Red Panda: A Novel Method for Detecting Variants in Single-Cell RNA Sequencing

BACKGROUND: Single-cell sequencing enables us to better understand genetic diseases, such as cancer or autoimmune disorders, which are often affected by changes in rare cells. Currently, no existing software is aimed at identifying single nucleotide variations or micro (1-50 bp) insertions and deletions in single-cell RNA sequencing (scRNA-seq) data. Generating high-quality variant data is vital to the study of the aforementioned diseases, among others. RESULTS: In this study, we report the design and implementation of Red Panda, a novel method to accurately identify variants in scRNA-seq data. Variants were called on scRNA-seq data from human articular chondrocytes, mouse embryonic fibroblasts (MEFs), and simulated data stemming from the MEF alignments. Red Panda had the highest Positive Predictive Value at 45.0%, while other tools-FreeBayes, GATK HaplotypeCaller, GATK UnifiedGenotyper, Monovar, and Platypus-ranged from 5.8-41.53%. From the simulated data, Red Panda had the highest sensitivity at 72.44%. CONCLUSIONS: We show that our method provides a novel and improved mechanism to identify variants in scRNA-seq as compared to currently existing software. However, methods for identification of genomic variants using scRNA-seq data can be still improved

Characterization of APE1 And DNA G-Quadruplex Interaction in Transcription Regulation and DNA Damage Repair

Human AP Endonuclease 1 (APE1) is the primary enzyme in the base excision repair (BER) pathway that repairs apurinic/apyrimidinic (AP) sites, the most frequently formed DNA lesions in the genome. Recently, through genome-wide mapping analysis, we have shown that APE1, as well as its post-translationally modified form, acetylated APE1 (AcAPE1), is enriched in the gene regulatory regions. The APE1/AcAPE1-enriched regions also harbor Guanine (G)-rich sequences that fold into DNA secondary structures called G-quadruplexes (G4s). Our lab has demonstrated a strong genome-wide correlation between the occurrence of G4 structures and the binding of APE1 and AcAPE1. However, it is unknown whether APE1/ AcAPE1 directly binds to the DNA G4 structures to regulate gene expression and DNA damage repair in the gene regulatory regions. This dissertation aims at characterizing APE1-G4 interaction and elucidating the role of this interaction in transcriptional regulation and DNA damage repair. To characterize the APE1-G4 interaction and its role in transcriptional regulation of genes, particularly oncogenes, we investigated the G4-mediated expression of KRAS in Pancreatic ductal adenocarcinoma (PDAC) cell lines. Using different cell biology, biochemistry, and biophysical methodologies, we show that APE1 is a DNA G4-binding protein and regulates KRAS expression by controlling the loading of transcription factors on the KRAS promoter G4. Our findings unravel a new role of APE1 in regulating stable G4 formation and KRAS expression in PDAC cells. We also observed that AcAPE1 remains bound to the chromatin throughout the cell cycle, including all the phases of mitosis. We found that AcAPE1 colocalizes with G4 structures on the condensed mitotic chromatin. We demonstrate that APE1-G4 interaction is important for modulating DNA damage repair to facilitate post-mitotic transcriptional reactivation in daughter cells as knockdown of APE1 significantly reduced nascent RNA production. Overall, our study has identified APE1 as a DNA G4-binding protein, highlighting the biological significance of APE1-G4 interaction in regulating transcription and endogenous DNA damage repair in gene regulatory regions

STAT3 Inhibition Attenuates MYC Expression by Modulating Co-Activator Recruitment and Suppresses Medulloblastoma Tumor Growth by Augmenting Cisplatin Efficacy In Vivo

MB is a common childhood malignancy of the central nervous system, with significant morbidity and mortality. Among the four molecular subgroups, MYC-amplified Group 3 MB is the most aggressive type and has the worst prognosis due to therapy resistance. The present study aimed to investigate the role of activated STAT3 in promoting MB pathogenesis and chemoresistance via inducing the cancer hallmark MYC oncogene. Targeting STAT3 function either by inducible genetic knockdown (KD) or with a clinically relevant small molecule inhibitor reduced tumorigenic attributes in MB cells, including survival, proliferation, anti-apoptosis, migration, stemness and expression of MYC and its targets. STAT3 inhibition attenuates MYC expression by affecting recruitment of histone acetyltransferase p300, thereby reducing enrichment of H3K27 acetylation in the MYC promoter. Concomitantly, it also decreases the occupancy of the bromodomain containing protein-4 (BRD4) and phosphoSer2-RNA Pol II (pSer2-RNAPol II) on MYC, resulting in reduced transcription. Importantly, inhibition of STAT3 signaling significantly attenuated MB tumor growth in subcutaneous and intracranial orthotopic xenografts, increased the sensitivity of MB tumors to cisplatin, and improved the survival of mice bearing high-risk MYC-amplified tumors. Together, the results of our study demonstrate that targeting STAT3 may be a promising adjuvant therapy and chemo-sensitizer to augment treatment efficacy, reduce therapy-related toxicity and improve quality of life in high-risk pediatric patients