H3K4me3 breadth is linked to cell identity and transcriptional consistency.

Trimethylation of histone H3 at lysine 4 (H3K4me3) is a chromatin modification known to mark the transcription start sites of active genes. Here, we show that H3K4me3 domains that spread more broadly over genes in a given cell type preferentially mark genes that are essential for the identity and function of that cell type. Using the broadest H3K4me3 domains as a discovery tool in neural progenitor cells, we identify novel regulators of these cells. Machine learning models reveal that the broadest H3K4me3 domains represent a distinct entity, characterized by increased marks of elongation. The broadest H3K4me3 domains also have more paused polymerase at their promoters, suggesting a unique transcriptional output. Indeed, genes marked by the broadest H3K4me3 domains exhibit enhanced transcriptional consistency and [corrected] increased transcriptional levels, and perturbation of H3K4me3 breadth leads to changes in transcriptional consistency. Thus, H3K4me3 breadth contains information that could ensure transcriptional precision at key cell identity/function genes.

Identification and validation of microtubule depolymerizing agent, CYT997, as a potential drug candidate for hepatocellular carcinoma.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is a typically fatal malignancy with limited treatment options and poor survival rates, despite recent FDA approvals of newer treatment options. We aim to address this unmet need by using a proprietary computational drug discovery platform that identifies drug candidates with the potential to advance rapidly and successfully through preclinical studies. METHODS: We generated an in silico model of HCC biology to identify the top 10 small molecules with predicted efficacy. The most promising candidate, CYT997, was tested for its in vitro effects on cell viability and cell death, colony formation, cell cycle changes, and cell migration/invasion in HCC cells. We used an HCC patient-derived xenograft (PDX) mouse model to assess its in vivo efficacy. RESULTS: CYT997 was significantly more cytotoxic against HCC cells than against primary human hepatocytes, and sensitized HCC cells to sorafenib. It arrested cell cycle at the G2/M phase with associated up-regulations of p21, p-MEK1/2, p-ERK, and down-regulation of cyclin B1. Cell apoptosis and senescence-like morphology were also observed. CYT997 inhibited HCC cell migration and invasion, and down-regulated the expressions of acetylated tubulins, ß-tubulin, glypican-3 (GPC3), ß-catenin, and c-Myc. In vivo, CYT997 (20 mg/kg, three times weekly by oral gavage) significantly inhibited PDX growth, while being non-toxic to mice. Immunohistochemistry confirmed the down-regulation of GPC3, c-Myc, and Ki-67, supporting its anti-proliferative effect. CONCLUSION: CYT997 is a potentially efficacious and non-toxic drug candidate for HCC therapy. Its ability to down-regulate GPC3, ß-catenin, and c-Myc highlights a novel mechanism of action.

Chromatin accessibility dynamics reveal novel functional enhancers in C. elegans.

Chromatin accessibility, a crucial component of genome regulation, has primarily been studied in homogeneous and simple systems, such as isolated cell populations or early-development models. Whether chromatin accessibility can be assessed in complex, dynamic systems in vivo with high sensitivity remains largely unexplored. In this study, we use ATAC-seq to identify chromatin accessibility changes in a whole animal, the model organism Caenorhabditis elegans, from embryogenesis to adulthood. Chromatin accessibility changes between developmental stages are highly reproducible, recapitulate histone modification changes, and reveal key regulatory aspects of the epigenomic landscape throughout organismal development. We find that over 5000 distal noncoding regions exhibit dynamic changes in chromatin accessibility between developmental stages and could thereby represent putative enhancers. When tested in vivo, several of these putative enhancers indeed drive novel cell-type- and temporal-specific patterns of expression. Finally, by integrating transcription factor binding motifs in a machine learning framework, we identify EOR-1 as a unique transcription factor that may regulate chromatin dynamics during development. Our study provides a unique resource for C. elegans, a system in which the prevalence and importance of enhancers remains poorly characterized, and demonstrates the power of using whole organism chromatin accessibility to identify novel regulatory regions in complex systems.

Mycobacterium smegmatis RoxY is a repressor of oxyS and contributes to resistance to oxidative stress and bactericidal ubiquitin-derived peptides.

The mycobactericidal properties of macrophages include the generation of reactive oxygen intermediates and the delivery of bacteria to a hydrolytic lysosome enriched in bactericidal ubiquitin-derived peptides (Ub-peptides). To better understand the interactions of ubiquitin-derived peptides with mycobacteria and identify putative mycobacterial intrinsic resistance mechanisms, we screened for transposon mutants with increased susceptibility to the bactericidal Ub-peptide Ub2. We isolated 27 Mycobacterium smegmatis mutants that were hypersusceptible to Ub2. Two mutants were isolated that possessed mutations in the msmeg_0166 gene, which encodes a transcriptional regulator. The msmeg_0166 mutants were also hypersusceptible to other host antimicrobial peptides and oxidative stress. In characterizing msmeg_0166, we found that it encodes a repressor of oxyS, and therefore we have renamed the gene roxY. We demonstrate that RoxY and OxyS contribute to M. smegmatis resistance to oxidative stress. An ahpD transposon mutant was also isolated in our screen for Ub-peptide hypersusceptibility. Overexpression of oxyS in M. smegmatis reduced transcription of the ahpCD genes, which encode a peroxide detoxification system. Our data indicate that RoxY, OxyS, and AhpD play a role in the mycobacterial oxidative stress response and are important for resistance to host antimicrobial peptides.

Role and regulation of iron-sulfur cluster biosynthesis genes in Shigella flexneri virulence.

Shigella flexneri, a causative agent of bacterial dysentery, possesses two predicted iron-sulfur cluster biosynthesis systems called Suf and Isc. S. flexneri strains containing deletion mutations in the entire suf operon (UR011) or the iscSUA genes (UR022) were constructed. Both mutants were defective in surviving exposure to oxidative stress. The suf mutant showed growth that was comparable to that of the parental strain in both iron-replete and iron-limiting media; however, the isc mutant showed reduced growth, relative to the parental strain, in both media. Although the suf mutant formed wild-type plaques on Henle cell monolayers, the isc mutant was unable to form plaques on Henle cell monolayers because the strain was noninvasive. Expression from both the suf and isc promoters increased in iron-limiting media and in the presence of hydrogen peroxide. Iron repression of the suf promoter was mediated by Fur, and increased suf expression in iron-limiting media was enhanced by the presence of IscR. Iron repression of the isc promoter was mediated by IscR. Hydrogen peroxide-dependent induction of suf expression, but not isc expression, was mediated by OxyR. Furthermore, IscR was a positive regulator of suf expression in the presence of hydrogen peroxide and a negative regulator of isc expression in the absence of hydrogen peroxide. Expression from the S. flexneri suf and isc promoters increased when Shigella was within Henle cells, and our data suggest that the intracellular signal mediating this increased expression is reduced iron levels.

Synergistic drug combinations from electronic health records and gene expression.

OBJECTIVE: Using electronic health records (EHRs) and biomolecular data, we sought to discover drug pairs with synergistic repurposing potential. EHRs provide real-world treatment and outcome patterns, while complementary biomolecular data, including disease-specific gene expression and drug-protein interactions, provide mechanistic understanding. METHOD: We applied Group Lasso INTERaction NETwork (glinternet), an overlap group lasso penalty on a logistic regression model, with pairwise interactions to identify variables and interacting drug pairs associated with reduced 5-year mortality using EHRs of 9945 breast cancer patients. We identified differentially expressed genes from 14 case-control human breast cancer gene expression datasets and integrated them with drug-protein networks. Drugs in the network were scored according to their association with breast cancer individually or in pairs. Lastly, we determined whether synergistic drug pairs found in the EHRs were enriched among synergistic drug pairs from gene-expression data using a method similar to gene set enrichment analysis. RESULTS: From EHRs, we discovered 3 drug-class pairs associated with lower mortality: anti-inflammatories and hormone antagonists, anti-inflammatories and lipid modifiers, and lipid modifiers and obstructive airway drugs. The first 2 pairs were also enriched among pairs discovered using gene expression data and are supported by molecular interactions in drug-protein networks and preclinical and epidemiologic evidence. CONCLUSIONS: This is a proof-of-concept study demonstrating that a combination of complementary data sources, such as EHRs and gene expression, can corroborate discoveries and provide mechanistic insight into drug synergism for repurposing.

The role of oxyR and soxRS in oxidative stress survival in Shigella flexneri.

Shigella flexneri, a facultative intracellular pathogen, is exposed to a variety of environments inside and outside of the human host. Some of these environments may contain significant oxidative stress. S. flexneri mutants were generated with deletions in the major oxidative stress regulators oxyR and/or soxRS to test their importance in Shigella biology. Strains that contained a deletion of oxyR had reduced growth and survival during aerobic growth, but not microaerobic growth. The mutants were also defective in surviving exposure to oxidative stress: oxyR mutants were sensitive to hydrogen peroxide, while soxRS mutants were sensitive to superoxide. Although the ΔsoxRS, ΔoxyR, and ΔoxyR/ΔsoxRS mutant Shigellae survived similarly to the parental strains within macrophages, the mutants formed plaques on Henle cell monolayers that were slightly smaller than the plaques formed by the wildtype strain.