The Wolbachia Genome of Brugia malayi: Endosymbiont Evolution within a Human Pathogenic Nematode

Complete genome DNA sequence and analysis is presented for Wolbachia, the obligate alpha-proteobacterial endosymbiont required for fertility and survival of the human filarial parasitic nematode Brugia malayi. Although, quantitatively, the genome is even more degraded than those of closely related Rickettsia species, Wolbachia has retained more intact metabolic pathways. The ability to provide riboflavin, flavin adenine dinucleotide, heme, and nucleotides is likely to be Wolbachia's principal contribution to the mutualistic relationship, whereas the host nematode likely supplies amino acids required for Wolbachia growth. Genome comparison of the Wolbachia endosymbiont of B. malayi (wBm) with the Wolbachia endosymbiont of Drosophila melanogaster (wMel) shows that they share similar metabolic trends, although their genomes show a high degree of genome shuffling. In contrast to wMel, wBm contains no prophage and has a reduced level of repeated DNA. Both Wolbachia have lost a considerable number of membrane biogenesis genes that apparently make them unable to synthesize lipid A, the usual component of proteobacterial membranes. However, differences in their peptidoglycan structures may reflect the mutualistic lifestyle of wBm in contrast to the parasitic lifestyle of wMel. The smaller genome size of wBm, relative to wMel, may reflect the loss of genes required for infecting host cells and avoiding host defense systems. Analysis of this first sequenced endosymbiont genome from a filarial nematode provides insight into endosymbiont evolution and additionally provides new potential targets for elimination of cutaneous and lymphatic human filarial disease

SynVisio: A Multiscale Tool to Explore Genomic Conservation

Comparative analysis of genomes is an important area in biological research that can shed light on an organism's internal functions and evolutionary history. It involves comparing two or more genomes to identify similar regions that can indicate shared ancestry and in turn conservation of genetic information. Due to rapid advancements in sequencing systems, high-resolution genome data is readily available for a wide range of species, and comparative analysis of this data can offer crucial evolutionary insights that can be applied in plant breeding and medical research. Visualizing the location, size, and orientation of conserved regions can assist biological researchers in comparative analysis as it is a tedious process that requires extensive manual interpretation and human judgement. However, visualization tools for the analysis of conserved regions have not kept pace with the increasing availability of information and are not designed to support the diverse use cases of researchers. To address this we gathered feedback from experts in the field, and designed improvements for these tools through novel interaction techniques and visual representations. We then developed SynVisio, a web-based tool for exploring conserved regions at multiple resolutions (genome, chromosome, or gene), with several visual representations and interactive features, to meet the diverse needs of genome researchers. SynVisio supports multi-resolution analysis and interactive filtering as researchers move deeper into the genome. It also supports revisitation to specific interface configurations, and enables loosely-coupled collaboration over the genomic data. An evaluation of the system with five researchers from three expert groups coupled with a longitudinal study of web traffic to the system provides evidence about the success of our system's novel features for interactive exploration of conservation

Telomeric DNA Damage and Repair Machineries in HIV Infection

In this thesis, we investigated T cell homeostasis and DNA damage repair machineries in HIV infection. We found that the frequencies of CD4T cells were low, which is associated with cell apoptosis in HIV patients compared to healthy subjects. Importantly, these events were closely correlated to the increase in T cell exhaustion, senescence, DNA damage, and telomere attrition. Mechanistically, while the DNA damage sensors Mer11, Rad50, and NBS1 (MRN) complexes remained intact, the ataxia-telangiectasia mutated (ATM) kinase and its downstream checkpoint kinase 2 (CHK2) were significantly inhibited during HIV infection. Additionally, telomeric repeat-binding factor 2 (TRF2) that functions to protect telomeres from unwanted DNA damage was also suppressed by HIV infection. These findings revealed an important mechanism by which telomeres undergo DNA damage that remained unrepaired due to ATM deficiency and TRF2 deprotection - a process that could promote T cell apoptosis, senescence, and cellular dysfunction in HIV infection

Anthoceros genomes illuminate the origin of land plants and the unique biology of hornworts

Hornworts comprise a bryophyte lineage that diverged from other extant land plants >400 million years ago and bears unique biological features, including a distinct sporophyte architecture, cyanobacterial symbiosis and a pyrenoid-based carbon-concentrating mechanism (CCM). Here, we provide three high-quality genomes of Anthoceros hornworts. Phylogenomic analyses place hornworts as a sister clade to liverworts plus mosses with high support. The Anthoceros genomes lack repeat-dense centromeres as well as whole-genome duplication, and contain a limited transcription factor repertoire. Several genes involved in angiosperm meristem and stomatal function are conserved in Anthoceros and upregulated during sporophyte development, suggesting possible homologies at the genetic level. We identified candidate genes involved in cyanobacterial symbiosis and found that LCIB, a Chlamydomonas CCM gene, is present in hornworts but absent in other plant lineages, implying a possible conserved role in CCM function. We anticipate that these hornwort genomes will serve as essential references for future hornwort research and comparative studies across land plants.</p

Comparative Study of the Distribution of Repetitive DNA in Model Organisms

Repetitive DNA elements are abundant in the genome of a wide range of organisms. In mammals, repetitive elements comprise about 40-50% of the total genomes. However, their biological functions remain largely unknown. Analysis of their abundance and distribution may shed some light on how they affect genome structure, function, and evolution. We conducted a detailed comparative analysis of repetitive DNA elements across ten different eukaryotic organisms, including chicken (G. gallus), zebrafish (D. rerio), Fugu (T. rubripes), fruit fly (D. melanogaster), and nematode worm (C. elegans), along with five mammalian organisms: human (H. sapiens), mouse (M. musculus), cow (B. taurus), rat (R. norvegicus), and rhesus (M. mulatta). Our results show that repetitive DNA content varies widely, from 7.3% in the Fugu genome to 52% in the zebrafish, based on RepeatMasker data. The most frequently observed transposable elements (TEs) in mammals are SINEs (Short Interspersed Nuclear Elements), followed by LINEs (Long Interspersed Nuclear Elements). In contrast, LINEs, DNA transposons, simple repeats, and low complexity repeats are the most frequently observed repeat classes in the chicken, zebrafish, fruit fly, and nematode worm genomes, respectively. LTRs (Long Terminal Repeats) have significant genomic coverage and diversity, which may make them suitable for regulatory roles. With the exception of the nematode worm and fruit fly, the frequency of the repetitive elements follows a log-normal distribution, characterized by a few highly prevalent repeats in each organism. In mammals, SINEs are enriched near genic regions, and LINEs are often found away from genes. We also identified many LTRs that are specifically enriched in promoter regions, some with a strong bias towards the same strand as the nearby gene. This raises the possibility that the LTRs may play a regulatory role. Surprisingly, most intronic repeats, with the exception of DNA transposons, have a strong tendency to be on the opposite DNA strand as the host gene. One possible explanation is that intronic RNAs which result from splicing may contribute to retrotransposition to the original intronic loci. Moreover, our observations of repetitive DNA elements enrichment near genic regions and, specifically, the promoter region of genes, raise the question as to whether repetitive DNA elements have a significant impact on gene expression in both human and mouse genomes. In order to investigate the impact of these repeats on gene expression, we calculate the total number of base pairs (bp) for these repeats in two different locations upstream from the genes — namely, the 2kbp and 20kbp promoter regions. In addition to that, we quantified the gene expression levels in both human and mouse tissues using RNAseq analysis. Then, we used different statistical modeling approaches to investigate the association between repetitive DNA elements and gene expression in two different promoter regions. Although most transposable elements are primarily involved in reduced gene expression, our model\u27s results showed that Alu elements in both human and mouse are significantly associated with higher average expression in the promoter region. Furthermore, we found that the B2 in both mouse 2kbp and 20kbp and hAT.Charlie elements in the human 20kbp, are also significantly associated with up-regulated gene expression in the 2kpb promoter. In addition to Alu and B2 in 2kbp, we found that the ERV1 have a significant association with higher average expression in the 20kbp promoter in mouse tissues. We also found that L1 and Simple_repeat elements are significantly associated with lower average expression in both human and mouse tissues. Furthermore, in the human, we found that the MIR is also associated with lower average expression. The effects of Alu elements in both human and mouse are stronger at 2kbp than at 20kbp. In contrast, the L1 effect at 20kbp is stronger than at 2kbp. Our results indicate that comparative studies of repetitive DNA elements in multiple organisms can provide insights into their evolution and expansion, and lead to the elucidation of their potential functions. The non-random distribution of repeats across multiple organisms adds to the existing evidence that some repetitive DNA elements are drivers of genome evolution, rather than just “junk” DNA

Telomere and Proximal Sequence Analysis Using High-Throughput Sequencing Reads

The telomere is a specialized simple sequence repeat found at the end of all linear chromosomes. It acts as a substrate for telomere binding factors that in coordination with other interacting elements form what is known as the shelterin complex to protect the end of the chromosome from the DNA damage repair machinery. The telomere shortens with each cell division, and once critically short is no longer able to perform this role. Short dysfunctional telomeres result in cellular senescence, apoptosis, or genome instability. Telomere length is regulated by many factors including cis-acting elements in the proximal sequence which is known as the subtelomere. The Riethman lab played a pivotal role in generating the reference sequence of the subtelomere in both the human and mouse genomes, providing an essential resource for this work. Short high throughput sequencing (HTS) reads generated from the simple repeat containing telomere or the segmental duplication rich subtelomere cannot be aligned to a reference genome uniquely. They are filtered and excluded from many HTS analysis methods. A ChIP-Seq analysis pipeline was developed to incorporate these multimapping reads to study DNA-protein interactions in the subtelomere. This pipeline was employed to search for factors regulating the expression TERRA, an essential long non-coding RNA, and to better characterize their transcription start sites. ChIP-seq analysis in the human subtelomere found colocalization of CTCF and Cohesin directly adjacent to the telomere and throughout the subtelomere specific repeats. Follow up functional studies showed this binding regulated TERRA transcription at these sites. Extending these analyses in the mouse genome showed very different patterns of CTCF and cohesin binding, with no evidence of binding at apparent sites of TERRA transcription. Mouse subtelomere sequence analysis showed the co-occurence of two repeats at sites of putative TERRA expression, MurSatRep1 and MMSAT4, one of which was previously shown to be expressed in lincRNAs. The Telomere Analysis from SEquencing Reads(TASER) pipeline was developed to capture telomere information from HTS data sets and used to investigate telomere changes that occur in prostate cancer. TASER analysis of 53 paired prostate tumor and normal samples revealed an overall decrease in telomere length in tumor samples relative to matched paired normal tissue, especially sequence containing the exact canonical telomere repeat. Multimapping reads contain important information, that when used properly, help elucidate understanding of telomere biology, cancer biology, and genome regulation and stability

Single Cell Analysis of the HIV-1 Latent Reservoir

Human immunodeficiency virus type 1 (HIV-1), the virus that causes acquired immune deficiency syndrome (AIDS), is one of the world’s most serious health and development challenges. Worldwide there are approximately 36.7 million people living with HIV, and tens of millions have died of AIDS-related causes since the beginning of the epidemic. Treatment of HIV-1 infection with combinations of antiretroviral drugs has significantly reduced the death rate and improved the quality of life of HIV-1 infected individuals. Despite over thirty years of HIV-1 research, however, both a cure and a vaccine remain elusive. Complete eradication of HIV-1 by antiretroviral drugs is prevented by the persistence of rare, long-lived, latently infected cells. These cells, called the latent reservoir, are thought to resist immune clearance and viral cytopathic effects by harboring a transcriptionally quiescent integrated HIV-1 provirus. As a result, interruption of suppressive therapy almost inevitably results in rapid viral rebound, which originates from these latently infected cells and prevents HIV-1 cure. It is thought that establishing the reservoir requires intact retroviral integration into the host cell genome and subsequent transcriptional silencing of the integrated provirus. These are rare events and these cells have no known distinguishing surface markers, which has made it difficult to define the precise cellular and molecular nature of the reservoir. The long half-life of the latent reservoir has been attributed to a stable pool of long-lived latently infected CD4+ T cells. An alternative explanation, consistent with the frequent occurrence of monotypic viral sequences, is that infected latent cells are maintained in part by cell proliferation. T cell division and productive HIV-1 transcription are mediated by shared metabolic and transcriptional pathways, and productive HIV-1 infection typically leads to CD4+ T cell death. Thus, how infected cells survive while dividing is unknown. I focused my thesis on characterizing this latent reservoir in virally suppressed, HIV-1 infected individuals and examining the mechanisms of HIV-1 latency. In the first part of this thesis, using a novel single-cell, high throughput integration site sequencing method, I demonstrate that HIV-1 infected cells are capable of cell division, but that the great majority of the largest expanded clones contain defective proviruses which cannot contribute to the replication competent rebound virus. In the second part of this thesis, using an assay to qualitatively and quantitatively characterize the latent reservoir, I suggest that the replication competent latent reservoir may, in fact, be maintained in part by rare cell division events. And finally, I developed a novel isolation strategy which allowed single cell characterization of recently reactivated latent cells. I was able to obtain reactivated latent T cells that produced intact, replication competent HIV-1. By sequencing the T cell receptors, I prove that these isolated latent cells are expanded T cell clones. Single cell gene expression analysis revealed that latent cells share a specific gene profile that prominently includes genes implicated in silencing the virus, T cell exhaustion markers, and genes that may aid in identification of specific CD4+ T cell subsets prone to latent infection. Together, the data supports a model for latency whereby infected T cells turn on a gene expression program that suppresses viral replication during cell division thereby preventing activation of the cell death pathways that are normally triggered by HIV-1 infection