Browsing by study line "Genetiikka ja genomiikka"

Intestinal stem cells maintain the regenerative potential of intestinal epithelium, which needs to be renewed constantly. Dysregulation of intestinal stem cell proliferation is associated with aging and intestinal diseases. The midgut of Drosophila melanogaster is a valuable model for studying intestinal stem cell driven tissue regeneration. It is similar in function to the mammalian small intestine but has a simpler cellular structure. Drosophila midgut is divided into five major regions with specialized physiological functions, characteristic morphological features and distinctive gene expression profiles. The midgut consists of a monolayer of absorptive enterocytes, small secreting enteroendocrine cells, intestinal stem cells and enteroblasts. Intestinal stem cells differentiate into enterocytes through a transient enteroblast phase. 5-hydroxytryptamine has been shown to activate proliferation of intestinal epithelium in mammals, but its mechanism of action is still unknown. Also, sex-specific differences in 5-hydroxytryptamine signalling have been recorded in mammals. 5-hydroxytryptamine signalling pathway has several downstream targets that have diverse downstream effect. Thus, 5-hydroxytryptamine signalling creates a complex and versatile regulatory network. The aim of my thesis is to study the effects of 5-hydroxytryptamine signalling on intestinal stem cell proliferation and cellular turnover in Drosophila midgut in both sexes. The effects of excessive amounts of 5-hydroxytryptamine are first studied by feeding Drosophila with 5-hydroxytryptophan, a product of the rate-limiting step in the 5-hydroxytryptamine synthesis pathway. The effects of 5-hydroxytryptamine signalling are further studied by knocking down and overexpressing a component of the 5-hydroxytryptamine signalling pathway in intestinal stem cells and enteroblasts. Dissected midguts are immunofluorescently stained, imaged and analysed both visually and with bioinformatics tools. The results indicate that 5-hydroxytryptamine signalling has both regional and sex-specific functions that affect intestinal stem cell proliferation and cellular turnover in Drosophila midgut. The most dramatic effects are seen in cellular turnover, which indicates that 5-hydroxytryptamine signalling plays a role in enteroblast differentiation. Furthermore, the results suggest that bidirectional signalling between enteroblasts and dying enterocytes facilitates cellular turnover in the midgut. As 5-hydroxytryptamine signalling is indicated in inflammatory bowel diseases such as Crohn’s disease, my results might help in the development of treatments for such conditions.

The Y chromosome has an essential role in the genetic sex determination in humans and other mammals. It contains a male-specific region (MSY) which escapes recombination and is inherited exclusively through the male line. The genetic variations inherited together on the MSY can be used in classifying Y chromosomes into haplogroups. Y-chromosomal haplogroups are highly informative of genetic ancestry, thus Y chromosomes have been widely used in tracing human population history. However, given the peculiar biology and analytical challenges specific to the Y chromosome, the chromosome is routinely excluded from genetic association studies. Consequently, potential impacts of Y-chromosomal variation on complex disease remain largely uncharacterized. Lately the access to large-scale biobank data has enabled to extend the Y-chromosomal genetic association studies. A recent UK Biobank study suggested links between Y-chromosomal haplogroup I1 and coronary artery disease (CAD) in the British population, but this result has not been validated in other datasets. Since Finland harbours a notable frequency of Y-chromosomal haplogroup I1, the relationship between haplogroup I1 and CAD can further be inferred in the Finnish population using data from the FinnGen project. The first aim of this thesis was to determine the prevalence of Y-chromosomal haplogroups in Finland and characterize their geographical distributions using genotyping array data from the FinnGen project. The second aim was to assess the role between Finnish Y-chromosomal haplogroups and coronary artery disease (CAD) by logistic regression. This thesis characterized the Y-chromosomal haplogroups in Finland for 24 160 males and evaluated the association between Y-chromosomal haplogroups and CAD in Finland. The dataset used in this study was extensive, providing an opportunity to study the Y-chromosomal variation geographically in Finland and its role in complex disease more accurately compared to previous studies. The geographical distribution of the Y-chromosomal haplogroups was characterized on 20 birth regions, and between eastern and western areas of Finland. Consistent with previous studies, the results demonstrated that two major Finnish Y-chromosomal haplogroup lineages, N1c1 and I1, displayed differing distributions within regions, especially between eastern and western Finland. Results from logistic regression analysis between CAD and Y-chromosomal haplogroups suggested no significant association between haplogroup I1 and CAD. Instead, the major Finnish Y-chromosomal haplogroup N1c1 displayed a decreased risk for CAD in the association analysis when compared against other haplogroups. Moreover, this thesis also demonstrated that the association results were not straightforwardly comparable between populations. For instance, haplogroup I1 displayed a decreased risk for CAD in the FinnGen dataset when compared against haplogroup R1b, whereas the same association was reported as risk increasing for CAD in the UK Biobank. Overall, this thesis demonstrates the possibility to study the genetics of Y chromosome using data from the FinnGen project, and highlights the value of including this part of the genome in the future complex disease studies.

Human induced pluripotent stem cells (hiPSCs) are derived from adult differentiated somatic cells and reprogrammed to an embryonic-like state. Pluripotent stem cells can be differentiated into almost any somatic cell type by using directed differentiation methods, but the differentiation efficiency often varies depending on the cell type. hiPSCs and cells differentiated from them can be used as a disease model carrying the patient’s phenotype and genotype. Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease where both upper and lower motor neurons degenerate, leading to paralysis. There is no curative treatment for ALS, and it leads to the death of the patient in 3 to 5 years on average from the first symptoms. The most common genetic cause of familial ALS is a hexanucleotide repeat expansion in C9orf72-gene. ALS pathology is strongly linked to endoplasmic reticulum (ER) stress, which affects cell homeostasis and proteostasis, and leads to apoptosis when prolonged. The primary aim of this research is to characterize the differentiation of four hiPSCs lines towards lower motor neurons and to study the neuroprotective effects of cerebral dopamine neurotrophic factor (CDNF) and CDNF-derived peptide on ER stress and cell viability. This experiment used two control cell lines from two healthy donors and two patient cell lines from two different ALS patients carrying the C9orf72-mutation. To evaluate the efficiency of the differentiation towards motor neurons, molecular markers for pluripotent and neural progenitor cells as well as for maturated motor neurons were analyzed. Relative gene expression levels were measured from weekly time points with qPCR. Immunocytochemical (ICC) antibody staining was performed during differentiation. Endogenic CDNF levels were analyzed from differentiating cells at weekly time points and the effect of CDNF on Thapsigargin (TG) -induced ER stress in motor neurons was analyzed. In addition, cell viability was analyzed in TG-CDNF treatment. All pluripotent and progenitor markers were downregulated in differentiated cells, and the expression of the mature motor neuron markers was upregulated. Mature motor neuron markers were also expressed at the protein level. The endogenous CDNF levels were highest at the progenitor cell stage. The ER stress response was upregulated in TG-treated cells, and there were no differences between treatments against ER stress. Furthermore, TG and growth factor treatments differentially affected the viability of the control and patient cell lines. Treatment decreased viability in control cell lines and increased viability in patient cell lines. Pluripotent stem cells were successfully differentiated toward motor neurons. The differentiation was performed twice, and the results were similar on both individual biological repeats. Analysis of endogenous CDNF expression levels was performed for the first time on hiPSCs lines. In this study, CDNF or its derivate didn’t reduce ER stress but it influenced cell viability, especially in patient cell lines with growth factor treatment. In the future, TG-treatment could be optimized regarding timing and growth factor treatment, or the toxin could be changed to another ER-stress inducing toxin. In addition, the C9orf72 pathology should be identified in order to use differentiated motor neurons as a pre-clinical disease model.

Enhancers are important regulatory elements of DNA, that are bound by transcription factors (TFs) to regulate gene expression. Enhancers control cell type specific gene expression and they can form structures called super-enhancers, that consist of multiple normal enhancers and are bound by high numbers and variety of transcription factors. These super-enhancers are important for defining cell identity and changes in the super-enhancer landscape have been linked to different cancers. In this project, characterization of super-enhancers and their transcription factors composition between primary and cancer cells were studied using genome-wide next-generation sequencing data from multiple assays, such as ChIP-seq, RNA-seq and ATAC-seq. The focus of the project was on the data processing and analysis to identify and characterize the super-enhancers. Analyses included GSEA, heatmap binding analysis, peak and super-enhancer calling and IGV analysis. This project used pancreatic HPDE cell line for primary cells and different cancers with endodermal origin as cancer cell lines. The goal of the thesis was to try show characteristic features of super-enhancers and their features in normal and cancer cells. Data analysis showed that distinct super-enhancers can be identified in cancer cells and defined super-enhancers had typical strong binding for specific transcription factor and histone modification such as histone 3 lysine 27 acetylation (H3K27ac) mark of active enhancers. Super-enhancer regions were located in highly accessible chromatin regions of the genome, and genes that were associated with HPDE super-enhancers could be shown to have association with cell identity. Peak and super-enhancer calling counts varied between cell lines for transcription factors, histone modifications and super-enhancers. Visualization of super-enhancers was successful and could show transcription factor binding and active enhancers that establish the super-enhancer structure. Comprehensive analyses allowed us to characterize typical features of super-enhancers and show differences in the numbers of super-enhancers between primary and cancer cell lines and cancer cell lines of different organ types. Analysis of the transcription factor binding showed unique peaks on some of the super-enhancers, and these peaks might have a role in inducing the super-enhancer structure.

Spinal muscular atrophy of Jokela type (SMAJ) is an autosomal dominant motor-neuron disease caused by a missense mutation c.197G>T, p.G66V in the gene CHCHD10. Coiled-coil-helix-coiled-coil-helix domain-containing protein 10 (CHCHD10) is a nuclear-encoded mitochondrial protein located in the intermembrane space (IMS) of mitochondria with an unknown exact function and disease-causing mechanism. In this project, the overarching aim was to correct a heterozygous SMAJ-causing mutation in patient myoblast cells with CRISPR-Cas9 genome editing. The goal was to create a genetically identical, isogenic, cell line to study only the effects of the mutation on cellular phenotype in vitro. Human myoblast cells isolated from patient biopsies provide the most pertinent experimental model to study neuromuscular atrophy-associated mutations in their natural genomic environment. More specific aims included genome editing optimization with myoblast cells, since it is not as widely conducted as with some other cell types, such as iPSCs. CRISPR-Cas9 ribonucleoprotein (RNP) complex and associated donor template were used to induce homology-directed repair (HDR) in the genome of patient-derived myoblast cells and correct the mutation. After optimization of electroporation conditions for myoblast cells, guide RNAs were designed and transfected into patient myoblasts. Clonal cell lines were made by utilizing techniques such as fluorescence adjusted cell sorting (FACS) and manual colony picking. The success and precision of genome editing were analyzed by Sanger sequencing, comparing the performance of the different guide RNAs with restriction enzyme analysis and Synthego ICE CRISPR web tool, and screening regions of potential off-target genome editing. A genome-edited myoblast cell line with the CHCHD10 c.197G>T mutation corrected, was successfully generated to provide an isogenic control for the patient myoblast cell line. Optimization of myoblast electroporation was successful and conditions used proved to be effective. Clonal cell line creation proved to be challenging with myoblast cells and work is still needed to improve the viability of single-cell clones after FACS. Nevertheless, the advances taken here regarding myoblast genome editing with CRISPR-Cas9 offer a fertile avenue for future research of myoblasts genome manipulation, myogenic disorders, and the role of CHCHD10 in skeletal muscle and SMAJ. Comparing the CHCHD10 protein level and mRNA expression between patient cells, corrected myoblasts, and differentiated myotubes is an area of future research. Future work also includes measuring the mitochondrial integrated stress response in both cell lines and co-culturing myotubes and iPSC derived motor neurons to study the effects of p.G66V on neuromuscular junction (NMJ) formation.

Uterine leiomyomas (ULs) are common benign tumors that originate from the smooth muscle cells of the uterine wall known as the myometrium. Around 70% of pre-menopausal women are affected by these tumors. The high prevalence of ULs is a significant public health issue and ULs are the leading cause for hysterectomy. Many tumors remain asymptomatic, but 15-30% of affected women develop symptoms ranging from pain and heavy menstrual bleeding to pregnancy complications and infertility. Despite their common occurrence, the underlying mechanisms of UL genesis are still largely unknown. Based on mutually exclusive recurring genetic alterations, ULs can be divided into molecular subclasses. Three main molecular subclasses have been established: MED12 mutated tumors, HMGA2 overexpressing tumors and tumors with biallelic FH inactivation. Combined, these three subclasses represent around 90% of ULs, indicating that additional smaller molecular subclasses also exist. Recently, novel mutations associated with ULs have been identified in genes encoding for subunits of the SRCAP chromatin remodeling complex that deposits histone variant H2A.Z onto chromatin. These included loss-of-function mutations in YEATS4, DMAP1 and ZNHIT1, and resulted in deficient H2A.Z loading in the tumors. The detailed functional consequences of these driver mutations need to be further investigated to fully understand their significance in UL genesis. This work aimed to elucidate the effects of YEATS4 mutations by characterizing previously established CRISPR-Cas9 edited immortalized human myometrial cell models carrying heterozygous mutations in YEATS4 using various molecular biology methods. Subcellular fractionation and western blot analysis was used to detect chromatin bound H2A.Z from cell lysates. Quantitative PCR was performed to determine relative YEATS4 expression levels in mutated and wild-type cells. No significant reduction of chromatin bound H2A.Z or YEATS4 expression was observed in the studied heterozygous mutants when compared to wild-type immortalized myometrial smooth muscle cells. Additional myometrial cell models were created by CRISPR-Cas9 gene editing. Objective was to achieve homozygous YEATS4 mutations to better reflect the changes previously reported in ULs. One homozygous YEATS4 mutant cell line was achieved. Understanding the detailed molecular mechanisms behind UL genesis will be instrumental for developing curative non-invasive therapies in the future. Insight into dysregulated pathways and identification of UL biomarkers could improve diagnostic accuracy and help design personalized targeted therapies effective for specific UL subclasses. Characterization of each molecular subclass offers a unique opportunity to understand UL genesis.

Cytokine release syndrome is a severe systematic inflammatory disease that can be triggered upon pharmaceuticals intake. Evaluating the potential risk levels of novel therapeutics with an optimal assay is therefore essential. In this study, we tried to set up and validate a cytokine release assay from human peripheral blood mononuclear cells (PBMCs) for its application in nonclinical immunotoxicity assessments. Fresh PBMCs were isolated from buffy coats obtained from 11 healthy donors of different characteristics. Freshly isolated PBMCs were treated with LPS, positive control antibodies (anti-CD28, anti-CD3) and their corresponding isotypes (negative control antibodies) in both aqueous and solid formats to assess their abilities to induce cytokine release. Similarly, cryopreserved frozen PBMCs were also incubated with LPS, the positive control antibodies and the negative control antibodies, and compared their cytokine releasing capacities with freshly isolated PBMCs. A nine-cytokine panel (IFNγ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, TNFα, IL-12) was screened to select four cytokines (IFNγ, IL-2, IL-6, TNFα) in the following experimental setup. Freshly isolated PBMCs appeared to have higher sensitivity in response to the treatments as shown by the higher level of cytokine release. However, similar trends of cytokine release were observed between freshly isolated and frozen PBMCs in both aqueous and solid assay formats. LPS and anti-CD3 strongly induced cytokine release in all donors. Conversely, anti-CD28 induced cytokine release in some, but not all donors, possibly due to donor specificity. In summary, we have successfully developed and optimized a cytokine release assay, and it can be used to test the potential risk of immune-modulating drug candidate in the preclinical safety studies.

Multiple myeloma (MM) is a heterogeneous plasma cell cancer that results from the excessive proliferation of mutated B cells in the bone marrow and the accumulation of ineffective antibodies, monoclonal proteins, in the blood. Despite recent advances in research and novel therapeutics, MM remains incurable, mainly due to the mechanisms underlying disease progression and drug resistance. Therefore, novel biomarkers and therapeutics for the treatment of relapsed and refractory MM are urgently needed. MicroRNAs (miRNAs), short non-coding RNA molecules that play a key role in post-transcriptional gene regulation, have been found to be associated with different hallmarks of MM. Previous studies have indicated that abnormally functioning miRNA-mediated gene regulation followed by oncogene activation and tumor suppressor gene silencing results in drastic alterations in cell proliferation, apoptosis, growth, and metabolism. These changes in cellular functions have been indicated to be associated with the pathogenesis, progression, and formation of drug resistance in MM. Therefore, the role and potential of miRNAs to act as biomarkers to predict MM progression and drug sensitivity should be further investigated to ultimately improve the survival rates of patients. The aim of this master’s thesis was to investigate the relationships between drug sensitivity, disease progression and miRNA regulation in MM patients. Bioinformatically predicted miRNAs identified to be associated with sensitivity to panobinostat, a novel histone deacetylase inhibitor, and MM progression were validated in MM patient samples by using real-time quantitative reverse transcription PCR (RT-qPCR). In addition, the specific gene targets of miRNAs involved in the regulation of drug responses and MM progression were predicted by identifying statistically significant, negatively correlated interactions between the miRNA and RNA sequencing data of 45 MM patients in pairwise comparative correlation analysis. Finally, the predicted miRNA targets genes were validated in MM patient samples using RT-qPCR. Based on the bioinformatic analyses and RT-qPCR validation, mir-424 expression was significantly increased in relapsed MM patients as compared to respective patient samples taken at diagnosis, suggesting a potential role of mir-424 in MM progression. Similarly, mir-4433b expression was significantly elevated in panobinostat-resistant patients compared to sensitive patients, suggesting a potential effect of mir-4433b on the regulation of panobinostat drug response in MM patients. In addition, the RT-qPCR validation demonstrated that the disease progression and drug sensitivity associated mir-92b, mir-363 and mir-221, would potentially regulate the expression of FGF2, MFF, and TMEM248, respectively, providing novel insights into the functional roles of miRNAs in MM pathways.

The intestinal stem cells (ISCs) adapt in response to environmental factors and continually proliferate to renew the mammalian intestinal epithelium due to its rapid turnover. Overall, intestinal homeostasis is maintained by the differentiation and self-renewal of ISCs, which are regulated by different mechanisms, including epigenetic histone modifications. Earlier studies in the host laboratory have shown that the histone methyltransferase Su(var)3-9 is essential in the nutrient-induced activation of intestinal stem cells. Su(var)3-9 specifically trimethylates histone H3 on lysine 9 (H3K9me3), which is a repressive histone mark, responsible for transcriptional silencing at heterochromatin regions. It influences stem cell maturation, lineage specification, and many other cellular processes. However, the precise mechanisms behind its function in ISCs remain unknown – that knowledge is important for understanding the development of many diseases, including cancer and metabolic disorders. This thesis aimed to investigate the distribution of the heterochromatin mark H3K9me3 in the intestine with an emphasis on ISCs, using the Drosophila midgut and mouse intestinal organoids as models. Confocal microscopy was used together with cell-type-specific fluorescent staining, to obtain the expression of the H3K9me3 specific histone methyltransferase Su(var)3-9, in the midgut. An antibody was used for the detection of H3K9me3 distribution along the anterior/posterior axis in Su(var)3-9 overexpressed flies. Additionally, DNA adenine methyltransferase identification (DamID) was applied in order to find target genes of the H3K9me3 regulation in the genome with the specific chromo domain of M-phase phosphoprotein 8 (MPHOSPH8) that binds to H3K9me3. The number of lineage-labeled differentiated enterocytes was shown to be locally higher in the Su(var)3-9 overexpressed flies compared with the control, although the flies were on starvation without nutrient-induced activation. Moreover, the number of lineage-labeled progenitor cells was not remarkably altered between the samples. However, the intensity of H3K9me3 was significantly higher throughout the whole midguts in the Su(var)3-9 overexpressed flies in comparison to the control. According to one replicate, the DamID in mouse intestinal organoids revealed that the peaks of H3K9me3 were divergent between the samples grown in different conditions. The first sample was assumed to contain more ISCs, whereas the other one was assumed to contain more differentiated intestinal cells. According to my results, the Su(var)3-9 overexpression drives the stem cells against the differentiation of enterocytes. Furthermore, the MPHOSPH8 chromo domain in the organoids was successfully applied in DamID; thus, more replicates should be prepared for additional analysis, because I found several potential target genes of H3K9me3. In the future, it is important to further study the epigenetic regulation of ISCs, for applying the epigenetic marks as targets for the treatment of many human pathophysiological conditions, such as cancer, obesity, and metabolic disorders.

The group has identified two rare, previously uncharacterized missense variants in the YBX3 gene in a Finnish patient presenting with an unusual form of nemaline myopathy. The patient also inherited two biallelic TPM3 variants, one RYR1 variant from the father and one SRPK3 variant from the mother. TPM3 and RYR1 are known nemaline myopathy causing genes and the other variants identified in the patients, including the YBX3 variants, are thought to have a modifying effect on the phenotype. YBX3 encodes Y-box binding protein 3 (YB-3) and, YB-3 is a member of the Y-box binding (YB) protein family, that in addition to YB-3 consists of YB-1 and YB-2. The YB-proteins have mainly been studied in the context of cancer, with most studies focusing on YB-1. Studies indicate the ability of YB-proteins to compensate for the loss of one homolog suggesting functional redundancy between YB-3 and YB-1, and YB-3 and YB-2. Compared to its homologs, YB-3 is highly expressed in skeletal muscle. The aim of this thesis was to try out a new cell culturing method when investigating the role of YB-3 in the differentiation of myoblasts into myotubes. MSY-3 is the murine orthologue of YB-3. MSY3-knockdown mouse C2C12 myoblast lines were established using GIPZ lentiviral short hairpin constructs and by selection with puromycin. The success of transfection was determined using qPCR. The myoblasts were differentiated for 20 days on a gelatin hydrogel surface to support long-term culture and to provide phenotypes of higher physiological relevance with improved contractile maturity. Myoblasts cultured on coverslips were immunofluorescently stained for MSY-3. HeLa cells were transfected with a construct encoding N-terminally FLAG-tagged human YB-3 in a pcDNA-vector. YB-3-FLAG was purified using anti-FLAG magnetic beads. The eluated immunoprecipitation sample was sent to N-terminal sequencing to obtain information on post-translational modifications, to support further experiments regarding the post-translational cleavage of YB-3. N-terminal sequencing revealed an enrichment of YB-3 and YB-1 in the immunoprecipitation sample but not of YB-2, and previously undescribed post-translational modifications were identified. The MSY3-knockdown myotubes exhibited no spontaneous twitching on the hydrogel, while the control C2C12 myotubes twitched frequently. Misalignment of the MSY3-knockdown myotubes and changes in morphology was also observed in one of the MSY3-knockdown cell lines. This suggests that differentiating myoblasts on gelatin hydrogel is a potential strategy for studying the functions of YB-3 in myoblast differentiation and to elucidate its role in skeletal muscle.