38 research outputs found
The Human Blood Transcriptome in a Large Population Cohort and Its Relation to Aging and Health
Background: The blood transcriptome is expected to provide a detailed picture of
an organism’s physiological state with potential outcomes for applications in medical
diagnostics and molecular and epidemiological research.We here present the analysis of
blood specimens of 3,388 adult individuals, together with phenotype characteristics such
as disease history, medication status, lifestyle factors, and body mass index (BMI). The
size and heterogeneity of this data challenges analytics in terms of dimension reduction,
knowledge mining, feature extraction, and data integration.
Methods: Self-organizing maps (SOM)-machine learning was applied to study
transcriptional states on a population-wide scale. This method permits a detailed
description and visualization of the molecular heterogeneity of transcriptomes and of
their association with different phenotypic features.
Results: The diversity of transcriptomes is described by personalized SOM-portraits,
which specify the samples in terms of modules of co-expressed genes of different
functional context. We identified two major blood transcriptome types where type
1 was found more in men, the elderly, and overweight people and it upregulated
genes associated with inflammation and increased heme metabolism, while type 2 was
predominantly found in women, younger, and normal weight participants and it was
associated with activated immune responses, transcriptional, ribosomal, mitochondrial,
and telomere-maintenance cell-functions. We find a striking overlap of signatures shared
by multiple diseases, aging, and obesity driven by an underlying common pattern, which
was associated with the immune response and the increase of inflammatory processes.
Conclusions: Machine learning applications for large and heterogeneous omics data
provide a holistic view on the diversity of the human blood transcriptome. It provides a
tool for comparative analyses of transcriptional signatures and of associated phenotypes
in population studies and medical applications
Generation of human induced pluripotent stem cells using non-synthetic mRNA
Here we describe some of the crucial steps to generate induced pluripotent stemcells (iPSCs) usingmRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribedmRNA. V. virus\'' 2′-O-Methyltransferase enzymecreates a cap1 structure found in higher eukaryotes and has higher translation efficiency compared to other methods. Use of the polymeric transfection reagent polyethylenimine proved superior to other transfection methods. The mRNA created via this method did not trigger an intracellular immune response via human IFN-gamma (hIFN-γ) or alpha (hIFN-α) release, thus circumventing the use of suppressors. Resulting mRNA and protein
were expressed at high levels for over 48 h, thus obviating daily transfections. Using this method, we demonstrated swift activation of pluripotency associated genes in human fibroblasts. Low oxygen conditions further facilitated colony formation. Differentiation into different germ layers was confirmed via teratoma assay. Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach
A keratin scaffold regulates epidermal barrier formation, mitochondrial lipid composition, and activity.
Keratin intermediate filaments (KIFs) protect the epidermis against mechanical force, support strong adhesion, help barrier formation, and regulate growth. The mechanisms by which type I and II keratins contribute to these functions remain incompletely understood. Here, we report that mice lacking all type I or type II keratins display severe barrier defects and fragile skin, leading to perinatal mortality with full penetrance. Comparative proteomics of cornified envelopes (CEs) from prenatal KtyI(-/-) and KtyII(-/-)(K8) mice demonstrates that absence of KIF causes dysregulation of many CE constituents, including downregulation of desmoglein 1. Despite persistence of loricrin expression and upregulation of many Nrf2 targets, including CE components Sprr2d and Sprr2h, extensive barrier defects persist, identifying keratins as essential CE scaffolds. Furthermore, we show that KIFs control mitochondrial lipid composition and activity in a cell-intrinsic manner. Therefore, our study explains the complexity of keratinopathies accompanied by barrier disorders by linking keratin scaffolds to mitochondria, adhesion, and CE formation
Correction: A keratin scaffold regulates epidermal barrier formation, mitochondrial lipid composition, and activity
Epigenetic Heterogeneity of B-Cell Lymphoma: DNA Methylation, Gene Expression and Chromatin States
Mature B-cell lymphoma is a clinically and biologically highly diverse disease. Its diagnosis and prognosis is a challenge due to its molecular heterogeneity and diverse regimes of biological dysfunctions, which are partly driven by epigenetic mechanisms. We here present an integrative analysis of DNA methylation and gene expression data of several lymphoma subtypes. Our study confirms previous results about the role of stemness genes during development and maturation of B-cells and their dysfunction in lymphoma locking in more proliferative or immune-reactive states referring to B-cell functionalities in the dark and light zone of the germinal center and also in plasma cells. These dysfunctions are governed by widespread epigenetic effects altering the promoter methylation of the involved genes, their activity status as moderated by histone modifications and also by chromatin remodeling. We identified four groups of genes showing characteristic expression and methylation signatures among Burkitt’s lymphoma, diffuse large B cell lymphoma, follicular lymphoma and multiple myeloma. These signatures are associated with epigenetic effects such as remodeling from transcriptionally inactive into active chromatin states, differential promoter methylation and the enrichment of targets of transcription factors such as EZH2 and SUZ12
Telomere Length Maintenance and Its Transcriptional Regulation in Lynch Syndrome and Sporadic Colorectal Carcinoma
Background: Activation of telomere maintenance mechanisms (TMMs) is a hallmark
of most cancers, and is required to prevent genome instability and to establish cellular
immortality through reconstitution of capping of chromosome ends. TMM depends on
the cancer type. Comparative studies linking tumor biology and TMM have potential
impact for evaluating cancer onset and development.
Methods: We have studied alterations of telomere length, their sequence composition
and transcriptional regulation in mismatch repair deficient colorectal cancers arising in
Lynch syndrome (LS-CRC) and microsatellite instable (MSI) sporadic CRC (MSI s-CRC),
and for comparison, in microsatellite stable (MSS) s-CRC and in benign colon
mucosa. Our study applied bioinformatics analysis of whole genome DNA and RNA
sequencing data and a pathway model to study telomere length alterations and the
potential effect of the “classical” telomerase (TEL-) and alternative (ALT-) TMM using
transcriptomic signatures.
Results: We have found progressive decrease of mean telomere length in all
cancer subtypes compared with reference systems. Our results support the view
that telomere attrition is an early event in tumorigenesis. TMM gets activated in
all tumors studied due to concerted overexpression of a large fraction of genes
with direct relation to telomere function, where only a very small fraction of them
showed recurrent mutations. TEL-related transcriptional state was dominating in all CRC
subtypes, showing, however, subtype-specific activation patterns; while contribution
of the ALT-TMM was slightly more prominent in the hypermutated MSI s-CRC
and LS-CRC. TEL-TMM is mainly activated by over-expression of DKC1 and/or
TERT genes and their interaction partners, where DKC1 is more prominent in MSS
than in MSI s-CRC and can serve as a transcriptomic marker of TMM activity.
Conclusions: Our results suggest that transcriptional patterns are indicative for TMM
pathway activation with subtle differences between TEL and ALT mechanisms in a CRC
subtype-specific fashion. Sequencing data potentially provide a suited measure to study
alterations of telomere length and of underlying transcriptional regulation. Further studies
are needed to improve this method
Epigenetic Heterogeneity of B-Cell Lymphoma: Chromatin Modifiers
We systematically studied the expression of more than fifty histone and DNA (de)methylating enzymes in lymphoma and healthy controls. As a main result, we found that the expression levels of nearly all enzymes become markedly disturbed in lymphoma, suggesting deregulation of large parts of the epigenetic machinery. We discuss the effect of DNA promoter methylation and of transcriptional activity in the context of mutated epigenetic modifiers such as EZH2 and MLL2. As another mechanism, we studied the coupling between the energy metabolism and epigenetics via metabolites that act as cofactors of JmjC-type demethylases. Our study results suggest that Burkitt’s lymphoma and diffuse large B-cell Lymphoma differ by an imbalance of repressive and poised promoters, which is governed predominantly by the activity of methyltransferases and the underrepresentation of demethylases in this regulation. The data further suggest that coupling of epigenetics with the energy metabolism can also be an important factor in lymphomagenesis in the absence of direct mutations of genes in metabolic pathways. Understanding of epigenetic deregulation in lymphoma and possibly in cancers in general must go beyond simple schemes using only a few modes of regulation