Laskennallisia menetelmiä säilyneiden geenisäätelyelementtien analyysiin ja paikallistamiseen DNA:sta

This thesis presents methods for locating and analyzing cis-regulatory DNA elements involved with the regulation of gene expression in multicellular organisms. The regulation of gene expression is carried out by the combined effort of several transcription factor proteins collectively binding the DNA on the cis-regulatory elements. Only sparse knowledge of the 'genetic code' of these elements exists today. An automatic tool for discovery of putative cis-regulatory elements could help their experimental analysis, which would result in a more detailed view of the cis-regulatory element structure and function. We have developed a computational model for the evolutionary conservation of cis-regulatory elements. The elements are modeled as evolutionarily conserved clusters of sequence-specific transcription factor binding sites. We give an efficient dynamic programming algorithm that locates the putative cis-regulatory elements and scores them according to the conservation model. A notable proportion of the high-scoring DNA sequences show transcriptional enhancer activity in transgenic mouse embryos. The conservation model includes four parameters whose optimal values are estimated with simulated annealing. With good parameter values the model discriminates well between the DNA sequences with evolutionarily conserved cis-regulatory elements and the DNA sequences that have evolved neutrally. In further inquiry, the set of highest scoring putative cis-regulatory elements were found to be sensitive to small variations in the parameter values. The statistical significance of the putative cis-regulatory elements is estimated with the Two Component Extreme Value Distribution. The p-values grade the conservation of the cis-regulatory elements above the neutral expectation. The parameter values for the distribution are estimated by simulating the neutral DNA evolution. The conservation of the transcription factor binding sites can be used in the upstream analysis of regulatory interactions. This approach may provide mechanistic insight to the transcription level data from, e.g., microarray experiments. Here we give a method to predict shared transcriptional regulators for a set of co-expressed genes. The EEL (Enhancer Element Locator) software implements the method for locating putative cis-regulatory elements. The software facilitates both interactive use and distributed batch processing. We have used it to analyze the non-coding regions around all human genes with respect to the orthologous regions in various other species including mouse. The data from these genome-wide analyzes is stored in a relational database which is used in the publicly available web services for upstream analysis and visualization of the putative cis-regulatory elements in the human genome.Kun ihmisen genomi saatiin sekvensoitua eli ihmisen geenit oli löydetty ja eritelty vuosituhannen alussa, tiedemiehet yllättyivät ihmisen geenien pienestä määrästä. Ihmisellä havaittiin olevan vain vähän enemmän geenejä kuin yksinkertaisella sukkulamadolla. Koska geenien lukumäärä ei pystykään selittämään ihmisen ja sukkulamadon ulkoisia eroavaisuuksia, selitystä ruvettiin etsimään geenien toiminnan eroista. Geenien toimintaa säädellään monisoluisissa eliöissä hyvin tarkasti tiettyyn paikkaan ja tiettyyn osaan ruumista. Tietyt proteiinit toteuttavat geenien säätelyä sitoutumalla tiettyihin kohtiin DNA:ta säädeltävän geenin läheisyydessä. Näiden DNA:han sitoutumiskohtien löytäminen genomista on kokeellisesti hyvin haastavaa: ne saattavat sijaita hyvin kaukana säädeltävästä geenistä eikä proteiinien sitoutumissääntöjä tunneta vielä kovin hyvin. Väitöstyössä on kehitetty laskennallisia menetelmiä geenisäätelyyn liittyvien DNA sitoutumiskohtien paikantamiseen eri nisäkkäiden genomeja vertailemalla. Esimerkiksi ihmisen ja hiiren genomeja vertailemalla voidaan paikantaa DNA:n pätkiä, jotka ovat olleet hiirien ja ihmisten viimeisessä yhteisessä esivanhemmassa noin 65 miljoonaa vuotta sitten ja lisäksi vaikuttavat mahdollisilta proteiinien sitoutumiskohdilta. Tällaisia mahdollisia DNA:han sitoutumiskohtia on löydetty ihmisen genomista tuhansia, ja osan niistä on kokeellisesti havaittu säätelevän lähellä sijaitsevaa geeniä. Sitoutumiskohtien analysointiin kehitettiin väitöstutkimuksessa menetelmä, jolla voidaan ennustaa geenijoukoille säätelyproteiineja. Nykyaikaiset tehoseulontamenetelmät löytävät nopeasti geenijoukkoja, joilla on jokin kiinnostava ominaisuus, jonka säätelystä ollaan kiinnostuneita. Kehitetyllä menetelmällä voidaan helposti ennustaa esimerkiksi tiettyyn sairauteen liittyvien geenien säätelijä. Kun mahdollinen säätelijäproteiini tunnetaan, sitä vastaan voidaan kehittää lääke. Työn tulokset antavat uusia menetelmiä erityisesti vaikeasti tutkittavien yksilönkehityksen aikana säädeltyjen geenien analyysiin. Kehitettyjen menetelmien lääketieteelliset sovellukset liittyvät esimerkiksi kudosspesifiin kasvun säätelyyn ja syöpägeenien kasvainspesifisyyteen. Nämä sovellukset pyrkivät selvittämään mm. syytä ihmisen suhteettoman suurille aivoille ja pienille lihaksille ja toisaalta pyrkivät avaamaan uusia lähestymistapoja esimerkiksi syövän diagnostiikkaan ja hoitoon

Integrating sequence, evolution and functional genomics in regulatory genomics

Finding transcription factor binding sites in regulatory regions of the genom

Vitamin C boosts DNA demethylation in TET2 mutation carriers

Background Accurate regulation of DNA methylation is necessary for normal cells to differentiate, develop and function. TET2 catalyzes stepwise DNA demethylation in hematopoietic cells. Mutations in the TET2 gene predispose to hematological malignancies by causing DNA methylation overload and aberrant epigenomic landscape. Studies on mice and cell lines show that the function of TET2 is boosted by vitamin C. Thus, by strengthening the demethylation activity of TET2, vitamin C could play a role in the prevention of hematological malignancies in individuals with TET2 dysfunction. We recently identified a family with lymphoma predisposition where a heterozygous truncating germline mutation in TET2 segregated with nodular lymphocyte-predominant Hodgkin lymphoma. The mutation carriers displayed a hypermethylation pattern that was absent in the family members without the mutation.Methods In a clinical trial of 1 year, we investigated the effects of oral 1 g/day vitamin C supplementation on DNA methylation by analyzing genome-wide DNA methylation and gene expression patterns from the family members.Results We show that vitamin C reinforces the DNA demethylation cascade, reduces the proportion of hypermethylated loci and diminishes gene expression differences between TET2 mutation carriers and control individuals.Conclusions These results suggest that vitamin C supplementation increases DNA methylation turnover and provide a basis for further work to examine the potential benefits of vitamin C supplementation in individuals with germline and somatic TET2 mutations.Peer reviewe

Detection of subclonal L1 transductions in colorectal cancer by long-distance inverse-PCR and Nanopore sequencing

Long interspersed nuclear elements-1 (L1s) are a large family of retrotransposons. Retrotransposons are repetitive sequences that are capable of autonomous mobility via a copy-and-paste mechanism. In most copy events, only the L1 sequence is inserted, however, they can also mobilize the flanking non-repetitive region by a process known as 3' transduction. L1 insertions can contribute to genome plasticity and cause potentially tumorigenic genomic instability. However, detecting the activity of a particular source L1 and identifying new insertions stemming from it is a challenging task with current methodological approaches. We developed a long-distance inverse PCR (LDI-PCR) based approach to monitor the mobility of active L1 elements based on their 3' transduction activity. LDI-PCR requires no prior knowledge of the insertion target region. By applying LDI-PCR in conjunction with Nanopore sequencing (Oxford Nanopore Technologies) on one L1 reported to be particularly active in human cancer genomes, we detected 14 out of 15 3' transductions previously identified by whole genome sequencing in two different colorectal tumour samples. In addition we discovered 25 novel highly subclonal insertions. Furthermore, the long sequencing reads produced by LDI-PCR/Nanopore sequencing enabled the identification of both the 5' and 3' junctions and revealed detailed insertion sequence information.Peer reviewe

Multiple clinical characteristics separate MED12-mutation-positive and -negative uterine leiomyomas

Up to 86% of uterine leiomyomas harbour somatic mutations in mediator complex subunit 12 (MED12). These mutations have been associated with conventional histology, smaller tumour size, and larger number of tumours within the uterus. Prior studies, with limited sample sizes, have failed to detect associations between other clinical features and MED12 mutations. Here, we prospectively collected 763 uterine leiomyomas and the corresponding normal myometrial tissue from 244 hysterectomy patients, recorded tumour characteristics, collected clinical data from medical records, and screened the tissue samples for MED12 mutations to assess potential associations between clinical variables and mutation status. Out of 763 leiomyomas, 599 (79%) harboured a MED12 mutation. In the analysis of tumour characteristics, positive MED12-mutation status was significantly associated with smaller tumour size, conventional histology, and subserous location, relative to intramural. In the analysis of clinical variables, the number of MED12-mutation-positive tumours showed an inverse association with parity, and the number of mutation-negative tumours showed a positive association with a history of pelvic inflammatory disease. This study confirmed the previously reported differences and discovered novel differentiating features for MED12-mutation-positive and -negative leiomyomas. These findings emphasise the relevance of specific driver mutations in genesis and presentation of uterine leiomyomas.Peer reviewe

No evidence of EMAST in whole genome sequencing data from 248 colorectal cancers

Microsatellite instability (MSI) is caused by defective DNA mismatch repair (MMR), and manifests as accumulation of small insertions and deletions (indels) in short tandem repeats of the genome. Another form of repeat instability, elevated microsatellite alterations at selected tetranucleotide repeats (EMAST), has been suggested to occur in 50% to 60% of colorectal cancer (CRC), of which approximately one quarter are accounted for by MSI. Unlike for MSI, the criteria for defining EMAST is not consensual. EMAST CRCs have been suggested to form a distinct subset of CRCs that has been linked to a higher tumor stage, chronic inflammation, and poor prognosis. EMAST CRCs not exhibiting MSI have been proposed to show instability of di- and trinucleotide repeats in addition to tetranucleotide repeats, but lack instability of mononucleotide repeats. However, previous studies on EMAST have been based on targeted analysis of small sets of marker repeats, often in relatively few samples. To gain insight into tetranucleotide instability on a genome-wide level, we utilized whole genome sequencing data from 227 microsatellite stable (MSS) CRCs, 18 MSI CRCs, 3 POLE-mutated CRCs, and their corresponding normal samples. As expected, we observed tetranucleotide instability in all MSI CRCs, accompanied by instability of mono-, di-, and trinucleotide repeats. Among MSS CRCs, some tumors displayed more microsatellite mutations than others as a continuum, and no distinct subset of tumors with the previously proposed molecular characters of EMAST could be observed. Our results suggest that tetranucleotide repeat mutations in non-MSI CRCs represent stochastic mutation events rather than define a distinct CRC subclass.Peer reviewe

Genetic predisposition to uterine leiomyoma is determined by loci for genitourinary development and genome stability

Uterine leiomyomas (ULs) are benign tumors that are a major burden to women's health. A genome-wide association study on 15,453 UL cases and 392,628 controls was performed, followed by replication of the genomic risk in six cohorts. Effects of the risk alleles were evaluated in view of molecular and clinical characteristics. 22 loci displayed a genome-wide significant association. The likely predisposition genes could be grouped to two biological processes. Genes involved in genome stability were represented by TERT, TERC, OBFC1 - highlighting the role of telomere maintenance - TP53 and ATM. Genes involved in genitourinary development, WNT4, WT1, SALL1, MED12, ESR1, GREB1, FOXO1, DMRT1 and uterine stem cell marker antigen CD44, formed another strong subgroup. The combined risk contributed by the 22 loci was associated with MED12 mutation-positive tumors. The findings link genes for uterine development and genetic stability to leiomyomagenesis, and in part explain the more frequent occurrence of UL in women of African origin.Peer reviewe

Exome and immune cell score analyses reveal great variation within synchronous primary colorectal cancers

BACKGROUND: Approximately 4% of colorectal cancer (CRC) patients have at least two simultaneous cancers in the colon. Due to the shared environment, these synchronous CRCs (SCRCs) provide a unique setting to study colorectal carcinogenesis. Understanding whether these tumours are genetically similar or distinct is essential when designing therapeutic approaches. METHODS: We performed exome sequencing of 47 primary cancers and corresponding normal samples from 23 patients. Additionally, we carried out a comprehensive mutational signature analysis to assess whether tumours had undergone similar mutational processes and the first immune cell score analysis (IS) of SCRC to analyse the interplay between immune cell invasion and mutation profile in both lesions of an individual. RESULTS: The tumour pairs shared only few mutations, favouring different mutations in known CRC genes and signalling pathways and displayed variation in their signature content. Two tumour pairs had discordant mismatch repair statuses. In majority of the pairs, IS varied between primaries. Differences were not explained by any clinicopathological variable or mutation burden. CONCLUSIONS: The study shows major diversity within SCRCs. Rather than rely on data from one tumour, our study highlights the need to evaluate both tumours of a synchronous pair for optimised targeted therapy.Peer reviewe

Genome-wide analysis of ETS-family DNA-binding in vitro and in vivo

Peer reviewe

Lack of an association between gallstone disease and bilirubin levels with risk of colorectal cancer : a Mendelian randomisation analysis

BACKGROUND: Epidemiological studies of the relationship between gallstone disease and circulating levels of bilirubin with risk of developing colorectal cancer (CRC) have been inconsistent. To address possible confounding and reverse causation, we examine the relationship between these potential risk factors and CRC using Mendelian randomisation (MR). METHODS: We used two-sample MR to examine the relationship between genetic liability to gallstone disease and circulating levels of bilirubin with CRC in 26,397 patients and 41,481 controls. We calculated the odds ratio per genetically predicted SD unit increase in log bilirubin levels (ORSD) for CRC and tested for a non-zero causal effect of gallstones on CRC. Sensitivity analysis was applied to identify violations of estimator assumptions. RESULTS: No association between either gallstone disease (P value = 0.60) or circulating levels of bilirubin (ORSD = 1.00, 95% confidence interval (CI) = 0.96-1.03, P value = 0.90) with CRC was shown. CONCLUSIONS: Despite the large scale of this study, we found no evidence for a causal relationship between either circulating levels of bilirubin or gallstone disease with risk of developing CRC. While the magnitude of effect suggested by some observational studies can confidently be excluded, we cannot exclude the possibility of smaller effect sizes and non-linear relationships.Peer reviewe