Asta Laiho

Comparison of software packages for detecting differential expression in RNA-seq studies

RNA-sequencing (RNA-seq) has rapidly become a popular tool to characterize transcriptomes. A fundamental research problem in many RNA-seq studies is the identification of reliable molecular markers that show differential expression between distinct sample groups. Together with the growing popularity of RNA-seq, a number of data analysis methods and pipelines have already been developed for this task. Currently, however, there is no clear consensus about the best practices yet, which makes the choice of an appropriate method a daunting task especially for a basic user without a strong statistical or computational background. To assist the choice, we perform here a systematic comparison of eight widely used software packages and pipelines for detecting differential expression between sample groups in a practical research setting and provide general guidelines for choosing a robust pipeline. In general, our results demonstrate how the data analysis tool utilized can markedly affect the outcome of the data analysis, highlighting the importance of this choice.

Transcriptome Profiling of the Murine Testis during the First Wave of Spermatogenesis

Correct gene expression patterns form the basis for male germ cell differentiation and male fertility. Although previous studies have elucidated the importance of testis specific gene expression, the exact transcripts and comprehensive gene expression patterns remain unknown. Large scale sequencing techniques have enabled cost effective analysis of gene expression and isoform studies. Using the SOLiD 4 next-generation sequencing platform we have investigated the gene expression patterns at five different time points during the first wave on murine spermatogenesis. Our results highlight the upregulation of spermatogenesis related biological processes and associated cellular components. Elucidation of differential gene expression at important time points during the sperm development emphasizes the importance of correct timing of gene expression within biological processes. Differential gene level expression was analyzed with R/Bioconductor’s Limma package and isoform analysis was conducted with the Cufflinks pipeline. At gene level total of 2494 differentially expressed genes were identified and Cufflinks characterized over 160 000 gene isoforms, of which 29% were novel transcripts assigned to known genes. Isoforms were detected for 57% of expressed genes and in a total over 26 000 genes were expressed in the testis. Differential promoter and transcription start site usage appears also to play a role in regulation of gene expression during spermatogenesis. Furthermore, we identified 947 upregulated long non-coding RNAs during the first wave of spermatogenesis. These RNAs appeared to be highly specific to different time points. Transcriptomic analysis of testis tissue samples is highly informative due to the large number of expressed genes and identified isoforms. Our study provides a very valuable basis for investigation of gene isoforms and regulation and factors contributing to male fertility.

Gut Microbiota as an Epigenetic Regulator: Pilot Study Based on Whole-Genome Methylation Analysis

ABSTRACT The core human gut microbiota contributes to the developmental origin of diseases by modifying metabolic pathways. To evaluate the predominant microbiota as an epigenetic modifier, we classified 8 pregnant women into two groups based on their dominant microbiota, i.e., Bacteroidetes, Firmicutes, and Proteobacteria. Deep sequencing of DNA methylomes revealed a clear association between bacterial predominance and epigenetic profiles. The genes with differentially methylated promoters in the group in which Firmicutes was dominant were linked to risk of disease, predominantly to cardiovascular disease and specifically to lipid metabolism, obesity, and the inflammatory response. This is one of the first studies that highlights the association of the predominant bacterial phyla in the gut with methylation patterns. Further longitudinal and in-depth studies targeting individual microbial species or metabolites are recommended to give us a deeper insight into the molecular mechanism of such epigenetic modifications. IMPORTANCE Epigenetics encompasses genomic modifications that are due to environmental factors and do not affect the nucleotide sequence. The gut microbiota has an important role in human metabolism and could be a significant environmental factor affecting our epigenome. To investigate the association of gut microbiota with epigenetic changes, we assessed pregnant women and selected the participants based on their predominant gut microbiota for a study on their postpartum methylation profile. Intriguingly, we found that blood DNA methylation patterns were associated with gut microbiota profiles. The gut microbiota profiles, with either Firmicutes or Bacteroidetes as a dominant group, correlated with differential methylation status of gene promoters functionally associated with cardiovascular diseases. Furthermore, differential methylation of gene promoters linked to lipid metabolism and obesity was observed. For the first time, we report here a position of the predominant gut microbiota in epigenetic profiling, suggesting one potential mechanism in obesity with comorbidities, if proven in further in-depth studies. Epigenetics encompasses genomic modifications that are due to environmental factors and do not affect the nucleotide sequence. The gut microbiota has an important role in human metabolism and could be a significant environmental factor affecting our epigenome. To investigate the association of gut microbiota with epigenetic changes, we assessed pregnant women and selected the participants based on their predominant gut microbiota for a study on their postpartum methylation profile. Intriguingly, we found that blood DNA methylation patterns were associated with gut microbiota profiles. The gut microbiota profiles, with either Firmicutes or Bacteroidetes as a dominant group, correlated with differential methylation status of gene promoters functionally associated with cardiovascular diseases. Furthermore, differential methylation of gene promoters linked to lipid metabolism and obesity was observed. For the first time, we report here a position of the predominant gut microbiota in epigenetic profiling, suggesting one potential mechanism in obesity with comorbidities, if proven in further in-depth studies.

Promoter ChIP-chip analysis in mouse testis reveals Y chromosome occupancy by HSF2

The mammalian Y chromosome is essential for spermatogenesis, which is characterized by sperm cell differentiation and chromatin condensation for acquisition of correct shape of the sperm. Deletions of the male-specific region of the mouse Y chromosome long arm (MSYq), harboring multiple copies of a few genes, lead to sperm head defects and impaired fertility. Using chromatin immunoprecipitation on promoter microarray (ChIP-chip) on mouse testis, we found a striking in vivo MSYq occupancy by heat shock factor 2 (HSF2), a transcription factor involved in spermatogenesis. HSF2 was also found to regulate the transcription of MSYq resident genes, whose transcriptional regulation has been unknown. Importantly, disruption of Hsf2 caused a similar phenotype as the 2/3 deletion of MSYq, i.e., altered expression of the multicopy genes and increased mild sperm head abnormalities. Consequently, aberrant levels of chromatin packing proteins and more frequent DNA fragmentation were detected, implying that HSF2 is required for correct chromatin organization in the sperm. Our findings define a physiological role for HSF2 in the regulation of MSYq resident genes and the quality of sperm.

Gene expression differences between Noccaea caerulescens ecotypes help to identify candidate genes for metal phytoremediation.

Populations of Noccaea caerulescens show tremendous differences in their capacity to hyperaccumulate and hypertolerate metals. To explore the differences that could contribute to these traits, we undertook SOLiD high-throughput sequencing of the root transcriptomes of three phenotypically well-characterized N. caerulescens accessions, i.e., Ganges, La Calamine, and Monte Prinzera. Genes with possible contribution to zinc, cadmium, and nickel hyperaccumulation and hypertolerance were predicted. The most significant differences between the accessions were related to metal ion (di-, trivalent inorganic cation) transmembrane transporter activity, iron and calcium ion binding, (inorganic) anion transmembrane transporter activity, and antioxidant activity. Analysis of correlation between the expression profile of each gene and the metal-related characteristics of the accessions disclosed both previously characterized (HMA4, HMA3) and new candidate genes (e.g., for nickel IRT1, ZIP10, and PDF2.3) as possible contributors to the hyperaccumulation/tolerance phenotype. A number of unknown Noccaea-specific transcripts also showed correlation with Zn(2+), Cd(2+), or Ni(2+) hyperaccumulation/tolerance. This study shows that N. caerulescens populations have evolved great diversity in the expression of metal-related genes, facilitating adaptation to various metalliferous soils. The information will be helpful in the development of improved plants for metal phytoremediation.

Heat shock transcription factor 1 localizes to sex chromatin during meiotic repression.

Heat shock factor 1 (HSF1) is an important transcription factor in cellular stress responses, cancer, aging, and developmental processes including gametogenesis. Disruption of Hsf1, together with another HSF family member, Hsf2, causes male sterility and complete lack of mature sperm in mice, but the specific role of HSF1 in spermatogenesis has remained unclear. Here, we show that HSF1 is transiently expressed in meiotic spermatocytes and haploid round spermatids in mouse testis. The Hsf1−/− male mice displayed regions of seminiferous tubules containing only spermatogonia and increased morphological abnormalities in sperm heads. In search for HSF1 target genes, we identified 742 putative promoters in mouse testis. Among them, the sex chromosomal multicopy genes that are expressed in postmeiotic cells were occupied by HSF1. Given that the sex chromatin mostly is repressed during and after meiosis, it is remarkable that HSF1 directly regulates the transcription of sex-linked multicopy genes during postmeiotic repression. In addition, our results show that HSF1 localizes to the sex body prior to the meiotic divisions and to the sex chromocenter after completed meiosis. To the best of our knowledge, HSF1 is the first known transcription factor found at the repressed sex chromatin during meiosis.

Differential Promoter Methylation of Macrophage Genes Is Associated With Impaired Vascular Growth in Ischemic Muscles of Hyperlipidemic and Type 2 Diabetic Mice Genome-Wide Promoter Methylation Study

RATIONALE Hyperlipidemia and type 2 diabetes mellitus (T2DM) severely impair adaptive vascular growth responses in ischemic muscles. This is largely attributed to dysregulated gene expression, although details of the changes are unknown. OBJECTIVE To define the role of promoter methylation in adaptive vascular growth in hyperlipidemia (LDLR(-/-)ApoB(100/100)) and T2DM (IGF-II/LDLR(-/-)ApoB(100/100)) mouse models of hindlimb ischemia. METHODS AND RESULTS Unilateral hindlimb ischemia was induced by ligating femoral artery. Perfusion was assessed using ultrasound, and capillary and arteriole parameters were assessed using immunohistochemistry. Genome-wide methylated DNA sequencing was performed with DNA isolated from ischemic muscle, tissue macrophages (Mϕs), and endothelial cells. Compared with the controls, hyperlipidemia and T2DM mice showed impaired perfusion recovery, which was associated with impaired angiogenesis and arteriogenesis. Genome-wide proximal promoter DNA methylation analysis suggested differential patterns of methylation in Mϕ genes in ischemic muscles. Classically activated M1-Mϕ gene promoters, including Cfb, Serping1, and Tnfsf15, were significantly hypomethylated, whereas alternatively activated M2-Mϕ gene promoters, including Nrp1, Cxcr4, Plxnd1, Arg1, Cdk18, and Fes, were significantly hypermethylated in Mϕs isolated from hyperlipidemia and T2DM ischemic muscles compared with controls. These results combined with mRNA expression and immunohistochemistry showed the predominance of proinflammatory M1-Mϕs, compared with anti-inflammatory and proangiogenic M2-Mϕs in hyperlipidemia and T2DM ischemic muscles. CONCLUSIONS We found significant promoter hypomethylation of genes typical for proinflammatory M1-Mϕs and hypermethylation of anti-inflammatory, proangiogenic M2-Mϕ genes in hyperlipidemia and T2DM ischemic muscles. Epigenetic alterations modify Mϕ phenotype toward proinflammatory M1 as opposed to anti-inflammatory, proangiogenic, and tissue repair M2 phenotype, which may contribute to the impaired adaptive vascular growth under these pathological conditions.

Fermentable fibres condition colon microbiota and promote diabetogenesis in NOD mice

Aims/hypothesisGut microbiota (GM) and diet both appear to be important in the pathogenesis of type 1 diabetes. Fermentable fibres (FFs), of which there is an ample supply in natural, diabetes-promoting diets, are used by GM as a source of energy. Our aim was to determine whether FFs modify GM and diabetes incidence in the NOD mouse.MethodsFemale NOD mice were weaned to a semisynthetic diet and the effects of FF supplementation on diabetes incidence and insulitis were evaluated. Real-time quantitative PCR was employed to determine the effects imposed to gene transcripts in the colon and lymph nodes. Changes to GM were analysed by next-generation sequencing.ResultsNOD mice fed semisynthetic diets free from FFs were largely protected from diabetes while semisynthetic diets supplemented with the FFs pectin and xylan (PX) resulted in higher diabetes incidence. Semisynthetic diet free from FFs altered GM composition significantly; addition of PX changed the composition of the GM towards that found in natural-diet-fed mice and increased production of FF-derived short-chain fatty acid metabolites in the colon. The highly diabetogenic natural diet was associated with expression of proinflammatory and stress-related genes in the colon, while the semisynthetic diet free from FFs promoted Il4, Il22, Tgfβ and Foxp3 transcripts in the colon and/or pancreatic lymph node. PX in the same diet counteracted these effects and promoted stress-related IL-18 activation in gut epithelial cells. 16S RNA sequencing revealed each diet to give rise to its particular GM composition, with different Firmicutes to Bacteroidetes ratios, and enrichment of mucin-degrading Ruminococcaceae following diabetes-protective FF-free diet.Conclusions/interpretationFFs condition microbiota, affect colon homeostasis and are important components of natural, diabetes-promoting diets in NOD mice.

Continuous hypoxic culturing of human embryonic stem cells enhances SSEA-3 and MYC levels.

Low oxygen tension (hypoxia) contributes critically to pluripotency of human embryonic stem cells (hESCs) by preventing spontaneous differentiation and supporting self-renewal. However, it is not well understood how hESCs respond to reduced oxygen availability and what are the molecular mechanisms maintaining pluripotency in these conditions. In this study we characterized the transcriptional and molecular responses of three hESC lines (H9, HS401 and HS360) on short (2 hours), intermediate (24 hours) and prolonged (7 days) exposure to low oxygen conditions (4% O2). In response to prolonged hypoxia the expression of pluripotency surface marker SSEA-3 was increased. Furthermore, the genome wide gene-expression analysis revealed that a substantial proportion (12%) of all hypoxia-regulated genes in hESCs, were directly linked to the mechanisms controlling pluripotency or differentiation. Moreover, transcription of MYC oncogene was induced in response to continuous hypoxia. At the protein level MYC was stabilized through phosphorylation already in response to a short hypoxic exposure. Total MYC protein levels remained elevated throughout all the time points studied. Further, MYC protein expression in hypoxia was affected by silencing HIF2α, but not HIF1α. Since MYC has a crucial role in regulating pluripotency we propose that induction of sustained MYC expression in hypoxia contributes to activation of transcriptional programs critical for hESC self-renewal and maintenance of enhanced pluripotent state.

Whole transcriptome characterization of the effects of dehydration and rehydration on Cladonia rangiferina, the grey reindeer lichen

BackgroundLichens are symbiotic organisms with a fungal and an algal or a cyanobacterial partner. Lichens inhabit some of the harshest climates on earth and most lichen species are desiccation-tolerant. Lichen desiccation-tolerance has been studied at the biochemical level and through proteomics, but the underlying molecular genetic mechanisms remain largely unexplored. The objective of our study was to examine the effects of dehydration and rehydration on the gene expression of Cladonia rangiferina.ResultsSamples of C. rangiferina were collected at several time points during both the dehydration and rehydration process and the gene expression intensities were measured using a custom DNA microarray. Several genes, which were differentially expressed in one or more time points, were identified. The microarray results were validated using qRT-PCR analysis. Enrichment analysis of differentially expressed transcripts was also performed to identify the Gene Ontology terms most associated with the rehydration and dehydration process.ConclusionsOur data identify differential expression patterns for hundreds of genes that are modulated during dehydration and rehydration in Cladonia rangiferina. These dehydration and rehydration events clearly differ from each other at the molecular level and the largest changes to gene expression are observed within minutes following rehydration. Distinct changes are observed during the earliest stage of rehydration and the mechanisms not appear to be shared with the later stages of wetting or with drying. Several of the most differentially expressed genes are similar to genes identified in previous studies that have investigated the molecular mechanisms of other desiccation-tolerant organisms. We present here the first microarray experiment for any lichen species and have for the first time studied the genetic mechanisms behind lichen desiccation-tolerance at the whole transcriptome level.