Identification of a ΔNp63-Dependent Basal-Like A Subtype-Specific Transcribed Enhancer Program (B-STEP) in Aggressive Pancreatic Ductal Adenocarcinoma.

A major hurdle to the application of precision oncology in pancreatic cancer is the lack of molecular stratification approaches and targeted therapy for defined molecular subtypes. In this work, we sought to gain further insight and identify molecular and epigenetic signatures of the Basal-like A pancreatic ductal adenocarcinoma (PDAC) subgroup that can be applied to clinical samples for patient stratification and/or therapy monitoring. We generated and integrated global gene expression and epigenome mapping data from patient-derived xenograft models to identify subtype-specific enhancer regions that were validated in patient-derived samples. In addition, complementary nascent transcription and chromatin topology (HiChIP) analyses revealed a Basal-like A subtype-specific transcribed enhancer program in PDAC characterized by enhancer RNA (eRNA) production that is associated with more frequent chromatin interactions and subtype-specific gene activation. Importantly, we successfully confirmed the validity of eRNA detection as a possible histologic approach for PDAC patient stratification by performing RNA-ISH analyses for subtype-specific eRNAs on pathologic tissue samples. Thus, this study provides proof-of-concept that subtype-specific epigenetic changes relevant for PDAC progression can be detected at a single-cell level in complex, heterogeneous, primary tumor material. IMPLICATIONS: Subtype-specific enhancer activity analysis via detection of eRNAs on a single-cell level in patient material can be used as a potential tool for treatment stratification.

Interactive enhancer hubs (iHUBs) mediate transcriptional reprogramming and adaptive resistance in pancreatic cancer.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) displays a remarkable propensity towards therapy resistance. However, molecular epigenetic and transcriptional mechanisms enabling this are poorly understood. In this study, we aimed to identify novel mechanistic approaches to overcome or prevent resistance in PDAC. DESIGN: We used in vitro and in vivo models of resistant PDAC and integrated epigenomic, transcriptomic, nascent RNA and chromatin topology data. We identified a JunD-driven subgroup of enhancers, called interactive hubs (iHUBs), which mediate transcriptional reprogramming and chemoresistance in PDAC. RESULTS: iHUBs display characteristics typical for active enhancers (H3K27ac enrichment) in both therapy sensitive and resistant states but exhibit increased interactions and production of enhancer RNA (eRNA) in the resistant state. Notably, deletion of individual iHUBs was sufficient to decrease transcription of target genes and sensitise resistant cells to chemotherapy. Overlapping motif analysis and transcriptional profiling identified the activator protein 1 (AP1) transcription factor JunD as a master transcription factor of these enhancers. JunD depletion decreased iHUB interaction frequency and transcription of target genes. Moreover, targeting either eRNA production or signaling pathways upstream of iHUB activation using clinically tested small molecule inhibitors decreased eRNA production and interaction frequency, and restored chemotherapy responsiveness in vitro and in vivo. Representative iHUB target genes were found to be more expressed in patients with poor response to chemotherapy compared with responsive patients. CONCLUSION: Our findings identify an important role for a subgroup of highly connected enhancers (iHUBs) in regulating chemotherapy response and demonstrate targetability in sensitisation to chemotherapy.

A novel thermophilic hemoprotein scaffold for rational design of biocatalysts.

Hemoproteins are commonly found in nature, and involved in many important cellular processes such as oxygen transport, electron transfer, and catalysis. Rational design of hemoproteins can not only inspire novel biocatalysts but will also lead to a better understanding of structure-function relationships in native hemoproteins. Here, the heme nitric oxide/oxygen-binding protein from Caldanaerobacter subterraneus subsp. tengcongensis (TtH-NOX) is used as a novel scaffold for oxidation biocatalyst design. We show that signaling protein TtH-NOX can be reengineered to catalyze H2O2 decomposition and oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) by H2O2. In addition, the role of the distal tyrosine (Tyr140) in catalysis is investigated. The mutation of Tyr140 to alanine hinders the catalysis of the oxidation reactions. On the other hand, the mutation of Tyr140 to histidine, which is commonly observed in peroxidases, leads to a significant increase of the catalytic activity. Taken together, these results show that, while the distal histidine plays an important role in hemoprotein reactions with H2O2, it is not always essential for oxidation activity. We show that TtH-NOX protein can be used as an alternative scaffold for the design of novel biocatalysts with desired reactivity or functionality. H-NOX proteins are homologous to the nitric oxide sensor soluble guanylate cyclase. Here, we show that the gas sensor protein TtH-NOX shows limited capacity for catalysis of redox reactions and it can be used as a novel scaffold in biocatalysis design.

Sensitive and specific detection of ligands using engineered riboswitches.

Riboswitches are RNA elements found in non-coding regions of messenger RNAs that regulate gene expression through a ligand-triggered conformational change. Riboswitches typically bind tightly and specifically to their ligands, so they have the potential to serve as highly effective sensors in vitro. In B. subtilis and other gram-positive bacteria, purine nucleotide synthesis is regulated by riboswitches that bind to guanine. We modified the xpt-pbuX guanine riboswitch for use in a fluorescence quenching assay that allowed us to specifically detect and quantify guanine in vitro. Using this assay, we reproducibly detected as little as 5 nM guanine. We then produced sensors for 2'-deoxyguanosine and cyclic diguanylate (c-diGMP) by appending the P1 stem of the guanine riboswitch to the ligand-binding domains of a 2'-deoxyguanosine riboswitch and a c-diGMP riboswitch. These hybrid sensors could detect 15 nM 2'-deoxyguanosine and 3 nM c-diGMP, respectively. Each sensor retained the ligand specificity of its corresponding natural riboswitch. In order to extend the utility of our approach, we developed a strategy for the in vitro selection of sensors with novel ligand specificity. Here we report a proof-of-principle experiment that demonstrated the feasibility of our selection strategy.