Biology subjects

Wang, A. B.

Publications and source records attributed to Wang, A. B..

3 recordsLinked to original sources

bioRxiv2024

A lineage-specific nascent RNA assay unveils principles of gene regulation in tissue biology

Gene regulatory mechanisms that modulate RNA Polymerase II activity are difficult to access in mammalian tissues composed of multiple cell lineages. Here, we develop a nascent RNA assay (PReCIS-seq) that measures lineage-specific transcriptionally-engaged Pol II on genes and DNA enhancer elements in intact mouse tissue. By employing keratinocytes as a prototype lineage, we unearth Pol II promoter-recruitment versus pause-release mechanisms operating in adult skin homeostasis. Moreover, we relate active enhancer proximity and transcription factor binding motifs on promoters to Pol II activity and promoter-proximal pausing level. Finally, we find Pol II firing rapidly into elongation on lineage identity genes and highly paused on cellular safeguarding genes in a context-dependent manner. Our work provides a basic platform to investigate mechanistic principles of gene regulation in individual lineages of complex mammalian tissues.

genomics↗

bioRxiv2022

A highly sensitive strand-specific multiplex RT-qPCR assay for quantitation of Zika virus replication

Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is widely used to quantify viral RNA genomes for diagnostics and research, yet conventional RT-qPCR protocols are unable to accurately distinguish between the different viral RNA species that exist during infection. Here we show that false-priming and self-priming occur during reverse transcription with several published Zika virus (ZIKV) primer sets. We developed a RT-qPCR assay using tagged primers and thermostable reverse transcriptase, which greatly reduced the occurrence of nonspecific cDNA products. Furthermore, we optimized the assay for use in multiplex qPCR which allows for simultaneous quantitative detection of positive-strand and negative-strand ZIKV RNA along with an internal control from both human and mosquito cells. Importantly, this assay is sensitive enough to study early stages of virus infection in vitro. Strikingly, using this assay, we detected ZIKV negative-strand RNA as early as 3 h post-infection in mammalian cell culture, at a time point prior to the onset of positive-strand RNA synthesis. Overall, the strand-specific RT-qPCR assay developed herein is a valuable tool to quantify ZIKV RNA and to study viral replication dynamics during infection. The application of these findings has the potential to increase accuracy of RNA detection methods for a variety of viral pathogens. HighlightsO_LISelf-primed cDNA is amplified by widely-used ZIKV qPCR primer sets C_LIO_LIUse of tagged primers and thermostable RT increases strand-specificity for RT-qPCR C_LIO_LIMultiplexed qPCR allows for simultaneous quantitation of (+) and (-) strand viral RNAs, and an internal control C_LIO_LIStrand-specific RT-qPCR can detect fewer than one copy of viral RNA per cell in human and mosquito cells C_LI

microbiology↗

bioRxiv2021

Structural Modeling of the TMPRSS Subfamily of Host Cell Proteases Reveals Potential Binding Sites

The transmembrane protease serine subfamily (TMPRSS) has at least eight members with known protein sequence: TMPRSS2, TMPRRS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, TMPRSS11, TMPRSS12 and TMPRSS13. A majority of these TMPRSS proteins have key roles in human hemostasis as well as promoting certain pathologies, including several types of cancer. In addition, TMPRSS proteins have been shown to facilitate the entrance of respiratory viruses into human cells, most notably TMPRSS2 and TMPRSS4 activate the spike protein of the SARS-CoV-2 virus. Despite the wide range of functions that these proteins have in the human body, none of them have been successfully crystallized. The lack of structural data has significantly hindered any efforts to identify potential drug candidates with high selectivity to these proteins. In this study, we present homology models for all members of the TMPRSS family including any known isoform (the homology model of TMPRSS2 is not included in this study as it has been previously published). The atomic coordinates for all homology models have been refined and equilibrated through molecular dynamic simulations. The structural data revealed potential binding sites for all TMPRSS as well as key amino acids that can be targeted for drug selectivity.

bioinformatics↗