Native single molecule sequencing and de novo assembly of Hepatitis B Virus to detect 5mCpG
Ontology highlight
ABSTRACT: Methylation of Hepatitis B Virus (HBV) DNA in a CpG context (5mCpG) can alter the expression patterns of viral genes related to infection and cellular transformation. Moreover, it may also provide clues to why certain infections are cleared, or persist with or without progression to cancer. The detection of 5mCpG often requires techniques that damage DNA or introduce bias through a myriad of limitations. Therefore, we developed a method for the detection of 5mCpG on the HBV genome that does not rely on bisulfite conversion or PCR. Moreover, using the developed technique, we have provided the first de novo assembly of native HBV DNA, as well as the first landscape of 5mCpG from native HBV sequences
Project description:Identity and plasticity of CD4 T helper (Th) cells are regulated in part by epigenetic mechanisms. Cytosine methylation in CpG context (5mCpG) and cytosine hydroxymethylation (5hmCpG) are DNA modifications that identify stable cell phenotypes. To assess transition states in Th cells, we developed a method based on Cas9-targeted single molecule nanopore sequencing and found that 5mCpG can be used as markers of cellular identity. Targeting as few as 10 mouse selected genomic loci, we were able to distinguish major differentiated T cell subtypes as well as intermediate phenotypes by their native DNA 5mCpG patterns. Moreover, by using off-target sequences we were able to infer transcription factor activities relevant to each cell subtype. Our data highlight the potential to exploit native DNA methylation profiling to study physiological and pathological Th transition states.
Project description:G-quadruplex (G4) are four‑stranded DNA secondary structures that form in guanine‑rich regions of the genome, which can enhance or repress gene expression. An R-loop is a special triple-stranded nucleic acid structure formed when nascent RNA invades double-stranded DNA (dsDNA) during transcription. G-loops are constituted by one or more DNA G4 on one strand and a stable RNA/DNA hybrid on the other. We developed the HepG4-seq for mapping the native G4s and the HBD-seq for mapping native R-loops. We combined the HepG4-seq and HBD-seq to profile the genomic native G-loops, which are regions co-occupied by both native G4s and R-loops, in both HEK293 cells and mouse embryonic stem cells (mESCs).
Project description:G-quadruplexes (G4s) are noncanonical DNA secondary structures formed through the self-association of guanines. They are distributed genome-widely and participate in multiple biological processes including gene transcription, and quadruplex-targeted ligands serve as potential therapeutic agents for DNA-targeted therapies. However, the roles of G-quadruplexes in transcriptional regulation remains elusive. Here, we establish a sensitive G4-CUT&Tag method for genome-wide profiling of native G-quadruplexes with high resolution and specificity. We find that native G-quadruplex signals are cell-type specific and are associated with transcriptional regulatory elements with active epigenetic modifications. Promoter-proximal RNA polymerase II pausing promotes native G-quadruplex formation, oppositely, G-quadruplex stabilization by quadruplex-targeted ligands globally reduces RNA polymerase II occupancy at gene promoters as well as nascent RNA synthesis. Moreover, G-quadruplex stabilization modulates chromatin states and impedes transcription initiation via inhibiting the loading of general transcription factors to promoters.Together, these studies reveal a reciprocal regulation between native G-quadruplex dynamics and gene transcription in the genome, which will deepen our knowledge of G-quadruplex biology towards considering therapeutically targeting G-quadruplexes in human diseases.
Project description:To understand widespread differences in the DNA methylation patterns of Conyza canadensis leaf samples from its native and non-native ranges. Using Whole Genome Bisulfite Sequencing, we found average read coverages in high mapped reads across native and non-native samples of Conyza canadensis. Using R bioconductor package, we found enrichment score of methylated sites in both native and non-native samples. while analyzing CG, CHG and CHH methylation, we found relatively low CG and CHG methylation across transcriptional units in natives over non-natives. However, differentially methylated regions were found to be 53% hypomethylated and 41% hypermethylated in non-natives on genic regions.
Project description:We investigated molecular profiles of CD8 T cells in persistent experimental Hepatitis B virus (HBV) infection. To this end, we established a preclinical in vivo model where HBV-replicating hepatocytes were cleared by virus-specific immunity after infection with 107 IU Ad-HBV, whereas persistent HBV replication developed after infection with 108 IU Ad-HBV, revealed by high serum HBeAg levels, high numbers of HBV genome copies and of HBcorepos hepatocytes in liver tissue. To overcome variable surface expression of the T cell receptor during chronic infection and unequivocally identify HBV-specific CD8 T cells, we adoptively transferred naïve CD45.1+HBcore-specific CD8 T cells from Cor93 transgenic mice (HBcoreCD8 T cells) the day before Ad-HBV infection. After clearance of HBV-replicating hepatocytes (d44 p.i.), liver CD45.1+HBcoreCD8 T cells expressed either CXCR6 or CX3CR1, and in the spleen only CX3CR1+ CD8 T cells were detected. Next, we evaluated the transcriptional profile of FACSorted HBcoreCD8 T cells after resolved compared to persistent infection employing SmartSeq2. The resulting dataset is reported here.
Project description:We previously found that a native lipoprotein mix with a high VLDL+LDL/HDL ratio causes a global de novoDNA methylation in THP-1 macrophages. In the present experiment we assessed the consequences of global lipoprotein-induced de novo DNA methylation on global gene expression in the same cells. Moreover, we sought to use gene expression array data to measure RNA expression levels for candidate factors mediating the epigenetic effects of lipoproteins. Experiment Overall Design: Human native VLDL, LDL and HDL lipoproteins were isolated from buffy coats, fractionated by ultracentrifugation, stored at -80deg., desalted to PBS before usage and kept at 4deg. for a maximum of 7d. THP-1 monocytes were differentiated to macrophages, stimulated for 24h with a mix of 68.8mg/ml VLDL, 32.1mg/ml LDL, 91.1mg/ml HDL or unstimulated (control), in serum-free medium with 2% BSA. Three independently isolated lipoprotein samples were used in triplicate.
Project description:We previously found that a native lipoprotein mix with a high VLDL+LDL/HDL ratio causes a global de novoDNA methylation in THP-1 macrophages. In the present experiment we assessed the consequences of global lipoprotein-induced de novo DNA methylation on global gene expression in the same cells. Moreover, we sought to use gene expression array data to measure RNA expression levels for candidate factors mediating the epigenetic effects of lipoproteins. Keywords: comparison treated vs. control
Project description:Only 59% of Alaska Native people have been adequately screened for colorectal cancer (CRC) despite having the highest reported incidence of CRC in the world. A new at-home multi-target stool DNA screening test (MT-sDNA; Cologuard) with high sensitivity for pre-cancerous polyps and CRC is now available. MT-sDNA has not been tested for feasibility or acceptability within the Alaska tribal health care delivery system, and it is unknown whether use of this new test will increase Alaska Native CRC screening rates. The long-term study goal is to improve screening and reduce CRC-attributable mortality. The objective of this application is to test the effectiveness of MT-sDNA for increasing CRC screening in Alaska Native communities using a mixed methods, community-based participatory research (CBPR) approach. The study will be conducted in collaboration with regional Tribal health organizations responsible for providing health care to geographically remote Alaska Native communities. Although the proposed implementation strategy is evidence-informed and promising, it is novel in that MT-sDNA has not been evaluated in the tribal health setting or among rural/remote populations. Using the Social Ecological Model, the research will be multi-level, examining influence on patients, providers, and tribal health organizations (THOs). This research study will pursue two specific aims: (1) Identify patient-, provider-, and system-level factors associated with CRC screening preferences, uptake, and follow-up; and (2) test the effectiveness of graded intensity MT-sDNA intervention in the Alaska Native community setting. For the first aim, focus groups with Alaska Native people who are not adherent to CRC screening guidelines and interviews with healthcare providers will be used to identify factors for future intervention. For the second aim, a three-arm cluster randomized controlled trial (high intensity with patient navigation, medium intensity with mailed reminders, usual care) will provide evidence on the MT-sDNA usefulness (MT-sDNA sample quality and neoplastic yield) as well as the first data on MT-sDNA follow up adherence rates in the Alaska Native population, which will inform plans to scale-up the intervention model. This research has the potential to sustainably improve public health by increasing CRC screening rates among a rural/remote tribal population as well as provide a model for other integrated health systems that provide care to high-risk or underserved populations in the U.S. and worldwide.