Abundance of G-Quadruplex Forming Sequences in the Hepatitis Delta Virus Genomes.

Hepatitis delta virus (HDV) is a highly unusual RNA satellite virus that depends on the presence of hepatitis B virus (HBV) to be infectious. Its compact and variable single-stranded RNA genome consists of eight major genotypes distributed unevenly across different continents. The significance of noncanonical secondary structures such as G-quadruplexes (G4s) is increasingly recognized at the DNA and RNA levels, particularly for transcription, replication, and translation. G4s are formed from guanine-rich sequences and have been identified in the vast majority of viral, eukaryotic, and prokaryotic genomes. In this study, we analyzed the G4 propensity of HDV genomes by using G4Hunter. Unlike HBV, which has a G4 density similar to that of the human genome, HDV displays a significantly higher number of potential quadruplex-forming sequences (PQS), with a density more than four times greater than that of the human genome. This finding suggests a critical role for G4s in HDV, especially given that the PQS regions are conserved across HDV genotypes. Furthermore, the prevalence of G4-forming sequences may represent a promising target for therapeutic interventions to control HDV replication.

Transactivation by partial function P53 family mutants is increased by the presence of G-quadruplexes at a promoter site.

The effect of mutations in the P53 family of transcription factors on their biological functions, including partial or complete loss of transcriptional activity, has been confirmed several times. At present, P53 family proteins showing partial loss of activity appear to be promising potential candidates for the development of novel therapeutic strategies which could restore their transcriptional activity. In this context, it is important to employ tools to precisely monitor their activity; in relation to this, non-canonical DNA secondary structures in promoters including G-quadruplexes (G4s) were shown to influence the activity of transcription factors. Here, we used a defined yeast assay to evaluate the impact of differently modeled G4 forming sequences on a panel of partial function P53 family mutant proteins. Specifically, a 22-mer G4 prone sequence (derived from the KSHV virus) and five derivatives that progressively mutate characteristic guanine stretches were placed upstream of a minimal promoter, adjacent to a P53 response element in otherwise isogenic yeast luciferase reporter strains. The transactivation ability of cancer-associated P53 (TA-P53α: A161T, R213L, N235S, V272L, R282W, R283C, R337C, R337H, and G360V) or Ectodermal Dyplasia syndromes-related P63 mutant proteins (ΔN-P63α: G134D, G134V and inR155) were tested. Our results show that the presence of G4 forming sequences can increase the transactivation ability of partial function P53 family proteins. These observations are pointing to the importance of DNA structural characteristics for accurate classification of P53 family proteins functionality in the context of the wide variety of TP53 and TP63 germline and somatic mutations.

Multiplexed RNA-FISH-guided Laser Capture Microdissection RNA Sequencing Improves Breast Cancer Molecular Subtyping, Prognostic Classification, and Predicts Response to Antibody Drug Conjugates.

On a retrospective cohort of 1,082 FFPE breast tumors, we demonstrated the analytical validity of a test using multiplexed RNA-FISH-guided laser capture microdissection (LCM) coupled with RNA-sequencing (mFISHseq), which showed 93% accuracy compared to immunohistochemistry. The combination of these technologies makes strides in i) precisely assessing tumor heterogeneity, ii) obtaining pure tumor samples using LCM to ensure accurate biomarker expression and multigene testing, and iii) providing thorough and granular data from whole transcriptome profiling. We also constructed a 293-gene intrinsic subtype classifier that performed equivalent to the research based PAM50 and AIMS classifiers. By combining three molecular classifiers for consensus subtyping, mFISHseq alleviated single sample discordance, provided near perfect concordance with other classifiers (κ > 0.85), and reclassified 30% of samples into different subtypes with prognostic implications. We also use a consensus approach to combine information from 4 multigene prognostic classifiers and clinical risk to characterize high, low, and ultra-low risk patients that relapse early (< 5 years), late (> 10 years), and rarely, respectively. Lastly, to identify potential patient subpopulations that may be responsive to treatments like antibody drug-conjugates (ADC), we curated a list of 92 genes and 110 gene signatures to interrogate their association with molecular subtype and overall survival. Many genes and gene signatures related to ADC processing (e.g., antigen/payload targets, endocytosis, and lysosome activity) were independent predictors of overall survival in multivariate Cox regression models, thus highlighting potential ADC treatment-responsive subgroups. To test this hypothesis, we constructed a unique 19-feature classifier using multivariate logistic regression with elastic net that predicted response to trastuzumab emtansine (T-DM1; AUC = 0.96) better than either ERBB2 mRNA or Her2 IHC alone in the T-DM1 arm of the I-SPY2 trial. This test was deployed in a research-use only format on 26 patients and revealed clinical insights into patient selection for novel therapies like ADCs and immunotherapies and de-escalation of adjuvant chemotherapy.

The presence of a G-quadruplex prone sequence upstream of a minimal promoter increases transcriptional activity in the yeast Saccharomyces cerevisiae.

Non-canonical secondary structures in DNA are increasingly being revealed as critical players in DNA metabolism, including modulating the accessibility and activity of promoters. These structures comprise the so-called G-quadruplexes (G4s) that are formed from sequences rich in guanine bases. Using a well-defined transcriptional reporter system, we sought to systematically investigate the impact of the presence of G4 structures on transcription in yeast Saccharomyces cerevisiae. To this aim, different G4 prone sequences were modeled to vary the chance of intramolecular G4 formation, analyzed in vitro by Thioflavin T binding test and circular dichroism and then placed at the yeast ADE2 locus on chromosome XV, downstream and adjacent to a P53 response element (RE) and upstream from a minimal CYC1 promoter and Luciferase 1 (LUC1) reporter gene in isogenic strains. While the minimal CYC1 promoter provides basal reporter activity, the P53 RE enables LUC1 transactivation under the control of P53 family proteins expressed under the inducible GAL1 promoter. Thus, the impact of the different G4 prone sequences on both basal and P53 family protein-dependent expression was measured after shifting cells onto galactose containing medium. The results showed that the presence of G4 prone sequences upstream of a yeast minimal promoter increased its basal activity proportionally to their potential to form intramolecular G4 structures; consequently, this feature, when present near the target binding site of P53 family transcription factors, can be exploited to regulate the transcriptional activity of P53, P63 and P73 proteins.

Dual mode of IFI16 binding to supercoiled and linear DNA: A closer insight.

IFI16 (Interferon inducible protein 16) is a DNA sensor responsible for innate immune response stimulation and a direct viral restriction by modulating gene expression and replication. Many IFI16-DNA binding properties were described - length-dependent and sequence-independent binding, oligomerization of IFI16 upon recognition, sliding on the DNA, and preference for supercoiled DNA. However, the question of the role of IFI16-DNA binding in distinct IFI16 functions remains unclear. Here we demonstrate two modes of IFI16 binding to DNA using atomic force microscopy and electrophoretic mobility shift assays. In our study, we show that IFI16 can bind to DNA in the form of globular complexes or oligomers depending on DNA topology and molar ratios. The stability of the complexes is different in higher salt concentrations. In addition, we observed no preferential binding with the HIN-A or HIN-B domains to supercoiled DNA, revealing the importance of the whole protein for this specificity. These results provide more profound insight into IFI16-DNA interactions and may be important in answering the question of self- and non-self-DNA binding by the IFI16 protein and potentially could shed light on the role of DNA binding in distinct IFI16 functions.