An interdependent network of functional enhancers regulates transcription and EZH2 loading at the INK4a/ARF locus.

The INK4a/ARF locus encodes important cell-cycle regulators p14ARF, p15INK4b, and p16INK4a. The neighboring gene desert to this locus is the most reproducible GWAS hotspot that harbors one of the densest enhancer clusters in the genome. However, how multiple enhancers that overlap with GWAS variants regulate the INK4a/ARF locus is unknown, which is an important step in linking genetic variation with associated diseases. Here, we show that INK4a/ARF promoters interact with a subset of enhancers in the cluster, independent of their H3K27ac and eRNA levels. Interacting enhancers transcriptionally control each other and INK4a/ARF promoters over long distances as an interdependent single unit. The deletion of even a single interacting enhancer results in an unexpected collapse of the entire enhancer cluster and leads to EZH2 enrichment on promoters in an ANRIL-independent manner. Dysregulated genes genome-wide mimic 9p21-associated diseases under these scenarios. Our results highlight intricate dependencies of promoter-interacting enhancers on each other.

Super-Enhancer Redistribution as a Mechanism of Broad Gene Dysregulation in Repeatedly Drug-Treated Cancer Cells.

Cisplatin is an antineoplastic drug administered at suboptimal and intermittent doses to avoid life-threatening effects. Although this regimen shortly improves symptoms in the short term, it also leads to more malignant disease in the long term. We describe a multilayered analysis ranging from chromatin to translation-integrating chromatin immunoprecipitation sequencing (ChIP-seq), global run-on sequencing (GRO-seq), RNA sequencing (RNA-seq), and ribosome profiling-to understand how cisplatin confers (pre)malignant features by using a well-established ovarian cancer model of cisplatin exposure. This approach allows us to segregate the human transcriptome into gene modules representing distinct regulatory principles and to characterize that the most cisplatin-disrupted modules are associated with underlying events of super-enhancer plasticity. These events arise when cancer cells initiate without ultimately ending the program of drug-stimulated death. Using a PageRank-based algorithm, we predict super-enhancer regulator ISL1 as a driver of this plasticity and validate this prediction by using CRISPR/dCas9-KRAB inhibition (CRISPRi) and CRISPR/dCas9-VP64 activation (CRISPRa) tools. Together, we propose that cisplatin reprograms cancer cells when inducing them to undergo near-to-death experiences.

A rare variant of African ancestry activates 8q24 lncRNA hub by modulating cancer associated enhancer.

Genetic variation at the 8q24 locus is linked with the greater susceptibility to prostate cancer in men of African ancestry. One such African ancestry specific rare variant, rs72725854 (A>G/T) (~6% allele frequency) has been associated with a ~2-fold increase in prostate cancer risk. However, the functional relevance of this variant is unknown. Here we show that the variant rs72725854 is present in a prostate cancer-specific enhancer at 8q24 locus. Chromatin-conformation capture and dCas9 mediated enhancer blocking establish a direct regulatory link between this enhancer and lncRNAs PCAT1, PRNCR1 and PVT1. The risk allele ('T') is associated with higher expression of PCAT1, PVT1 and c-myc in prostate tumors. Further, enhancer with the risk allele gains response to androgen stimulation by recruiting the transcription factor SPDEF whereas, non-risk alleles remain non-responsive. Elevated expression of these lncRNAs and c-myc in risk allele carriers may explain their greater susceptibility to prostate cancer.

Phase separation of ligand-activated enhancers licenses cooperative chromosomal enhancer assembly.

A crucial feature of differentiated cells is the rapid activation of enhancer-driven transcriptional programs in response to signals. The potential contributions of physicochemical properties of enhancer assembly in signaling events remain poorly understood. Here we report that in human breast cancer cells, the acute 17ß-estradiol-dependent activation of functional enhancers requires assembly of an enhancer RNA-dependent ribonucleoprotein (eRNP) complex exhibiting properties of phase-separated condensates. Unexpectedly, while acute ligand-dependent assembly of eRNPs resulted in enhancer activation sensitive to chemical disruption of phase separation, chronically activated enhancers proved resistant to such disruption, with progressive maturation of eRNPs to a more gel-like state. Acute, but not chronic, stimulation resulted in ligand-induced, condensin-dependent changes in spatial chromatin conformation based on homotypic enhancer association, resulting in cooperative enhancer-activation events. Thus, distinct physicochemical properties of eRNP condensates on enhancers serve as determinants of rapid ligand-dependent alterations in chromosomal architecture and cooperative enhancer activation.

Isolation of Nuclear RNA-Associated Protein Complexes.

Noncoding RNAs, being at the center stage of the organismal development and homeostasis, warrant a detailed analysis to utilize their full therapeutic potential. They form complexes with various proteins that enable the noncoding RNAs to acquire specific cellular functions such as the transcriptional outcomes that are controlled in a spatio-temporal manner. In this protocol, we describe a method to isolate such known (and unknown) protein complexes bound to a nuclear noncoding RNA.

Prostate Cancer Susceptibility in Men of African Ancestry at 8q24.

The 8q24 region harbors multiple risk variants for distinct cancers, including >8 for prostate cancer. In this study, we conducted fine mapping of the 8q24 risk region (127.8-128.8Mb) in search of novel associations with common and rare variation in 4853 prostate cancer case patients and 4678 control subjects of African ancestry. All statistical tests were two-sided. We identified three independent associations at P values of less than 5.00×10(-8), all of which were replicated in studies from Ghana and Uganda (combined sample = 5869 case patients, 5615 control subjects; rs114798100: risk allele frequency [RAF] = 0.04, per-allele odds ratio [OR] = 2.31, 95% confidence interval [CI] = 2.04 to 2.61, P = 2.38×10(-40); rs72725879: RAF = 0.33, OR = 1.37, 95% CI = 1.30 to 1.45, P = 3.04×10(-27); and rs111906932: RAF = 0.03, OR = 1.79, 95% CI = 1.53 to 2.08, P = 1.39×10(-13)). Risk variants rs114798100 and rs111906923 are only found in men of African ancestry, with rs111906923 representing a novel association signal. The three variants are located within or near a number of prostate cancer-associated long noncoding RNAs (lncRNAs), including PRNCR1, PCAT1, and PCAT2. These findings highlight ancestry-specific risk variation and implicate prostate-specific lncRNAs at the 8q24 prostate cancer susceptibility region.

Screening of Bacterial Strains for Polygalacturonase Activity: Its Production by Bacillus sphaericus (MTCC 7542).

At present almost all the pectinolytic enzymes used for industrial applications are produced by fungi. There are a few reports of pectinase production by bacterial strains. Therefore, in the present study, seventy-four bacterial strains, isolated from soil and rotten vegetable samples, were screened for polygalacturonase production. The strain PG-31, which gave maximum activity, was identified as Bacillus sphaericus (MTCC 7542). Maximal quantities of polygalacturonase were produced when a 16-hours-old inoculum was used at 7.5% (v/v) in production medium and incubated in shaking conditions (160 rpm) for 72 hours. The optimal temperature and pH for bacterial growth and polygalacturonase production were found to be 30°C and 6.8, respectively. Maximum enzyme production resulted when citrus pectin was used as the carbon source at a concentration of 1.25% (w/v), whereas other carbon sources led to a decrease (30%-70%) in enzyme production. Casein hydrolysate and yeast extract used together as organic nitrogen source gave best results, and ammonium chloride was found to be the most suitable inorganic nitrogen source. The supplementation of media with 0.9% (w/v) D-galacturonic acid led to a 23% increase in activity. Bacillus sphaericus, a bacterium isolated from soil, produced good amount of polygalacturonase activity at neutral pH; hence, it would be potentially useful to increase the yield of banana, grape, or apple juice.