Chromatin accessibility: methods, mechanisms, and biological insights.

Access to DNA is a prerequisite to the execution of essential cellular processes that include transcription, replication, chromosomal segregation, and DNA repair. How the proteins that regulate these processes function in the context of chromatin and its dynamic architectures is an intensive field of study. Over the past decade, genome-wide assays and new imaging approaches have enabled a greater understanding of how access to the genome is regulated by nucleosomes and associated proteins. Additional mechanisms that may control DNA accessibility in vivo include chromatin compaction and phase separation - processes that are beginning to be understood. Here, we review the ongoing development of accessibility measurements, we summarize the different molecular and structural mechanisms that shape the accessibility landscape, and we detail the many important biological functions that are linked to chromatin accessibility.

DRUL for school: Opening Pre-K with safe, simple, sensitive saliva testing for SARS-CoV-2.

To address the need for simple, safe, sensitive, and scalable SARS-CoV-2 tests, we validated and implemented a PCR test that uses a saliva collection kit use at home. Individuals self-collected 300 µl saliva in vials containing Darnell Rockefeller University Laboratory (DRUL) buffer and extracted RNA was assayed by RT-PCR (the DRUL saliva assay). The limit of detection was confirmed to be 1 viral copy/µl in 20 of 20 replicate extractions. Viral RNA was stable in DRUL buffer at room temperature up to seven days after sample collection, and safety studies demonstrated that DRUL buffer immediately inactivated virus at concentrations up to 2.75x106 PFU/ml. Results from SARS-CoV-2 positive nasopharyngeal (NP) swab samples collected in viral transport media and assayed with a standard FDA Emergency Use Authorization (EUA) test were highly correlated with samples placed in DRUL buffer. Direct comparison of results from 162 individuals tested by FDA EUA oropharyngeal (OP) or NP swabs with co-collected saliva samples identified four otherwise unidentified positive cases in DRUL buffer. Over six months, we collected 3,724 samples from individuals ranging from 3 months to 92 years of age. This included collecting weekly samples over 10 weeks from teachers, children, and parents from a pre-school program, which allowed its safe reopening while at-risk pods were quarantined. In sum, we validated a simple, sensitive, stable, and safe PCR-based test using a self-collected saliva sample as a valuable tool for clinical diagnosis and screening at workplaces and schools.

Genomic Copy-Number Loss Is Rescued by Self-Limiting Production of DNA Circles.

Copy-number changes generate phenotypic variability in health and disease. Whether organisms protect against copy-number changes is largely unknown. Here, we show that Saccharomyces cerevisiae monitors the copy number of its ribosomal DNA (rDNA) and rapidly responds to copy-number loss with the clonal amplification of extrachromosomal rDNA circles (ERCs) from chromosomal repeats. ERC formation is replicative, separable from repeat loss, and reaches a dynamic steady state that responds to the addition of exogenous rDNA copies. ERC levels are also modulated by RNAPI activity and diet, suggesting that rDNA copy number is calibrated against the cellular demand for rRNA. Last, we show that ERCs reinsert into the genome in a dosage-dependent manner, indicating that they provide a reservoir for ultimately increasing rDNA array length. Our results reveal a DNA-based mechanism for rapidly restoring copy number in response to catastrophic gene loss that shares fundamental features with unscheduled copy-number amplifications in cancer cells.

Condensin and Hmo1 Mediate a Starvation-Induced Transcriptional Position Effect within the Ribosomal DNA Array.

Repetitive DNA arrays are important structural features of eukaryotic genomes that are often heterochromatinized to suppress repeat instability. It is unclear, however, whether all repeats within an array are equally subject to heterochromatin formation and gene silencing. Here, we show that in starving Saccharomyces cerevisiae, silencing of reporter genes within the ribosomal DNA (rDNA) array is less pronounced in outer repeats compared with inner repeats. This position effect is linked to the starvation-induced contraction of the nucleolus. We show that the chromatin regulators condensin and Hmo1 redistribute within the rDNA upon starvation; that Hmo1, like condensin, is required for nucleolar contraction; and that the position effect partially depends on both proteins. Starvation-induced nucleolar contraction and differential desilencing of the outer rDNA repeats may provide a mechanism to activate rDNA-encoded RNAPII transcription units without causing general rDNA instability.

Promoters recognized by forkhead proteins exist for individual 21U-RNAs.

C. elegans 21U-RNAs are equivalent to the piRNAs discovered in other metazoans and have important roles in gametogenesis and transposon control. The biogenesis and molecular function of 21U-RNAs and piRNAs are poorly understood. Here, we demonstrate that transcription of each 21U-RNA is regulated separately through a conserved upstream DNA motif. We use genomic analysis to show that this motif is associated with low nucleosome occupancy, a characteristic of many promoters that drive expression of protein-coding genes, and that RNA polymerase II is localized to this nucleosome-depleted region. We establish that the most conserved 8-mer sequence in the upstream region of 21U-RNAs, CTGTTTCA, is absolutely required for their individual expression. Furthermore, we demonstrate that the 8-mer is specifically recognized by Forkhead family (FKH) transcription factors and that 21U-RNA expression is diminished in several FKH mutants. Our results suggest that thousands of small noncoding transcription units are regulated by FKH proteins.

A conserved PHD finger protein and endogenous RNAi modulate insulin signaling in Caenorhabditis elegans.

Insulin signaling has a profound effect on longevity and the oxidative stress resistance of animals. Inhibition of insulin signaling results in the activation of DAF-16/FOXO and SKN-1/Nrf transcription factors and increased animal fitness. By studying the biological functions of the endogenous RNA interference factor RDE-4 and conserved PHD zinc finger protein ZFP-1 (AF10), which regulate overlapping sets of genes in Caenorhabditis elegans, we identified an important role for these factors in the negative modulation of transcription of the insulin/PI3 signaling-dependent kinase PDK-1. Consistently, increased expression of pdk-1 in zfp-1 and rde-4 mutants contributed to their reduced lifespan and sensitivity to oxidative stress and pathogens due to the reduction in the expression of DAF-16 and SKN-1 targets. We found that the function of ZFP-1 in modulating pdk-1 transcription was important for the extended lifespan of the age-1(hx546) reduction-of-function PI3 kinase mutant, since the lifespan of the age-1; zfp-1 double mutant strain was significantly shorter compared to age-1(hx546). We further demonstrate that overexpression of ZFP-1 caused an increased resistance to oxidative stress in a DAF-16-dependent manner. Our findings suggest that epigenetic regulation of key upstream signaling components in signal transduction pathways through chromatin and RNAi may have a large impact on the outcome of signaling and expression of numerous downstream genes.