Recruitment and allosteric stimulation of a histone-deubiquitinating enzyme during heterochromatin assembly.

Heterochromatin formation in budding yeast is regulated by the silent information regulator (SIR) complex. The SIR complex comprises the NAD-dependent deacetylase Sir2, the scaffolding protein Sir4, and the nucleosome-binding protein Sir3. Transcriptionally active regions present a challenge to SIR complex-mediated de novo heterochromatic silencing due to the presence of antagonistic histone post-translational modifications, including acetylation and methylation. Methylation of histone H3K4 and H3K79 is dependent on monoubiquitination of histone H2B (H2B-Ub). The SIR complex cannot erase H2B-Ub or histone methylation on its own. The deubiquitinase (DUB) Ubp10 is thought to promote heterochromatic silencing by maintaining low H2B-Ub at sub-telomeres. Here, we biochemically characterized the interactions between Ubp10 and the SIR complex machinery. We demonstrate that a direct interaction between Ubp10 and the Sir2/4 sub-complex facilitates Ubp10 recruitment to chromatin via a co-assembly mechanism. Using hydrolyzable H2B-Ub analogs, we show that Ubp10 activity is lower on nucleosomes compared with H2B-Ub in solution. We find that Sir2/4 stimulates Ubp10 DUB activity on nucleosomes, likely through a combination of targeting and allosteric regulation. This coupling mechanism between the silencing machinery and its DUB partner allows erasure of active PTMs and the de novo transition of a transcriptionally active DNA region to a silent chromatin state.

Proteomic profiling of yeast heterochromatin connects direct physical and genetic interactions.

Heterochromatin domains are stably repressed chromatin structures composed of a core assembly of silencing proteins that condense adjacent nucleosomes. The minimal heterochromatin structure can serve as a platform for recruitment of complementary regulatory factors. We find that a reconstituted budding yeast heterochromatin domain can act as a platform to recruit multiple factors that play a role in regulating heterochromatin function. We uncover the direct interaction between the SIR heterochromatin complex and a chromosomal boundary protein that restricts the spread of heterochromatin. We find that the SIR complex relieves a mechanism of auto-inhibition within the boundary protein Yta7, allowing the Yta7 bromodomain to engage chromatin. Our results suggest that budding yeast shares with other eukaryotes the ability to establish complex heterochromatin domains that coordinate multiple mechanisms of silencing regulation through physical interactions.

The interplay of histone H2B ubiquitination with budding and fission yeast heterochromatin.

Mono-ubiquitinated histone H2B (H2B-Ub) is important for chromatin regulation of transcription, chromatin assembly, and also influences heterochromatin. In this review, we discuss the effects of H2B-Ub from nucleosome to higher-order chromatin structure. We then assess what is currently known of the role of H2B-Ub in heterochromatic silencing in budding and fission yeasts (S. cerevisiae and S. pombe), which have distinct silencing mechanisms. In budding yeast, the SIR complex initiates heterochromatin assembly with the aid of a H2B-Ub deubiquitinase, Ubp10. In fission yeast, the RNAi-dependent pathway initiates heterochromatin in the context of low H2B-Ub. We examine how the different silencing machineries overcome the challenge of H2B-Ub chromatin and highlight the importance of using these microorganisms to further our understanding of H2B-Ub in heterochromatic silencing pathways.

THE HISTONE CHAPERONE SPN1 PRESERVES SUBNUCLEOSOMAL STRUCTURES AT PROMOTERS AND NUCLEOSOME POSITIONING IN OPEN READING FRAMES.

Spn1 is a multifunctional histone chaperone essential for life in eukaryotes. While previous work has elucidated regions of the protein important for its many interactions, it is unknown how these domains contribute to the maintenance of chromatin structure. Here, we employ digestion by micrococcal nuclease followed by single-stranded library preparation and sequencing (MNase-SSP) to characterize chromatin structure in yeast expressing wild-type or mutants of Spn1. We mapped nucleosome and subnucleosomal protections genome-wide, and surprisingly, we observed a genome-wide loss of subnucleosomal protection over nucleosome-depleted regions (NDRs) in the Spn1-K192N-containing strain, indicating critical functions of Spn1 in maintaining normal chromatin architecture in promoter regions. Additionally, alterations in nucleosome and hexasome positioning were observed in markedly different mutant Spn1 strains, demonstrating that multiple functions of Spn1 are required to maintain proper chromatin structure in open reading frames, particularly at higher expressed and longer genes. Taken together, our results reveal a previously unknown role of Spn1 in the maintenance of NDR architecture and deepen our understanding of Spn1-dependent chromatin maintenance over transcribed regions.

Atlas Salmonella detection method using transcription mediated amplification (TMA) to detect Salmonella enterica in a variety of foods and select surfaces.

The Atlas Salmonella detection assay was compared to the reference culture methods for 12 foods and three surfaces. Comparison of the Atlas method to the U.S. Food and Drug Administration Bacteriological Analytical Manual (FDA/BAM) and U.S. Department of Agriculture-Food Safety and Inspection Service/Microbiology Laboratory Guidebook (USDA-FSIS/MLG) reference methods required an unpaired approach. Each method had a total of 320 samples inoculated with an S. enterica strain. Each food and surface was inoculated with a different strain of S. enterica at two different levels/ method. Meat and egg products were compared to the USDA-FSIS/MLG 4.05 method. All other foods were compared to the FDA/BAM-5 method. The Atlas method had 148 positives out of 320 total inoculated samples, compared to 119 positives for the reference methods. Overall, the probability of detection analysis of the results showed equivalent or better performance by the Atlas Salmonella detection method compared to the reference methods. The Atlas Salmonella detection assay detected all 100 inclusive organisms and none of the 30 exclusive organisms. The lot-to-lot and kit stability studies showed no statistical differences between lots or over the term of the shelf-life. Instrument-to-instrument testing showed no statistical difference between instruments. Finally, the robustness study showed no difference when the sample volume added to the Atlas Salmonella detection assay varied by 10%, storage time was extended up to 5 days before analysis, or enrichment times were varied from 12 to 24 h.

Roka Listeria detection method using transcription mediated amplification to detect Listeria species in select foods and surfaces. Performance Tested Method(SM) 011201.

The Roka Listeria Detection Assay was compared to the reference culture methods for nine select foods and three select surfaces. The Roka method used Half-Fraser Broth for enrichment at 35 +/- 2 degrees C for 24-28 h. Comparison of Roka's method to reference methods requires an unpaired approach. Each method had a total of 545 samples inoculated with a Listeria strain. Each food and surface was inoculated with a different strain of Listeria at two different levels per method. For the dairy products (Brie cheese, whole milk, and ice cream), our method was compared to AOAC Official Method(SM) 993.12. For the ready-to-eat meats (deli chicken, cured ham, chicken salad, and hot dogs) and environmental surfaces (sealed concrete, stainless steel, and plastic), these samples were compared to the U.S. Department of Agriculture/Food Safety and Inspection Service-Microbiology Laboratory Guidebook (USDA/FSIS-MLG) method MLG 8.07. Cold-smoked salmon and romaine lettuce were compared to the U.S. Food and Drug Administration/Bacteriological Analytical Manual, Chapter 10 (FDA/BAM) method. Roka's method had 358 positives out of 545 total inoculated samples compared to 332 positive for the reference methods. Overall the probability of detection analysis of the results showed better or equivalent performance compared to the reference methods.

Transcription factor-nucleosome dynamics from plasma cfDNA identifies ER-driven states in breast cancer.

Genome-wide binding profiles of estrogen receptor (ER) and FOXA1 reflect cancer state in ER+ breast cancer. However, routine profiling of tumor transcription factor (TF) binding is impractical in the clinic. Here, we show that plasma cell-free DNA (cfDNA) contains high-resolution ER and FOXA1 tumor binding profiles for breast cancer. Enrichment of TF footprints in plasma reflects the binding strength of the TF in originating tissue. We defined pure in vivo tumor TF signatures in plasma using ER+ breast cancer xenografts, which can distinguish xenografts with distinct ER states. Furthermore, state-specific ER-binding signatures can partition human breast tumors into groups with significantly different ER expression and mortality. Last, TF footprints in human plasma samples can identify the presence of ER+ breast cancer. Thus, plasma TF footprints enable minimally invasive mapping of the regulatory landscape of breast cancer in humans and open vast possibilities for clinical applications across multiple tumor types.

Phenotypes from cell-free DNA.

Cell-free DNA (cfDNA) has the potential to enable non-invasive detection of disease states and progression. Beyond its sequence, cfDNA also represents the nucleosomal landscape of cell(s)-of-origin and captures the dynamics of the epigenome. In this review, we highlight the emergence of cfDNA epigenomic methods that assess disease beyond the scope of mutant tumour genotyping. Detection of tumour mutations is the gold standard for sequencing methods in clinical oncology. However, limitations inherent to mutation targeting in cfDNA, and the possibilities of uncovering molecular mechanisms underlying disease, have made epigenomics of cfDNA an exciting alternative. We discuss the epigenomic information revealed by cfDNA, and how epigenomic methods exploit cfDNA to detect and characterize cancer. Future applications of cfDNA epigenomic methods to act complementarily and orthogonally to current clinical practices has the potential to transform cancer management and improve cancer patient outcomes.