The spermatogonial stem cell (SSC) that supports spermatogenesis throughout adult life resides within the GFRα1-expressing A type undifferentiated spermatogonia. The decision to commit to ...spermatogenic differentiation coincides with the loss of GFRα1 and reciprocal gain of Ngn3 (Neurog3) expression. Through the analysis of the piRNA factor
(
), we identify a novel population of Ngn3-expressing spermatogonia that are essential for efficient testicular regeneration after injury. Depletion of
-expressing cells results in a transient impact on testicular homeostasis, with this population behaving strictly as transit amplifying cells under homeostatic conditions. However, upon injury,
-expressing cells are essential for the efficient regenerative capacity of the testis, and also display facultative stem activity in transplantation assays. In summary, the mouse testis has adopted a regenerative strategy to expand stem cell activity by incorporating a transit-amplifying population to the effective stem cell pool, thus ensuring rapid and efficient tissue repair.
DNA replication is temporally and spatially organized in all eukaryotes, yet the molecular control and biological function of the replication-timing program are unclear. Rif1 is required for normal ...genome-wide regulation of replication timing, but its molecular function is poorly understood. Here we show that in mouse embryonic stem cells, Rif1 coats late-replicating domains and, with Lamin B1, identifies most of the late-replicating genome. Rif1 is an essential determinant of replication timing of non-Lamin B1-bound late domains. We further demonstrate that Rif1 defines and restricts the interactions between replication-timing domains during the G1 phase, thereby revealing a function of Rif1 as organizer of nuclear architecture. Rif1 loss affects both number and replication-timing specificity of the interactions between replication-timing domains. In addition, during the S phase, Rif1 ensures that replication of interacting domains is temporally coordinated. In summary, our study identifies Rif1 as the molecular link between nuclear architecture and replication-timing establishment in mammals.
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•Most late-replicating regions are marked by Rif1 (RADs)•Late replication in RADs is differentially regulated where Lamin B1 is stably bound•Rif1 constrains inter-domain contacts within the same replication timing in G1•Rif1 coordinates the replication timing of interacting domains
At replication-timing establishment, chromatin domains are forced to interact only with regions sharing the same replication timing. Foti et al. demonstrate that Rif1 is responsible for this constraint because Rif1 deletion leads to loss of spatial limitations followed by replication-timing program disruption. Therefore, Rif1 links nuclear architecture and replication-timing establishment.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Recent advances in single-cell transcriptomics techniques have opened the door to the study of gene regulatory networks (GRNs) at the single-cell level. Here, we studied the GRNs controlling the ...emergence of hematopoietic stem and progenitor cells from mouse embryonic endothelium using a combination of single-cell transcriptome assays. We found that a heptad of transcription factors (Runx1, Gata2, Tal1, Fli1, Lyl1, Erg and Lmo2) is specifically co-expressed in an intermediate population expressing both endothelial and hematopoietic markers. Within the heptad, we identified two sets of factors of opposing functions: one (Erg/Fli1) promoting the endothelial cell fate, the other (Runx1/Gata2) promoting the hematopoietic fate. Surprisingly, our data suggest that even though Fli1 initially supports the endothelial cell fate, it acquires a pro-hematopoietic role when co-expressed with Runx1. This work demonstrates the power of single-cell RNA-sequencing for characterizing complex transcription factor dynamics.
Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the ...nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Early and accurate pre-clinical and clinical biomarkers of hepatotoxicity facilitate the drug development process and the safety monitoring in clinical studies. We selected eight known model ...compounds to be administered to male Wistar rats to identify biomarkers of drug induced liver injury (DILI) using transcriptomics, metabolite profiling (metabolomics) and conventional endpoints. We specifically explored early biomarkers in serum and liver tissue associated with histopathologically evident acute hepatotoxicity. A tailored data analysis strategy was implemented to better differentiate animals with no treatment-related findings in the liver from animals showing evident hepatotoxicity as assessed by histopathological analysis. From the large number of assessed parameters, our data analysis strategy allowed us to identify five metabolites in serum and five in liver tissue, 58 transcripts in liver tissue and seven clinical chemistry markers in serum that were significantly associated with acute hepatotoxicity. The identified markers comprised metabolites such as taurocholic acid and putrescine (measured as sum parameter together with agmatine), classical clinical chemistry markers like AST (aspartate aminotransferase), ALT (alanine aminotransferase), and bilirubin, as well as gene transcripts like Igfbp1 (insulin-like growth factor-binding protein 1) and Egr1 (early growth response protein 1). The response pattern of the identified biomarkers was concordant across all types of parameters and sample matrices. Our results suggest that a combination of several of these biomarkers could significantly improve the robustness and accuracy of an early diagnosis of hepatotoxicity.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Pediatric tumors frequently arise from embryonal cells, often displaying a stem cell-like ("small round blue") morphology in tissue sections. Because recently "stemness" has been associated with a ...poor immune response in tumors, we investigated the association of prognostic gene expression, stemness and the immune microenvironment systematically using transcriptomes of 4068 tumors occurring mostly at the pediatric and young adult age. While the prognostic landscape of gene expression (PRECOG) and infiltrating immune cell types (CIBERSORT) is similar to that of tumor entities occurring mainly in adults, the patterns are distinct for each diagnostic entity. A high stemness score (mRNAsi) correlates with clinical and morphologic subtype in Wilms tumors, neuroblastomas, synovial sarcomas, atypical teratoid rhabdoid tumors and germ cell tumors. In neuroblastomas, a high mRNAsi is associated with shortened overall survival. In Wilms tumors a high mRNAsi correlates with blastemal morphology, whereas tumors with predominant epithelial or stromal differentiation have a low mRNAsi and a high percentage of M2 type macrophages. This could be validated in Wilms tumor tissue (
= 78). Here, blastemal areas are low in M2 macrophage infiltrates, while nearby stromal differentiated areas contain abundant M2 macrophages, suggesting local microanatomic regulation of the immune response.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Bone marrow (BM) fibrosis in myeloproliferative neoplasms (MPNs) is associated with a poor prognosis. The development of myelofibrosis and differentiation of mesenchymal stromal cells to profibrotic ...myofibroblasts depends on macrophages. Here, we compared macrophage frequencies in BM biopsies of MPN patients and controls (patients with non-neoplastic processes), including primary myelofibrosis (PMF,
n
= 18), essential thrombocythemia (ET,
n
= 14), polycythemia vera (PV,
n
= 12), and Philadelphia chromosome–positive chronic myeloid leukemia (CML,
n
= 9). In PMF, CD68-positive macrophages were greatly increased compared to CML (
p
= 0.017) and control BM (
p
< 0.001). Similar findings were observed by CD163 staining (PMF vs. CML:
p
= 0.017; PMF vs. control:
p
< 0.001). Moreover, CD68-positive macrophages were increased in PV compared with ET (
p
= 0.009) and reactive cases (
p
< 0.001). PMF had higher frequencies of macrophages than PV (CD68:
p
< 0.001; CD163:
p
< 0.001) and ET (CD68:
p
< 0.001; CD163:
p
< 0.001). CD163 and CD68 were often co-expressed in macrophages with stellate morphology in Philadelphia chromosome–negative MPN, resulting in a sponge-like reticular network that may be a key regulator of unbalanced hematopoiesis in the BM space and may explain differences in cellularity and clinical course.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Female mammals achieve dosage compensation by inactivating one of their two X chromosomes during development, a process entirely dependent on Xist, an X-linked long non-coding RNA (lncRNA). At the ...onset of X chromosome inactivation (XCI), Xist is up-regulated and spreads along the future inactive X chromosome. Contextually, it recruits repressive histone and DNA modifiers that transcriptionally silence the X chromosome. Xist regulation is tightly coupled to differentiation and its expression is under the control of both pluripotency and epigenetic factors. Recent evidence has suggested that chromatin remodelers accumulate at the X Inactivation Center (XIC) and here we demonstrate a new role for Chd8 in Xist regulation in differentiating ES cells, linked to its control and prevention of spurious transcription factor interactions occurring within Xist regulatory regions. Our findings have a broader relevance, in the context of complex, developmentally-regulated gene expression.
Identification of a Common Gene Expression Signature in Dilated Cardiomyopathy Across Independent Microarray Studies
Andreas S. Barth, Ruprecht Kuner, Andreas Buness, Markus Ruschhaupt, Sylvia Merk, ...Ludwig Zwermann, Stefan Kääb, Eckart Kreuzer, Gerhard Steinbeck, Ulrich Mansmann, Annemarie Poustka, Michael Nabauer, Holger Sültmann
Although microarrays have emerged as a powerful tool for delineating complex disease patterns, differences in platform technology, tissue heterogeneity, and small sample sizes hamper a comprehensive interpretation of microarray studies in heart failure. Therefore, we performed 2 cDNA and oligonucleotide microarray studies with septal and left ventricular tissue from nonfailing and DCM hearts (each n = 20). Immune response processes displayed the most pronounced regulation on both microarray platforms. Furthermore, a robust set of 27 genes was identified that classified DCM and NF samples in our own microarray studies as well as in 2 additional publicly available datasets (total of 108 samples) with >90% accuracy.
This study was designed to identify a common gene expression signature in dilated cardiomyopathy (DCM) across different microarray studies.
Dilated cardiomyopathy is a common cause of heart failure in Western countries. Although gene expression arrays have emerged as a powerful tool for delineating complex disease patterns, differences in platform technology, tissue heterogeneity, and small sample sizes obscure the underlying pathophysiologic events and hamper a comprehensive interpretation of different microarray studies in heart failure.
We accounted for tissue heterogeneity and technical aspects by performing 2 genome-wide expression studies based on cDNA and short-oligonucleotide microarray platforms which comprised independent septal and left ventricular tissue samples from nonfailing (NF) (n = 20) and DCM (n = 20) hearts.
Concordant results emerged for major gene ontology classes between cDNA and oligonucleotide microarrays. Notably, immune response processes displayed the most pronounced down-regulation on both microarray types, linking this functional gene class to the pathogenesis of end-stage DCM. Furthermore, a robust set of 27 genes was identified that classified DCM and NF samples with >90% accuracy in a total of 108 myocardial samples from our cDNA and oligonucleotide microarray studies as well as 2 publicly available datasets.
For the first time, independent microarray datasets pointed to significant involvement of immune response processes in end-stage DCM. Moreover, based on 4 independent microarray datasets, we present a robust gene expression signature of DCM, encouraging future prospective studies for the implementation of disease biomarkers in the management of patients with heart failure.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
A relevant part of the genetic architecture of complex traits is still unknown; despite the discovery of many disease-associated common variants. Polygenic risk score (PRS) models are based on the ...evaluation of the additive effects attributable to common variants and have been successfully implemented to assess the genetic susceptibility for many phenotypes. In contrast, burden tests are often used to identify an enrichment of rare deleterious variants in specific genes. Both kinds of genetic contributions are typically analyzed independently. Many studies suggest that complex phenotypes are influenced by both low effect common variants and high effect rare deleterious variants. The aim of this paper is to integrate the effect of both common and rare functional variants for a more comprehensive genetic risk modeling. We developed a framework combining gene-based scores based on the enrichment of rare functionally relevant variants with genome-wide PRS based on common variants for association analysis and prediction models. We applied our framework on UK Biobank dataset with genotyping and exome data and considered 28 blood biomarkers levels as target phenotypes. For each biomarker, an association analysis was performed on full cohort using gene-based scores (GBS). The cohort was then split into 3 subsets for PRS construction and feature selection, predictive model training, and independent evaluation, respectively. Prediction models were generated including either PRS, GBS or both (combined). Association analyses of the cohort were able to detect significant genes that were previously known to be associated with different biomarkers. Interestingly, the analyses also revealed heterogeneous effect sizes and directionality highlighting the complexity of the blood biomarkers regulation. However, the combined models for many biomarkers show little or no improvement in prediction accuracy compared to the PRS models. This study shows that rare variants play an important role in the genetic architecture of complex multifactorial traits such as blood biomarkers. However, while rare deleterious variants play a strong role at an individual level, our results indicate that classical common variant based PRS might be more informative to predict the genetic susceptibility at the population level.
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