Browsing by Subject "Gene Expression Profiling"

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Aberrant DNA Methylation and Cancer: A Global Analysis of Promoter Hypermethylation in Human Lung Cancers
(2006-12-20) Shames, David S.; Minna, John D.
Tumor-acquired alterations in DNA methylation include both genome-wide hypomethylation and locus specific hypermethylation. Global loss of DNA methylation destabilizes chromatin architecture, augments genomic instability, and may reactivate repetitive element expression. Promoter hypermethylation often coincides with loss of heterozygosity at the same loci, and together these events can result in loss of function of the gene in tumor cells. The "rules" governing which genes are methylated during the pathogenesis of individual cancers are unknown; however, it is known that certain genes are methylated with high frequency in selected tumors, whereas others are methylated across most types of tumors. The objective of the work described below was to use global profiling platforms (RNA and DNA) to identify epigenetically modulated genes that may be involved in cancer pathogenesis and bring these to the point where they could be developed as targets for diagnostic and treatment strategies. Using a global expression profiling approach and pharmacological inhibition of the DNA methyltransferases, 132 genes were identified that have 5' CpG islands, are induced from undetectable levels by 5-aza-2'-deoxycytidine (5-aza) in multiple non-small cell lung cancer cell lines, and are expressed in untreated immortalized human bronchial epithelial cells. Methylation analysis of a subset (45/132) of these promoter regions in primary lung cancer (N=20) and adjacent non-malignant tissue showed that 31 genes had acquired methylation in the tumors, but did not show methylation in normal lung or peripheral blood cells. Promoter methylation of eight of these genes were studied in breast cancers (N=37), colon cancers (N=24), and prostate cancers (N=24) along with counterpart non-malignant tissues. We found that seven loci were frequently methylated in both breast and lung cancers, with four showing extensive methylation in all four epithelial tumors. The data presented below suggest that while tumors differ in their molecular genetic phenotypes and pathogenesis, there may be underlying similarities in the pathways they follow toward malignancy. Some of these similarities may be reflected in the methylation programs tumor cells engage, which in turn, provides an opportunity to exploit for therapeutic applications and diagnosis. The approaches described herein entail a systematic and reproducible method to identify novel methylation markers in a variety of cancers, and the results of these studies provide a basis for developing a generic set of methylation markers for early detection screening across common epithelial cancers.
Analysis of Interrelationships Among NAD+, PARP1, ADP-Ribosylation, and Splicing in Murine Embryonic Stem Cells
(2022-05) Jones, Aarin; Banaszynski, Laura; DeBerardinis, Ralph J.; Wang, Yingfei; Kraus, W. Lee
The differentiation of embryonic stem cells (ESC) into a lineage-committed state is a dynamic process involving changes in epigenetic modifications, gene expression, RNA processing, and cellular metabolism. Previous studies have implicated poly(ADP-ribose) polymerase 1 (PARP1), an abundant nuclear enzyme that plays key roles in a variety of nuclear processes, in ESC self-renewal and lineage commitment. Given the diverse molecular functions of PARP1, I sought to determine the potential regulatory role of PARP1 in determining ESC state. PARP1 functions both as an enzyme, through its NAD+-dependent ADP-ribosyltransferase catalytic activity, and as a structural protein, through its NAD+-independent nucleic acid binding activity. I observed a dramatic induction of PARP1 catalytic activity during the early stages of mESC differentiation (e.g., within 12 hours of LIF removal) leading me to query the regulation and outcome of PARP1-mediated ADP-ribosylation in mESCs. NAD+ is synthesized through three main pathways - De novo, Salvage, and Preiss-Handler - and is constrained within cellular compartments. I found that both pathway usage and subcellular localization were dynamic during differentiation in a PARP1-dependent manner, with transition from De novo to Salvage pathway usage and increases in nuclear NAD+ levels upon differentiation feeding PARP1 catalytic activity. Using an NAD+ analog-sensitive PARP (asPARP) chemical biology approach, I characterized the PARP1-mediated ADP-ribosylated proteome during mESC differentiation. PARP1-modified proteins in mESCs are enriched for biological processes related to stem cell maintenance, transcriptional regulation, and RNA processing. The PARP1 substrates include core spliceosome components, such as U2AF35 and U2AF65, whose splicing functions are modulated by PARP1-mediated site-specific ADP-ribosylation. In addition, I observed a genome-wide dysregulation of splicing events upon loss of PARP1 in transcriptomic analysis. These results demonstrate a role for the NAD+-PARP1 axis in the maintenance of mESC cell state, specifically in the splicing program during differentiation.
The Analysis of the MCF7 Cancer Model System and the Effects of 5-AZA-2'-Deoxycytidine Treatment on the Chromantin State Using a Novel Microarray-Based Technology for High Resolution Global Chromatin State Measurement
(2006-07-10) Weil, Michael Ryan; Garner, Harold R.
A microarray method to measure the global chromatin state of the human genome was developed in order to provide a novel view of gene regulation. The 'chromatin array' employs traditional methods of chromatin isolation, microarray technology, and advanced data analysis, and was applied to a cancer model system. Chromatin is first separated by its condensation state using chromatin fractionation. By probing with a comparative genomic hybridization-style microarray, the chromatin condensation state of thousands of individual loci in an MCF7 tumor model cell line was determined and correlated with transcriptional activity. The chromatin array showed a significant portion (>3,000) of the genes were in a condensation state that was neither condensed or relaxed as a result of heterogeneity in the condensation states in the population. The utility of the chromatin array in deciphering gene regulation was demonstrated in a MCF7 cell line treated with 5 Aza dC, which disrupts genome methylation, and as a result causes global relaxation of chromatin structure. 5 Aza dC treatment results in strong changes in expression, and a normalized global chromatin relaxation of two-fold. A significant subset of 378 genes was condensed by 5 Aza dC treatment, indicating that a mechanism of chromatin regulation exists that can resist the effects of 5 Aza dC treatment. The genes with the largest changes in response to 5 Aza dC treatment showed a strong correlation with CpG island-based regulation (p < 0.0001), and a restoration of transcription patterns associated with normal mammary tissue. Analysis using splice-form specific microarray probes demonstrated that the chromatin state was not uniform across a gene. These findings indicate that certain gene regions exhibit differential sensitivity to 5 Aza dC treatment, and therefore may be regulated independently. Using functional annotation, expression microarray, and comparative genomic hybridization data, this work should provide a framework through which the biological implications of the relationship between chromatin accessibility and expression may be deciphered.
Bayesian Spatial Analysis of High Throughput Sequencing Data
(2021-05-01T05:00:00.000Z) Zhang, Minzhe; Mendell, Joshua T.; Nijhawan, Deepak; Zhan, Xiaowei; Xie, Yang; Xiao, Guanghua
The past decade has witnessed the development and wide use of high-throughput sequencing data in biology. The recent advancement of RNA Sequencing (RNA-Seq) coupled with other molecular technologies such as methylated RNA immunoprecipitation (MeRIP) and spatial barcoding has delivered more specialized platform to investigate certain cellular process and spatial molecular profiling. However, the development of associated analysis tools capable of accommodating the unique features of these new sequencing technologies is still lacking or unsatisfied. For the past few years, I have been devoting to the methodology development of MeRIP-Seq and spatial molecular profiling data. The proposed BaySeqPeak and BOOST-GP methods demonstrated good accuracy, sensitivity and robustness in identifying methylated RNA region and spatial variable genes in both the simulation study and real data analysis.
Functional Analysis of the Human Cytomegalovirus Ul82 Gene Product PP71 Protein During Virus Replication
(2007-08-08) Hagemeier, Stacy Cantrell; Bresnahan, Wade A.
Human cytomegalovirus (HCMV) is a beta-herpesvirus that infects the majority of the human population. Although primary infection is usually asymptomatic in immunocompetent individuals, HCMV infection can lead to severe disease in immunosuppressed individuals including neonates, transplant recipients, and AIDS patients. The HCMV UL82 gene encodes for the tegument protein pp71. It has previously been shown that pp71 is required for efficient virus replication at low multiplicities of infection and that it is a regulator of immediate-early gene expression. However, the mechanism whereby pp71 regulates IE gene expression and/or virus replication has not been elucidated. pp71 has also been shown to bind a number of cellular proteins including Rb familiy member proteins and the cellular protein hDaxx. This dissertation focused on determining if pp71's interaction with either of these proteins is important for viral replication and immediate-early gene expression in the context of a viral infection. We demonstrate that pp71's ability to target Rb family member proteins for degradation is not required for efficient viral replication. However, pp71's ability to interact with hDaxx is required for efficient viral replication and immediate-early gene expression. hDaxx has been identified as a transcriptional regulatory protein and is thought to regulate transcription through its interaction with histone deacetylases and core histones. This dissertation further defines the mechanism by which pp71 enhances viral replication by demonstrating that hDaxx functions to repress HCMV replication and IE gene expression. Importantly, we also demonstrated that the severe growth defect associated with the pp71 deletion mutant could be fully restored following infection of hDaxx knock-down cells. Experiments examining the mechanism by which pp71 relieves hDaxx mediated repression suggest that pp71 interacts with hDaxx to block histone deacetylase activity and therefore promotes the association of acetylated histones with viral immediate-early promoters. Taken together, these results demonstrate that pp71's interaction with hDaxx is critical for efficient virus replication and supports the hypothesis that pp71's interaction with hDaxx is important to "kick-start" the HCMV replication cycle by preventing host cell-mediated repression of viral immediate-early gene expression.
Genetic Analysis of Adipose Lineage and Development
(2008-05-13) Tang, Wei; Graff, Jonathan M.
Adipose tissues protect t against traumatic and thermal insults, and regulate lifespan, reproduction and metabolism. The importance of forming the appropriate number of adipocytes is highlighted by the significant metabolic disturbances that accompany too few (lipodystrophy) or too many (obesity) adipocytes. Most of our current understanding about adipocyte formation come from in vitro culture studies. Little is known about adipose development in vivo because of the lack of genetic tools. To this end, I generated a few knock-in mice that offer both spatial and temporal controls to manipulate gene expression in adipose tissues. Here I demonstrate the application of one of the tools, PPARgamma tTA, in exploring some important aspects of adipose development, such as the adipose depot specification, the identity of adipocyte progenitor cells and their anatomical niche. Adipose tissues form throughout the body in various places in a stereotypical pattern, with each adipose depot displaying distinctive properties. As the first step to understand depot specificity, I used the PPARgamma tTA mice for lineage tracing on adipose tissues, and found that each adipose depot is specified at very distinctive developmental stages, suggesting that different adipose depots are derived from distinct origins. With new genetic tools, I also marked and isolated adipogenic progenitors. I found that the majority of adipocytes descend from a pool of PPARgamma -expressing proliferateing progenitors already commited early in post-natal life, prior to the development of most adipocytes. These progenitors are morphologically and moleculary distinct from adipocytes, have high potential to undergo adipogenesis both in vitro and in vivo after transplantation. Interestingly, some progenitors reside in the mural cell compartment of blood vessels that supply adipose depots and not in vessels of other tissues. The identification of the adipocyte progenitor and localization to the blood vessel wall indicate the presence of a vascular niche in adipose development and provide a basis to examine the interplay between adipogenesis and angiogenesis that could be exploited as a new avenue for obesity and diabetes therapies.
High-Performance Software Development for Genomic Sequence Alignment and Analysis
(2023-05-01T05:00:00.000Z) Zhang, Yun; Zhan, Xiaowei; Kim, Daehwan; Li, Bo; Wang, Tao; Hon, Gary C.
Nucleic acid sequencing technology is a powerful tool for understanding genetic information. Genomic data analysis software is critical for transforming complex sequencing results into meaningful biological information. Emerging sequencing technologies help scientists to understand biological processes from multiple angles, but they also raise the challenge of developing new sequence analysis tools, especially new alignment methods, to support these techniques. In this dissertation, I developed a rapid and accurate sequence alignment software, HISAT-3N, to solve the alignment problem of nucleotide conversion sequencing (NC) technologies. NC technologies, such as BS-seq and SLAM seq, involve converting one type of nucleotide to another, which allows researchers to identify specific chemical modifications in DNA or RNA molecules. However, the conversions generated in these NC technologies make it difficult to align the reads back to the reference genome. To solve this issue, I implemented the 3-letter alignment algorithm into HISAT2, which was developed by our lab previously, to create HISAT-3N. I thoroughly tested HISAT-3N and demonstrated that it is more than seven times faster and more accurate than widely used sequence aligners, and can support all types of nucleotide conversion sequencing technologies, including those that have not yet been developed. Additionally, to generalize the process of developing new alignment methods to support new sequencing technologies, I created a platform that allows for the modularized design of sequence alignment software. This platform incorporates algorithms from HISAT2, STAR, and BWA, providing greater efficiency for developers to create novel sequence alignment software and more flexibility for users to analyze different types of data in a variety of computational environments. Finally, I developed a metagenomics analysis pipeline that effectively organizes and manages multiple well-known sequence analysis software for rapid and accurate soil microbial analysis. The successful development and implementation of these tools demonstrate the robustness of a well-designed bioinformatics software and pipeline framework in bioinformatics analysis. Overall, my work emphasizes the significance of continuously improving genomics data analysis tools. This is important to support emerging sequencing technologies and deliver more precise results, which assist researchers in revealing valuable genetic information.
Human evolution in the 21st century: a novel genetic variant reveals a contemporary heterozygous advantage
(2024-05-17) Vaughan, Douglas E.
In Vivo Gene Expression Profiling of the Plague Bacillus, Yersinia Pestis
(2006-08-11) Lawson, Jonathan Neale; Johnston, Stephen A.
Yersinia pestis, the causative agent of plague, can be transmitted by infected-flea bite or inhaled aerosol. Both routes of infection have a high mortality rate, and pneumonic infections of Yersinia pestis represent a significant fear as a tool of bioterrorism. Understanding the transcriptional program of this pathogen during pulmonary infection is valuable to better understand plague pathogenesis. Improved understanding of the mechanisms employed by Y. pestis to cause illness may lead to new vaccines or therapies. Using a long-oligonucleotide microarray to the plague bacillus I evaluated the expression profiles of in vitro and in vivo models of Y. pestis. The change in temperature from ambient (e.g. aerosol or flea midgut) to the mammalian host has been used as a model to understand the effects of temperature as a signal for virulence gene expression. I have profiled transcription during an aerosol delivered mouse infection. By amplifying the Y. pestis RNA from individual mouse lungs, I was able to map the transcriptional profile of plague at post-infection days 1 to 3. My data suggest a dramatically altered transcriptional profile relative to the in vitro model, suggesting Y. pestis is responding to a variety of host signals during infection. Of note was the number of genes found in genomic regions with altered %GC content that are up-regulated within the mouse lung environment. These data suggest these regions may provide promising targets for future therapy design and vaccine target discovery.