Molecular Evolution of Metazoan Hypoxia-inducing Factors

Most metazoans rely on aerobic energy production, which is dependent on adequate oxygen supply. In the case of reduced oxygen supply (hypoxia), the most profound changes in gene expression are mediated by transcription factors named hypoxia-inducible factors (HIF alpha). These proteins are post-translationally regulated by prolyl-4-hydroxylase (PHD) enzymes that are direct “sensors” of cellular oxygen levels. This thesis examines the molecular evolution of metazoan HIF systems. In early metazoans the HIF system emerged from pre-existing PHD oxygen sensors and early bHLH-PAS transcription factors. In invertebrates our analysis revealed an unexpected diversity of PHD genes and HIF alpha sequence characteristics. An early branching vertebrate, the epaulette shark (Hemiscyllium ocellatum) was chosen for sequencing and hypoxia preconditioning studies of HIF alpha and PHD genes. As no quantitative PCR reference genes were available, this thesis includes the first study of reference genes in cartilaginous fish species. Applying multiple statistical analysis we also discoveredthat commonly used reference gene software may perform poorly with some data sets. Novel reference genes allowed accurate measurements of the mRNAlevels of the studied target genes. Cartilaginous fishes have three genomic duplicates of both HIF alpha and PHD genes like mammals and teleost fishes. Combining functional divergence and selection analyses it was possible to describe how sequence changes in both HIF alpha and PHD duplicates may have contributed to the differential oxygen sensitivityof HIF alphas. Additionally, novel teleost HIF-1 alpha sequences were produced and used to reveal the molecular evolution of HIF-1 alpha in this lineage rich with hypoxia tolerant species.Siirretty Doriast

Computational strategies for single-cell multi-omics integration

Single-cell omics technologies are currently solving biological and medical problems that earlier have remained elusive, such as discovery of new cell types, cellular differentiation trajectories and communication networks across cells and tissues. Current advances especially in single-cell multi-omics hold high potential for breakthroughs by integration of multiple different omics layers. To pair with the recent biotechnological developments, many computational approaches to process and analyze single-cell multi-omics data have been proposed. In this review, we first introduce recent developments in single-cell multi-omics in general and then focus on the available data integration strategies. The integration approaches are divided into three categories: early, intermediate, and late data integration. For each category, we describe the underlying conceptual principles and main characteristics, as well as provide examples of currently available tools and how they have been applied to analyze single-cell multi-omics data. Finally, we explore the challenges and prospective future directions of single-cell multi-omics data integration, including examples of adopting multi-view analysis approaches used in other disciplines to single-cell multi-omics.</p

Histone H3K4me3 breadth in hypoxia reveals endometrial core functions and stress adaptation linked to endometriosis

Trimethylation of histone H3 at lysine 4 (H3K4me3) is a marker of active promoters. Broad H3K4me3 promoter domains have been associated with cell type identity, but H3K4me3 dynamics upon cellular stress have not been well characterized. We assessed this by exposing endometrial stromal cells to hypoxia, which is a major cellular stress condition. We observed that hypoxia modifies the existing H3K4me3 marks and that promoter H3K4me3 breadth rather than height correlates with transcription. Broad H3K4me3 domains mark genes for endometrial core functions and are maintained or selectively extended upon hypoxia. Hypoxic extension of H3K4me3 breadth associates with stress adaptation genes relevant for the survival of endometrial cells including transcription factor KLF4, for which we found increased protein expression in the stroma of endometriosis lesions. These results substantiate the view on broad H3K4me3 as a marker of cell identity genes and reveal participation of H3K4me3 extension in cellular stress adaptation

Overexpression of Human Estrogen Biosynthetic Enzyme Hydroxysteroid (17beta) Dehydrogenase Type 1 Induces Adenomyosis-like Phenotype in Transgenic Mice

Hydroxysteroid (17beta) dehydrogenase type 1 (HSD17B1) is an enzyme that converts estrone to estradiol, while adenomyosis is an estrogen-dependent disease with poorly understood pathophysiology. In the present study, we show that mice universally over-expressing human estrogen biosynthetic enzyme HSD17B1 (HSD17B1TG mice) present with adenomyosis phenotype, characterized by histological and molecular evaluation. The first adenomyotic changes with endometrial glands partially or fully infiltrated into the myometrium appeared at the age of 5.5 months in HSD17B1TG females and became more prominent with increasing age. Preceding the phenotype, increased myometrial smooth muscle actin positivity and increased amount of glandular myofibroblast cells were observed in HSD17B1TG uteri. This was accompanied by transcriptomic upregulation of inflammatory and estrogen signaling pathways. Further, the genes upregulated in the HSD17B1TG uterus were enriched with genes previously observed to be induced in the human adenomyotic uterus, including several genes of the NFKB pathway. A 6-week-long HSD17B1 inhibitor treatment reduced the occurrence of the adenomyotic changes by 5-fold, whereas no effect was observed in the vehicle-treated HSD17B1TG mice, suggesting that estrogen is the main upstream regulator of adenomyosis-induced uterine signaling pathways. HSD17B1 is considered as a promising drug target to inhibit estrogen-dependent growth of endometrial disorders. The present data indicate that HSD17B1 over-expression in TG mice results in adenomyotic changes reversed by HSD17B1 inhibitor treatment and HSD17B1 is, thus, a potential novel drug target for adenomyosis.</p

Evolutionary origins of oxygen sensing in animals

Oxygen is required for aerobic energy production but its levels have to be tightly regulated to avoid deleterious effects. Thus, animals have evolved mechanisms to monitor and respond to fluctuations in oxygen availability. Here, the evolution of the HIF system is discussed in light of a report that reveals its presence in the simplest animal

Molecular evolution of the metazoan PHD-HIF oxygen-sensing system

Metazoans rely on aerobic energy production, which requires an adequate oxygen supply. During reduced oxygen supply (hypoxia), the most profound changes in gene expression are mediated by transcription factors known as hypoxia-inducible factors (HIFs). HIF alpha proteins are commonly posttranslationally regulated by prolyl-4-hydroxylase (PHD) enzymes, which are direct ‘‘sensors’ ’ of cellular oxygen levels. We examined the molecular evolution of the metazoan PHD– HIF oxygen-sensing system by constructing complete phylogenies for PHD and HIF alpha genes and used computational tools to characterize the molecular changes underlying the functional divergence of PHD and HIF alpha duplicates. The presence of PHDs in metazoan genomes predates the emergence of HIF alphas. Our analysis revealed an unexpected diversity of PHD genes and HIF alpha sequence characteristics in invertebrates, suggesting that the simple oxygen-sensing systems of Caenorhabditis and Drosophila may not be typical of other invertebrate bilaterians. We studied the early vertebrate evolution of the system by sequencing these genes in early-diverging cartilaginous fishes, elasmobranchs. Cartilaginous fishes appear to have three paralogs of both PHD and HIF alpha. The novel sequences were used as outgroups for a detailed molecular analysis of PHD and HIF alpha duplicates in a major air-breathing vertebrate lineage, the mammals, and a major water-breathing vertebrate lineage, the teleosts. In PHDs, functionally divergent amino acid sites were detected near the HIF alpha-binding channel and beta2beta3 loop that defines its substrate specificity. In HIF alphas

Overexpression of Human Estrogen Biosynthetic Enzyme Hydroxysteroid (17beta) Dehydrogenase Type 1 Induces Adenomyosis-like Phenotype in Transgenic Mice

Hydroxysteroid (17beta) dehydrogenase type 1 (HSD17B1) is an enzyme that converts estrone to estradiol, while adenomyosis is an estrogen-dependent disease with poorly understood pathophysiology. In the present study, we show that mice universally over-expressing human estrogen biosynthetic enzyme HSD17B1 (HSD17B1TG mice) present with adenomyosis phenotype, characterized by histological and molecular evaluation. The first adenomyotic changes with endometrial glands partially or fully infiltrated into the myometrium appeared at the age of 5.5 months in HSD17B1TG females and became more prominent with increasing age. Preceding the phenotype, increased myometrial smooth muscle actin positivity and increased amount of glandular myofibroblast cells were observed in HSD17B1TG uteri. This was accompanied by transcriptomic upregulation of inflammatory and estrogen signaling pathways. Further, the genes upregulated in the HSD17B1TG uterus were enriched with genes previously observed to be induced in the human adenomyotic uterus, including several genes of the NFKB pathway. A 6-week-long HSD17B1 inhibitor treatment reduced the occurrence of the adenomyotic changes by 5-fold, whereas no effect was observed in the vehicle-treated HSD17B1TG mice, suggesting that estrogen is the main upstream regulator of adenomyosis-induced uterine signaling pathways. HSD17B1 is considered as a promising drug target to inhibit estrogen-dependent growth of endometrial disorders. The present data indicate that HSD17B1 over-expression in TG mice results in adenomyotic changes reversed by HSD17B1 inhibitor treatment and HSD17B1 is, thus, a potential novel drug target for adenomyosis