Allele frequencies of variants in Ultra Conserved Elements identify selective pressure on transcription factor binding

Ultra-conserved genes or elements (UCGs/UCEs) in the human genome are extreme examples of conservation. We characterized natural variations in 2884 UCEs and UCGs in two distinct populations ; Singaporean Chinese (n=280) and Italian (n=501) by using a pooled sample, targeted capture, sequencing approach. We identify, with high confidence, in these regions the abundance of rare SNVs (MAF<0.5%) of which 75% is not present in dbSNP137. UCEs association studies for complex human traits can use this information to model expected background variation and thus necessary power for association studies. By combining our data with 1000 Genome Project data, we show in three independent datasets that prevalent UCE variants (MAF>5%) are more often found in relatively less-conserved nucleotides within UCEs, compared to rare variants. Moreover, prevalent variants are less likely to overlap transcription factor binding site. Using SNPfold we found no significant influence of RNA secondary structure on UCE conservation. All together, these results suggest UCEs are not under selective pressure as a stretch of DNA but are under differential evolutionary pressure on the single nucleotide level

Veise papilloomiviiruse tüüp 1 segregatsioonimehhanismi uurimine

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Papilloomiviirused (PV) on DNA genoomiga viirused, mis nakatavad epiteelkoe rakke. PV-te peremeestering on väga lai. Lisaks lindudele ja roomajatele on kirjeldatud enam kui 100 erinevat tüüpi PV-i, mis nakatavad inimesi (HPV- human papillomavirus). Väga suure tõenäosusega mingil elu etapil iga inimene nakatub ühe või enama HPV-ga. Nendele viirustele on iseloomulik pikaajaline vaikoleku võime. Seega, HPV võib inimese rakkudes püsida aastaid põhjustamata ühtegi haigust. Perioodiliselt siiski võivad HPV-d nakatunud kudedes põhjustada healoomulisi vohandeid- soolatüükaid ja kondüloome. Kuid lisaks soolatüügastele ja kondüloomidele võivad mõned pahaloomulised HPV-d põhjustada ka emakakaela kasvajat, mis üle maailma põhjustavad ligikaudu 250000 surmajuhtu igal aastal. See fakt muudab PV-d meditsiiniliselt olulisteks objektideks. Kaua oli saladuseks, kuidas PV-d suudavad aastaid rakkudes püsida ja rakkust rakku liikuda. Nüüdseks on teada, et viiruse genoomid on viiruselise valgu E2 vahendusel seotud peremeesraku kromosoomidele ning kui toimub kromosoomide jagunemine peremeesraku pooldumise ajal, siis viiruse genoomid nö. transporditakse uude tütarrakku koos peremehe kromosoomidega. Oma doktoritöös olengi keskendunud viirusvalgu E2 poolt vahendatud rakust rakku liikumise mehhanismi uurimisele. Meil õnnestus konstrueerida kahest viirusest hübriidsed DNA molekulid, mis E2 valgu juuresolekul olid võimelised rakkudes pikk aega säilima. See töö näitas, et PV vaikolekut tagav element on funktsionaalne ka koos teistest viirustest pärit elementidega. Praeguseks on selle töö tulemused ka patenteeritud ning leidnud tööstuslikku kasutamist. Väga paljud maailmas kasutatavad ravimid on valgud, kuid valkude tootmine on aeganõudev ja kulukas. Meie poolt patenteeritud tehnoloogia kasutab ära PV vaikoleku elementi, et muuta valgutootmine kiiremaks ja ka odavamaks. Tehnoloogia arendamine toimub praeguseks firmas Icosagen Cell Factory OÜ ning mitmed kuulsad ravimifirmad nagu Bayer Healthcare, Novartis, Wyeth, GE Healthcare on omandanud selle tehnoloogia litsentsi. Lisaks rakenduslikule poolele, uurisin oma doktoritöös rakulise valgu Brd4 mõju E2 valgu funktsioonidele ning püüdsin kaardistada need E2-e osad, mis on vaikoleku tagamisel olulised.Papillomaviruses (PVs) are diverse group DNA viruses that have been found in more than 20 different mammalian species, as well as in birds and reptiles. PVs have their life cycle exclusively in body surface tissues such as the skin, or the mucosal surfaces of the genitals, anus, and mouth. Visible symptoms of PV infection are benign tumors- warts, papillomas, condylomas. To date, more then 100 human papillomavirus types (HPV) has been isolated. Many PV occur preferentially in a latent life cycle without causing any visible symptoms. However, some HPVs are responsible for causing cervical cancer, which is source for 250000 deaths every year. It has been longstanding challenge to discover the mechanism for PV stable maintenance- how PVs are maintained in the cells and how they are moving from cell to cell? Nowadays it is known that PV encoded protein E2 tethers viral genomes to the host chromatin. This robust tethering mechanism ensures that viral genomes are more evenly distributed between daughter cells, thus ensuring genome maintenance in dividing cells and persistent viral infection. In current thesis I have focused to E2 mediated segregation process. We succeeded to construct hybrid DNA molecules, which in the presence of E2 protein are stably maintained. We were first to demonstrate that PV stable maintenance element is functional in hetereologues configuration. These findings have been patented and already used in biotechnology. Modern biotechnology and pharmaceutical industry need lots of amounts of pure proteins for screenings in drug development. In addition, many drugs currently on market contain protein component. However, protein production is very time consuming and usually very expensive. Our patented technology enables shorten the time period for protein production and also makes it more inexpensive. Currently the technology is further developed by company Icosagne Cell Factory OÜ and it has already been licensed for such well-known pharmaceutical companies as Bayer Healthcare, Novartis, Wyeth and GE Healthcare. In addition, I studied cellular protein Brd4 influences to E2 protein functions and I characterized E2 domains that are required in stable maintenance process

Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿ †

Effective segregation of the bovine papillomavirus type 1 (BPV1), Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated human herpesvirus type 8 (KSHV) genomes into daughter cells is mediated by a single viral protein that tethers viral genomes to host mitotic chromosomes. The linker proteins that mediate BPV1, EBV, and KSHV segregation are E2, LANA1, and EBNA1, respectively. The N-terminal transactivation domain of BPV1 E2 is responsible for chromatin attachment and subsequent viral genome segregation. Because E2 transcriptional activation and chromatin attachment functions are not mutually exclusive, we aimed to determine the requirement of these activities during segregation by analyzing chimeric E2 proteins. This approach allowed us to separate the two activities. Our data showed that attachment of the segregation protein to chromatin is not sufficient for proper segregation. Rather, formation of a segregation-competent complex which carries multiple copies of the segregation protein is required. Complementation studies of E2 functional domains indicated that chromatin attachment and transactivation functions must act in concert to ensure proper plasmid segregation. These data indicate that there are specific interactions between linker molecules and transcription factors/complexes that greatly increase segregation-competent complex formation. We also showed, using hybrid E2 molecules, that restored segregation function does not involve interactions with Brd4

Brd4 Is Involved in Multiple Processes of the Bovine Papillomavirus Type 1 Life Cycle

Brd4 protein has been proposed to act as a cellular receptor for the bovine papillomavirus type 1 (BPV1) E2 protein in the E2-mediated chromosome attachment and mitotic segregation of viral genomes. Here, we provide data that show the involvement of Brd4 in multiple early functions of the BPV1 life cycle, suggest a Brd4-dependent mechanism for E2-dependent transcription activation, and indicate the role of Brd4 in papillomavirus and polyomavirus replication as well as cell-specific utilization of Brd4-linked features in BPV1 DNA replication. Our data also show the potential therapeutic value of the disruption of the E2-Brd4 interaction for the development of antiviral drugs

Episomal Maintenance of Plasmids with Hybrid Origins in Mouse Cells

Bovine papillomavirus type 1 (BPV1), Epstein-Barr virus (EBV), and human herpesvirus 8 genomes are stably maintained as episomes in dividing host cells during latent infection. The mitotic segregation/partitioning function of these episomes is dependent on single viral protein with specific DNA-binding activity and its multimeric binding sites in the viral genome. In this study we show that, in the presence of all essential viral trans factors, the segregation/partitioning elements from both BPV1 and EBV can provide the stable maintenance function to the mouse polyomavirus (PyV) core origin plasmids but fail to do so in the case of complete PyV origin. Our study is the first which follows BPV1 E2- and minichromosome maintenance element (MME)-dependent stable maintenance function with heterologous replication origins. In mouse fibroblast cell lines expressing PyV large T antigen (LT) and either BPV1 E2 or EBV EBNA1, the long-term episomal replication of plasmids carrying the PyV minimal origin together with the MME or family of repeats (FR) element can be monitored easily for 1 month under nonselective conditions. Our data demonstrate clearly that the PyV LT-dependent replication function and the segregation/partitioning function of the BPV1 or EBV are compatible in certain, but not all, configurations. The quantitative analysis indicates a loss rate of 6% per cell, doubling in the case of MME-dependent plasmids, and 13% in the case of FR-dependent plasmids in nonselective conditions. Our data clearly indicate that maintenance functions from different viruses are principally interexchangeable and can provide a segregation/partitioning function to different heterologous origins in a variety of cells

The long noncoding RNA NEAT1_1 is seemingly dispensable for normal tissue homeostasis and cancer cell growth

NEAT1 is one of the most studied lncRNAs, in part because its silencing in mice causes defects in mammary gland development and corpus luteum formation and protects them from skin cancer development. Moreover, depleting NEAT1 in established cancer cell lines reduces growth and sensitizes cells to DNA damaging agents. However, NEAT1 produces two isoforms and because the short isoform, NEAT1_1, completely overlaps the 5' part of the long NEAT1_2 isoform; the respective contributions of each of the isoforms to these phenotypes has remained unclear. Whereas NEAT1_1 is highly expressed in most tissues, NEAT1_2 is the central architectural component of paraspeckles, which are nuclear bodies that assemble in specific tissues and cells exposed to various forms of stress. Using dual RNA-FISH to detect both NEAT1_1 outside of the paraspeckles and NEAT1_2/NEAT1 inside this nuclear body, we report herein that NEAT1_1 levels are dynamically regulated during the cell cycle and targeted for degradation by the nuclear RNA exosome. Unexpectedly, however, cancer cells engineered to lack NEAT1_1, but not NEAT1_2, do not exhibit cell cycle defects. Moreover, Neat1_1-specific knockout mice do not exhibit the phenotypes observed in Neat1-deficient mice. We propose that NEAT1 functions are mainly, if not exclusively, attributable to NEAT1_2 and, by extension, to paraspeckles.status: publishe

Dual agonistic and antagonistic roles of ZC3H18 provide for co-activation of distinct nuclear RNA decay pathways

Summary: The RNA exosome is a versatile ribonuclease. In the nucleoplasm of mammalian cells, it is assisted by its adaptors the nuclear exosome targeting (NEXT) complex and the poly(A) exosome targeting (PAXT) connection. Via its association with the ARS2 and ZC3H18 proteins, NEXT/exosome is recruited to capped and short unadenylated transcripts. Conversely, PAXT/exosome is considered to target longer and adenylated substrates via their poly(A) tails. Here, mutational analysis of the core PAXT component ZFC3H1 uncovers a separate branch of the PAXT pathway, which targets short adenylated RNAs and relies on a direct ARS2-ZFC3H1 interaction. We further demonstrate that similar acidic-rich short linear motifs of ZFC3H1 and ZC3H18 compete for a common ARS2 epitope. Consequently, while promoting NEXT function, ZC3H18 antagonizes PAXT activity. We suggest that this organization of RNA decay complexes provides co-activation of NEXT and PAXT at loci with abundant production of short exosome substrates

General characterization of SNVs in the UCEs.

<p>(<b>A</b>) Number of SNVs per mega base (Mb) of UCE sequence per sample. SNVs from three data sources- Singaporean Chinese cohort (SG-CHN), Italian cohort (ITA) and 1000 Genome Project (1 KG) were used. SNVs are discriminated according to their minor allele frequency (MAF). Numbers in the parentheses represent sample size used in this study (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110692#s2" target="_blank">Materials and Methods</a>). Random set represents random genomic regions that have the same total length as the UCEs set. Y-axis represents SNVs per Mb divided by sample count in the analyzed population. (<b>B–D</b>) Shared and distinct SNVs between SG-CHN, ITA and 1 KG populations. Venn diagrams of (<b>B</b>) all, (<b>C</b>) prevalent (MAF>0.5%) and rare (<b>D</b>) (MAF<0.5%) SNVs from three analyzed population. Numbers in the parentheses indicate analyzed SNVs in the corresponding population.</p