Probes for TP53

Aging-associated muscle wasting is regulated by multiple molecular processes, whereby aberrant mRNA processing regulation induces muscle wasting. The poly(A)-binding protein nuclear 1 (PABPN1) regulates polyadenylation site (PAS) utilization, in the absence of PABPN1 the alternative PAS (APA) is utilized. Reduced PABPN1 levels induce muscle wasting where the expression of cellular processes regulating protein homeostasis, the ubiquitin-proteasome system, and translation, are robustly dysregulated. Translation is impacted by mRNA levels, but PABPN1 impact on translation is not fully understood. Here we show that a persistent reduction in PABPN1 levels led to a significant loss of translation efficiency. RNA sequencing of rRNA-depleted libraries from polysome traces revealed reduced mRNA abundance across ribosomal fractions, as well as reduced levels of small RNAs. We show that the abundance of translated mRNAs in the polysomes correlated with PAS switches at the 3'-UTR. Those mRNAs are enriched in cellular processes that are essential for proper muscle function. This study suggests that the effect of PABPN1 on translation efficiency impacts protein homeostasis in aging-associated muscle atrophy.

Genomic DNA anomalies such as copy number variations (gene duplication, amplification, deletion) and gene rearrangements are important biomarkers and drug targets in many cancer types. DNA in-situ hybridization (ISH) is the gold standard method to directly visualize these molecular alterations in formalin-fixed paraffin-embedded (FFPE) tumor tissues at single-cell resolution within a histological section. However, currently available fluorescent ISH (FISH) assays provide limited morphological detail due to the use of fluorescent nuclear staining compared to chromogenic staining. Furthermore, FISH techniques rely on expensive fluorescence microscopes, risk loss of fluorescent signal over time and involve tedious imaging at high magnifications (100X). There is thus an unmet need for a sensitive and robust chromogenic DNA-ISH assay that can enable high-resolution detection of genomic DNA targets with the ease of bright-field microscopy. We present here DNAscope - a novel chromogenic DNA-ISH assay - for detecting and visualizing genomic DNA targets under a standard light microscope. DNAscope is based on the widely used RNAscope double-Z probe design and signal amplification technology and provides unparalleled sensitivity and specificity with large signal dots readily visualized at 40X magnification and with full morphological context. Furthermore, DNAscope ensures specific DNA detection without interference from RNA due to the use of a novel RNA removal method. Using a duplex chromogenic detection assay in red and blue, we demonstrate highly specific and efficient detection of gene rearrangements (ALK, ROS1, RET and NTRK1), gene amplification (ERBB2, EGFR, MET) and deletion (TP53 and CDKN2A). The DNAscope assay has been carefully optimized for probe signal size and color contrast to enable easy interpretation of signal patterns under conventional light microscopy or digital pathology. Compared to conventional FISH assays, DNAscope probes are standard oligos that are designed in silico to be free of any repetitive sequences and can be rapidly synthesized for any DNA target. In conclusion, the DNAscope assay provides a powerful and convenient alternative to commonly used FISH assays in many cancer research applications.

Multiciliated cells (MCCs) in the brain reside in the ependyma and the choroid plexus (CP) epithelia. The CP secretes cerebrospinal fluid that circulates within the ventricular system, driven by ependymal cilia movement. Tumors of the CP are rare primary brain neoplasms mostly found in children. CP tumors exist in three forms: CP papilloma (CPP), atypical CPP, and CP carcinoma (CPC). Though CPP and atypical CPP are generally benign and can be resolved by surgery, CPC is a particularly aggressive and little understood cancer with a poor survival rate and a tendency for recurrence and metastasis. In contrast to MCCs in the CP epithelia, CPCs in humans are characterized by solitary cilia, frequent TP53 mutations, and disturbances to multiciliogenesis program directed by the GMNC-MCIDAS transcriptional network. GMNC and MCIDAS are early transcriptional regulators of MCC fate differentiation in diverse tissues. Consistently, components of the GMNC-MCIDAS transcriptional program are expressed during CP development and required for multiciliation in the CP, while CPC driven by deletion of Trp53 and Rb1 in mice exhibits multiciliation defects consequent to deficiencies in the GMNC-MCIDAS program. Previous studies revealed that abnormal NOTCH pathway activation leads to CPP. Here we show that combined defects in NOTCH and Sonic Hedgehog signaling in mice generates tumors that are similar to CPC in humans. NOTCH-driven CP tumors are monociliated, and disruption of the NOTCH complex restores multiciliation and decreases tumor growth. NOTCH suppresses multiciliation in tumor cells by inhibiting the expression of GMNC and MCIDAS, while Gmnc-Mcidas overexpression rescues multiciliation defects and suppresses tumor cell proliferation. Taken together, these findings indicate that reactivation of the GMNC-MCIDAS multiciliogenesis program is critical for inhibiting tumorigenesis in the CP, and it may have therapeutic implications for the treatment of CPC.