with chromatin and can substantially reduce global levels of H3 acetylation, but how SIRT6 is able to accomplish this feat is unknown. Here, we describe an exquisitely tight interaction between SIRT6 and nucleosome core particles, in which a 2:1 enzyme:nucleosome complex assembles via asymmetric binding with distinct affinities. While both SIRT6 molecules associate with the acidic patch on the nucleosome, we find that the intrinsically disordered SIRT6 C-terminus promotes binding at the higher affinity site through recognition of nucleosomal DNA. Together, multivalent interactions couple productive binding to efficient deacetylation of histones on endogenous chromatin. Unique among histone deacetylases, SIRT6 possesses the intrinsic capacity to tightly interact with nucleosomes for efficient activity