Publications by authors named "Michael B Stadler"

86 Publications

Cooperation between HDAC3 and DAX1 mediates lineage restriction of embryonic stem cells.

EMBO J 2021 Jun 28;40(12):e106818. Epub 2021 Apr 28.

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

Mouse embryonic stem cells (mESCs) are biased toward producing embryonic rather than extraembryonic endoderm fates. Here, we identify the mechanism of this barrier and report that the histone deacetylase Hdac3 and the transcriptional corepressor Dax1 cooperatively limit the lineage repertoire of mESCs by silencing an enhancer of the extraembryonic endoderm-specifying transcription factor Gata6. This restriction is opposed by the pluripotency transcription factors Nr5a2 and Esrrb, which promote cell type conversion. Perturbation of the barrier extends mESC potency and allows formation of 3D spheroids that mimic the spatial segregation of embryonic epiblast and extraembryonic endoderm in early embryos. Overall, this study shows that transcriptional repressors stabilize pluripotency by biasing the equilibrium between embryonic and extraembryonic lineages that is hardwired into the mESC transcriptional network.
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http://dx.doi.org/10.15252/embj.2020106818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8204867PMC
June 2021

The NFIB-ERO1A axis promotes breast cancer metastatic colonization of disseminated tumour cells.

EMBO Mol Med 2021 Apr 10;13(4):e13162. Epub 2021 Mar 10.

Department of Biomedicine, Department of Surgery, University Hospital Basel, University of Basel, Basel, Switzerland.

Metastasis is the main cause of deaths related to solid cancers. Active transcriptional programmes are known to regulate the metastatic cascade but the molecular determinants of metastatic colonization remain elusive. Using an inducible piggyBac (PB) transposon mutagenesis screen, we have shown that overexpression of the transcription factor nuclear factor IB (NFIB) alone is sufficient to enhance primary mammary tumour growth and lung metastatic colonization. Mechanistically and functionally, NFIB directly increases expression of the oxidoreductase ERO1A, which enhances HIF1α-VEGFA-mediated angiogenesis and colonization, the last and fatal step of the metastatic cascade. NFIB is thus clinically relevant: it is preferentially expressed in the poor-prognostic group of basal-like breast cancers, and high expression of the NFIB/ERO1A/VEGFA pathway correlates with reduced breast cancer patient survival.
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http://dx.doi.org/10.15252/emmm.202013162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8033524PMC
April 2021

Molecular clues in the regulation of mini-puberty involve neuronal DNA binding transcription factor NHLH2.

Basic Clin Androl 2021 Mar 18;31(1). Epub 2021 Mar 18.

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

Gonadotropin releasing hormone agonist (GnRHa) treatment following surgery to correct cryptorchidism restores mini-puberty via endocrinological and transcriptional effects and prevents adult infertility in most cases. Several genes are important for central hypogonadotropic hypogonadism in mammals, including many that are transcribed in both the brain and testis. However, the expression of these genes in prepubertal gonads has not been studied systematically, and little is known about the effect of hormone therapy on their testicular and neuronal expression levels. In this review, we interpret histological sections, data on hormone levels, and RNA profiling data from adult normal testes compared to pre-pubertal low infertility risk (LIR) and high infertility risk (HIR) patients randomly treated with surgery in combination with GnRHa or only surgery. We organize 31 target genes relevant for idiopathic hypogonadotropic hypogonadism and cryptorchidism into five classes depending on their expression levels in HIR versus LIR samples and their response to GnRHa treatment. Nescient-helix-loop-helix 2 (NHLH2) was the only gene showing a decreased mRNA level in HIR patients and an increase after GnRHa treatment. This phenomenon may reflect a broader effect of hormone treatment on gene expression in both testicular and central nervous system tissues, which could explain why the hypothalamus-pituitary-testicular axis is permanently restored by the administration of GnRHa.
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http://dx.doi.org/10.1186/s12610-021-00124-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7971961PMC
March 2021

A unique bipartite Polycomb signature regulates stimulus-response transcription during development.

Nat Genet 2021 03 18;53(3):379-391. Epub 2021 Feb 18.

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

Rapid cellular responses to environmental stimuli are fundamental for development and maturation. Immediate early genes can be transcriptionally induced within minutes in response to a variety of signals. How their induction levels are regulated and their untimely activation by spurious signals prevented during development is poorly understood. We found that in developing sensory neurons, before perinatal sensory-activity-dependent induction, immediate early genes are embedded into a unique bipartite Polycomb chromatin signature, carrying active H3K27ac on promoters but repressive Ezh2-dependent H3K27me3 on gene bodies. This bipartite signature is widely present in developing cell types, including embryonic stem cells. Polycomb marking of gene bodies inhibits mRNA elongation, dampening productive transcription, while still allowing for fast stimulus-dependent mark removal and bipartite gene induction. We reveal a developmental epigenetic mechanism regulating the rapidity and amplitude of the transcriptional response to relevant stimuli, while preventing inappropriate activation of stimulus-response genes.
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http://dx.doi.org/10.1038/s41588-021-00789-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610396PMC
March 2021

Mammalian SWI/SNF continuously restores local accessibility to chromatin.

Nat Genet 2021 03 8;53(3):279-287. Epub 2021 Feb 8.

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

Chromatin accessibility is a hallmark of regulatory regions, entails transcription factor (TF) binding and requires nucleosomal reorganization. However, it remains unclear how dynamic this process is. In the present study, we use small-molecule inhibition of the catalytic subunit of the mouse SWI/SNF remodeler complex to show that accessibility and reduced nucleosome presence at TF-binding sites rely on persistent activity of nucleosome remodelers. Within minutes of remodeler inhibition, accessibility and TF binding decrease. Although this is irrespective of TF function, we show that the activating TF OCT4 (POU5F1) exhibits a faster response than the repressive TF REST. Accessibility, nucleosome depletion and gene expression are rapidly restored on inhibitor removal, suggesting that accessible chromatin is regenerated continuously and in a largely cell-autonomous fashion. We postulate that TF binding to chromatin and remodeler-mediated nucleosomal removal do not represent a stable situation, but instead accessible chromatin reflects an average of a dynamic process under continued renewal.
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http://dx.doi.org/10.1038/s41588-020-00768-wDOI Listing
March 2021

OBF1 and Oct factors control the germinal center transcriptional program.

Blood 2021 May;137(21):2920-2934

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

OBF1 is a specific coactivator of the POU family transcription factors OCT1 and OCT2. OBF1 and OCT2 are B cell-specific and indispensable for germinal center (GC) formation, but their mechanism of action is unclear. Here, we show by chromatin immunoprecipitation-sequencing that OBF1 extensively colocalizes with OCT1 and OCT2. We found that these factors also often colocalize with transcription factors of the ETS family. Furthermore, we showed that OBF1, OCT2, and OCT1 bind widely to the promoters or enhancers of genes involved in GC formation in mouse and human GC B cells. Short hairpin RNA knockdown experiments demonstrated that OCT1, OCT2, and OBF1 regulate each other and are essential for proliferation of GC-derived lymphoma cell lines. OBF1 downregulation disrupts the GC transcriptional program: genes involved in GC maintenance, such as BCL6, are downregulated, whereas genes related to exit from the GC program, such as IRF4, are upregulated. Ectopic expression of BCL6 does not restore the proliferation of GC-derived lymphoma cells depleted of OBF1 unless IRF4 is also depleted, indicating that OBF1 controls an essential regulatory node in GC differentiation.
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http://dx.doi.org/10.1182/blood.2020010175DOI Listing
May 2021

Preprocessing choices affect RNA velocity results for droplet scRNA-seq data.

PLoS Comput Biol 2021 01 11;17(1):e1008585. Epub 2021 Jan 11.

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

Experimental single-cell approaches are becoming widely used for many purposes, including investigation of the dynamic behaviour of developing biological systems. Consequently, a large number of computational methods for extracting dynamic information from such data have been developed. One example is RNA velocity analysis, in which spliced and unspliced RNA abundances are jointly modeled in order to infer a 'direction of change' and thereby a future state for each cell in the gene expression space. Naturally, the accuracy and interpretability of the inferred RNA velocities depend crucially on the correctness of the estimated abundances. Here, we systematically compare five widely used quantification tools, in total yielding thirteen different quantification approaches, in terms of their estimates of spliced and unspliced RNA abundances in five experimental droplet scRNA-seq data sets. We show that there are substantial differences between the quantifications obtained from different tools, and identify typical genes for which such discrepancies are observed. We further show that these abundance differences propagate to the downstream analysis, and can have a large effect on estimated velocities as well as the biological interpretation. Our results highlight that abundance quantification is a crucial aspect of the RNA velocity analysis workflow, and that both the definition of the genomic features of interest and the quantification algorithm itself require careful consideration.
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http://dx.doi.org/10.1371/journal.pcbi.1008585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822509PMC
January 2021

Phenotypic landscape of intestinal organoid regeneration.

Nature 2020 10 7;586(7828):275-280. Epub 2020 Oct 7.

Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland.

The development of intestinal organoids from single adult intestinal stem cells in vitro recapitulates the regenerative capacity of the intestinal epithelium. Here we unravel the mechanisms that orchestrate both organoid formation and the regeneration of intestinal tissue, using an image-based screen to assay an annotated library of compounds. We generate multivariate feature profiles for hundreds of thousands of organoids to quantitatively describe their phenotypic landscape. We then use these phenotypic fingerprints to infer regulatory genetic interactions, establishing a new approach to the mapping of genetic interactions in an emergent system. This allows us to identify genes that regulate cell-fate transitions and maintain the balance between regeneration and homeostasis, unravelling previously unknown roles for several pathways, among them retinoic acid signalling. We then characterize a crucial role for retinoic acid nuclear receptors in controlling exit from the regenerative state and driving enterocyte differentiation. By combining quantitative imaging with RNA sequencing, we show the role of endogenous retinoic acid metabolism in initiating transcriptional programs that guide the cell-fate transitions of intestinal epithelium, and we identify an inhibitor of the retinoid X receptor that improves intestinal regeneration in vivo.
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http://dx.doi.org/10.1038/s41586-020-2776-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116869PMC
October 2020

ExploreModelMatrix: Interactive exploration for improved understanding of design matrices and linear models in R.

F1000Res 2020 4;9:512. Epub 2020 Jun 4.

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

Linear and generalized linear models are used extensively in many scientific fields, to model observed data and as the basis for hypothesis tests. The use of such models requires specification of a design matrix, and subsequent formulation of contrasts representing scientific hypotheses of interest. Proper execution of these steps requires a thorough understanding of the meaning of the individual coefficients, and is a frequent source of uncertainty for end users. Here, we present an R/Bioconductor package, , which enables interactive exploration of design matrices and linear model diagnostics. Given a sample data table and a desired design formula, the package displays how the model coefficients are combined to give the fitted values for each combination of predictor variables, which allows users to both extract the interpretation of each individual coefficient, and formulate desired linear contrasts. In addition, the interactive interface displays informative characteristics for the regular linear model corresponding to the provided design, such as variance inflation factors and the pseudoinverse of the design matrix. We envision the package and the built-in collection of common types of linear model designs to be useful for teaching and self-learning purposes, as well as for assisting more experienced users in the interpretation of complex model designs.
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http://dx.doi.org/10.12688/f1000research.24187.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359746PMC
March 2021

Cell Types of the Human Retina and Its Organoids at Single-Cell Resolution.

Cell 2020 09;182(6):1623-1640.e34

Bio Engineering Laboratory, Department of Biosystems Science and Engineering of ETH Zurich, 4058 Basel, Switzerland.

Human organoids recapitulating the cell-type diversity and function of their target organ are valuable for basic and translational research. We developed light-sensitive human retinal organoids with multiple nuclear and synaptic layers and functional synapses. We sequenced the RNA of 285,441 single cells from these organoids at seven developmental time points and from the periphery, fovea, pigment epithelium and choroid of light-responsive adult human retinas, and performed histochemistry. Cell types in organoids matured in vitro to a stable "developed" state at a rate similar to human retina development in vivo. Transcriptomes of organoid cell types converged toward the transcriptomes of adult peripheral retinal cell types. Expression of disease-associated genes was cell-type-specific in adult retina, and cell-type specificity was retained in organoids. We implicate unexpected cell types in diseases such as macular degeneration. This resource identifies cellular targets for studying disease mechanisms in organoids and for targeted repair in human retinas.
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http://dx.doi.org/10.1016/j.cell.2020.08.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7505495PMC
September 2020

Testicular expression of long non-coding RNAs is affected by curative GnRHa treatment of cryptorchidism.

Basic Clin Androl 2019 27;29:18. Epub 2019 Dec 27.

4Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

Background: Cryptorchidism is a frequent endocrinopathy in boys that has been associated with an increased risk of developing testicular cancer and infertility. The condition is curable by combined surgery and hormonal treatment during early pre-pubertal stages using gonadotropin releasing hormone agonist (GnRHa). However, whether the treatment also alters the expression of testicular long non-coding RNAs (lncRNAs) is unknown. To gain insight into the effect of GnRHa on testicular lncRNA levels, we re-analyzed an expression dataset generated from testicular biopsies obtained during orchidopexy for bilateral cryptorchidism.

Results: We identified , , and as potentially relevant for the stimulation of cell proliferation mediated by GnRHa based on their direct or indirect association with rapidly dividing cells in normal and pathological tissues. Surgery alone failed to alter the expression of these transcripts.

Conclusion: Given that lncRNAs can cooperate with chromatin-modifying enzymes to promote epigenetic regulation of genes, GnRHa treatment may act as a surrogate for mini-puberty by triggering the differentiation of Ad spermatogonia via lncRNA-mediated epigenetic effects. Our work provides additional molecular evidence that infertility and azoospermia in cryptorchidism, resulting from defective mini-puberty cannot be cured with successful orchidopexy alone.
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http://dx.doi.org/10.1186/s12610-019-0097-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6933710PMC
December 2019

Zfp281 orchestrates interconversion of pluripotent states by engaging Ehmt1 and Zic2.

EMBO J 2020 01 29;39(2):e102591. Epub 2019 Nov 29.

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

Developmental cell fate specification is a unidirectional process that can be reverted in response to injury or experimental reprogramming. Whether differentiation and de-differentiation trajectories intersect mechanistically is unclear. Here, we performed comparative screening in lineage-related mouse naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), and identified the constitutively expressed zinc finger transcription factor (TF) Zfp281 as a bidirectional regulator of cell state interconversion. We showed that subtle chromatin binding changes in differentiated cells translate into activation of the histone H3 lysine 9 (H3K9) methyltransferase Ehmt1 and stabilization of the zinc finger TF Zic2 at enhancers and promoters. Genetic gain-of-function and loss-of-function experiments confirmed a critical role of Ehmt1 and Zic2 downstream of Zfp281 both in driving exit from the ESC state and in restricting reprogramming of EpiSCs. Our study reveals that cell type-invariant chromatin association of Zfp281 provides an interaction platform for remodeling the cis-regulatory network underlying cellular plasticity.
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http://dx.doi.org/10.15252/embj.2019102591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6960450PMC
January 2020

Genes located in Y-chromosomal regions important for male fertility show altered transcript levels in cryptorchidism and respond to curative hormone treatment.

Basic Clin Androl 2019 3;29. Epub 2019 Jun 3.

Cryptorchidism Research Institute, Kindermedizinisches Zentrum Liestal, 4410 Liestal, Switzerland.

Background: Undescended (cryptorchid) testes in patients with defective mini-puberty and low testosterone levels contain gonocytes that fail to differentiate normally, which impairs the development of Ad spermatogonia and ultimately leads to adult infertility. Treatment with the gonadotropin-releasing hormone agonist GnRHa increases luteinizing hormone and testosterone and rescues fertility in the majority of pathological cryptorchid testes. Several Y-chromosomal genes in the male-specific Y region (MSY) are essential for spermatogenesis, testis development and function, and are associated with azoospermia, infertility and cryptorchidism. In this study, we analyzed the expression of MSY genes in testes with Ad spermatogonia (low infertility risk patients) as compared to testes lacking Ad spermatogonia (high infertility risk) before and after curative GnRHa treatment, and in correlation to their location on the Y-chromosome.

Results: Twenty genes that are up- or down-regulated in the Ad- group are in the X-degenerate or the ampliconic region, respectively. GnRHa treatment increases mRNA levels of 14 genes in the ampliconic region and decreases mRNA levels of 10 genes in the X-degenerate region.

Conclusion: Our findings implicate Y-chromosomal genes, including , some of which are known to be important for spermatogenesis, in the curative hormonal treatment of cryptorchidism-induced infertility.
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http://dx.doi.org/10.1186/s12610-019-0089-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545630PMC
June 2019

Self-organization and symmetry breaking in intestinal organoid development.

Nature 2019 05 24;569(7754):66-72. Epub 2019 Apr 24.

Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland.

Intestinal organoids are complex three-dimensional structures that mimic the cell-type composition and tissue organization of the intestine by recapitulating the self-organizing ability of cell populations derived from a single intestinal stem cell. Crucial in this process is a first symmetry-breaking event, in which only a fraction of identical cells in a symmetrical sphere differentiate into Paneth cells, which generate the stem-cell niche and lead to asymmetric structures such as the crypts and villi. Here we combine single-cell quantitative genomic and imaging approaches to characterize the development of intestinal organoids from single cells. We show that their development follows a regeneration process that is driven by transient activation of the transcriptional regulator YAP1. Cell-to-cell variability in YAP1, emerging in symmetrical spheres, initiates Notch and DLL1 activation, and drives the symmetry-breaking event and formation of the first Paneth cell. Our findings reveal how single cells exposed to a uniform growth-promoting environment have the intrinsic ability to generate emergent, self-organized behaviour that results in the formation of complex multicellular asymmetric structures.
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http://dx.doi.org/10.1038/s41586-019-1146-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544541PMC
May 2019

Mammalian ISWI and SWI/SNF selectively mediate binding of distinct transcription factors.

Nature 2019 05 17;569(7754):136-140. Epub 2019 Apr 17.

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

Chromatin remodelling complexes evict, slide, insert or replace nucleosomes, which represent an intrinsic barrier for access to DNA. These remodellers function in most aspects of genome utilization including transcription-factor binding, DNA replication and repair. Although they are frequently mutated in cancer, it remains largely unclear how the four mammalian remodeller families (SWI/SNF, ISWI, CHD and INO80) orchestrate the global organization of nucleosomes. Here we generated viable embryonic stem cells that lack SNF2H, the ATPase of ISWI complexes, enabling study of SNF2H cellular function, and contrast it to BRG1, the ATPase of SWI/SNF. Loss of SNF2H decreases nucleosomal phasing and increases linker lengths, providing in vivo evidence for an ISWI function in ruling nucleosomal spacing in mammals. Systematic analysis of transcription-factor binding reveals that these remodelling activities have specific effects on binding of different transcription factors. One group critically depends on BRG1 and contains the transcriptional repressor REST, whereas a non-overlapping set of transcription factors, including the insulator protein CTCF, relies on SNF2H. This selectivity readily explains why chromosomal folding and insulation of topologically associated domains requires SNF2H, but not BRG1. Collectively, this study shows that mammalian ISWI is critical for nucleosomal periodicity and nuclear organization and that transcription factors rely on specific remodelling pathways for correct genomic binding.
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http://dx.doi.org/10.1038/s41586-019-1115-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522387PMC
May 2019

Lysosomal Signaling Licenses Embryonic Stem Cell Differentiation via Inactivation of Tfe3.

Cell Stem Cell 2019 02 27;24(2):257-270.e8. Epub 2018 Dec 27.

Friedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland. Electronic address:

Self-renewal and differentiation of pluripotent murine embryonic stem cells (ESCs) is regulated by extrinsic signaling pathways. It is less clear whether cellular metabolism instructs developmental progression. In an unbiased genome-wide CRISPR/Cas9 screen, we identified components of a conserved amino-acid-sensing pathway as critical drivers of ESC differentiation. Functional analysis revealed that lysosome activity, the Ragulator protein complex, and the tumor-suppressor protein Folliculin enable the Rag GTPases C and D to bind and seclude the bHLH transcription factor Tfe3 in the cytoplasm. In contrast, ectopic nuclear Tfe3 represses specific developmental and metabolic transcriptional programs that are associated with peri-implantation development. We show differentiation-specific and non-canonical regulation of Rag GTPase in ESCs and, importantly, identify point mutations in a Tfe3 domain required for cytoplasmic inactivation as potentially causal for a human developmental disorder. Our work reveals an instructive and biomedically relevant role of metabolic signaling in licensing embryonic cell fate transitions.
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http://dx.doi.org/10.1016/j.stem.2018.11.021DOI Listing
February 2019

Histone Methyltransferase mRNA Levels Increase in Response to Curative Hormone Treatment for Cryptorchidism-Dependent Male Infertility.

Genes (Basel) 2018 Aug 1;9(8). Epub 2018 Aug 1.

Swiss Institute of Bioinformatics, 4058 Basel, Switzerland.

There is a correlation between cryptorchidism and an increased risk of testicular cancer and infertility. During orchidopexy, testicular biopsies are performed to confirm the presence of type A dark (Ad) spermatogonia, which are a marker for low infertility risk (LIR). The Ad spermatogonia are absent in high infertility risk (HIR) patients, who are treated with a gonadotropin-releasing hormone agonist (GnRHa) to significantly lower the risk of infertility. Despite its prevalence, little is known about the molecular events involved in cryptorchidism. Previously, we compared the transcriptomes of LIR versus HIR patients treated with and without hormones. Here, we interpreted data regarding members of the positive regulatory domain-containing () family; some of which encoded histone methyltransferases that are important for reproduction. We found there were lower levels of , , , , and mRNA in the testes of HIR patients compared with LIR patients, and that , , and were significantly induced after GnRHa treatment. Furthermore, we observed protein staining in the cytoplasm of germ cells in the testes from LIR and HIR patients, indicating that the mRNA and protein levels corresponded. This result indicated that the curative hormonal therapy for cryptorchidism involved conserved chromatin modification enzymes.
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http://dx.doi.org/10.3390/genes9080391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6116052PMC
August 2018

A Novel Inducible Mouse Model of -driven Mixed-lineage Acute Leukemia.

Hemasphere 2018 Aug 12;2(4):e51. Epub 2018 Jun 12.

Department of Biomedicine, University Children's Hospital of Basel, Basel, Switzerland.

Previous retroviral and knock-in approaches to model human t(11;19) acute mixed-lineage leukemia in mice resulted in myeloproliferation and acute myeloid leukemia not fully recapitulating the human disease. The authors established a doxycycline (DOX)-inducible transgenic mouse model "" in which induction in long-term hematopoietic stem cells, lymphoid primed multipotent progenitor cells, multipotent progenitors (MPP4) but not in more committed myeloid granulocyte-macrophage progenitors led to a fully reversible acute leukemia expressing myeloid and B-cell markers. leukemic cells generally expressed lower mRNA than those obtained after retroviral transduction. Disease induction was associated with levels exceeding the endogenous at mRNA and protein levels. In leukemic cells from t(11;19) leukemia patients, mRNA also exceeded the endogenous levels suggesting a critical threshold for transformation. Expression profiling of acute leukemia revealed gene signatures that segregated t(11;19) leukemia patients from those without an MLL translocation. Importantly, B220 leukemic cells showed a higher in vivo leukemia initiation potential than coexisting B220 cells. Collectively, characterization of a novel transgenic mouse model indicates that the cell-of-origin and the fusion gene expression levels are both critical determinants for -driven acute leukemia.
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http://dx.doi.org/10.1097/HS9.0000000000000051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6745998PMC
August 2018

The CSR-1 endogenous RNAi pathway ensures accurate transcriptional reprogramming during the oocyte-to-embryo transition in Caenorhabditis elegans.

PLoS Genet 2018 03 26;14(3):e1007252. Epub 2018 Mar 26.

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

Endogenous RNAi (endoRNAi) is a conserved mechanism for fine-tuning gene expression. In the nematode Caenorhabditis elegans, several endoRNAi pathways are required for the successful development of reproductive cells. The CSR-1 endoRNAi pathway promotes germ cell development, primarily by facilitating the expression of germline genes. In this study, we report a novel function for the CSR-1 pathway in preventing premature activation of embryonic transcription in the developing oocytes, which is accompanied by a general Pol II activation. This CSR-1 function requires its RNase activity, suggesting that, by controlling the levels of maternal mRNAs, CSR-1-dependent endoRNAi contributes to an orderly reprogramming of transcription during the oocyte-to-embryo transition.
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http://dx.doi.org/10.1371/journal.pgen.1007252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886687PMC
March 2018

Cell-autonomous and cell non-autonomous downregulation of tumor suppressor DAB2IP by microRNA-149-3p promotes aggressiveness of cancer cells.

Cell Death Differ 2018 07 22;25(7):1224-1238. Epub 2018 Mar 22.

National Laboratory CIB (LNCIB), AREA Science Park, 34149, Trieste, Italy.

The tumor suppressor DAB2IP contributes to modulate the network of information established between cancer cells and tumor microenvironment. Epigenetic and post-transcriptional inactivation of this protein is commonly observed in multiple human malignancies, and can potentially favor progression of tumors driven by a variety of genetic mutations. Performing a high-throughput screening of a large collection of human microRNA mimics, we identified miR-149-3p as a negative post-transcriptional modulator of DAB2IP. By efficiently downregulating DAB2IP, this miRNA enhances cancer cell motility and invasiveness, facilitating activation of NF-kB signaling and promoting expression of pro-inflammatory and pro-angiogenic factors. In addition, we found that miR-149-3p secreted by prostate cancer cells induces DAB2IP downregulation in recipient vascular endothelial cells, stimulating their proliferation and motility, thus potentially remodeling the tumor microenvironment. Finally, we found that inhibition of endogenous miR-149-3p restores DAB2IP activity and efficiently reduces tumor growth and dissemination of malignant cells. These observations suggest that miR-149-3p can promote cancer progression via coordinated inhibition of DAB2IP in tumor cells and in stromal cells.
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http://dx.doi.org/10.1038/s41418-018-0088-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030048PMC
July 2018

Curative GnRHa treatment has an unexpected repressive effect on Sertoli cell specific genes.

Basic Clin Androl 2018 9;28. Epub 2018 Feb 9.

Cryptorchidism Research Institute, Kindermedizinisches Zentrum Liestal, 4410 Liestal, Switzerland.

Background: Follicle stimulating hormone and testosterone stimulate Sertoli cells to support germ cell function and differentiation. During mini-puberty, when gonadotropin (GnRH) stimulates increases in plasma luteinizing hormone (LH) and testosterone levels, gonocytes are transformed into Ad spermatogonia. In cryptorchidism, impaired gonadotropin secretion during mini-puberty results in insufficient LH and testosterone secretion, impaired gonocyte transition to Ad spermatogonia, and perturbed Sertoli cell proliferation. Treatment with a gonadotropin-releasing hormone agonist (GnRHa/Buserelin) induced gonocytes to differentiate into Ad spermatogonia and rescued fertility. The present study evaluated the impact of low LH secretion on Sertoli cell function by comparing differential gene expression data between testes with low LH that lacked Ad spermatogonia (Ad-) and testes that completed mini-puberty (Ad+). Furthermore, we analyzed changes in the transcription of selected Sertoli cell specific genes in response to GnRHa treatment.

Results: Ad- testes showed reduced expression of nine out of 40 selected Sertoli cell specific genes compared to Ad+ testes. GnRHa treatment repressed most of the Sertoli cell specific genes, including the inhibins, but it increased the expression of genes that regulate apoptosis () and proliferation ().

Conclusions: Impaired-minipuberty with decreased LH and testosterone levels affected Ad and Sertoli cell development through positive and negative regulation of morphoregulatory and apoptotic genes. GnRHa treatment had a repressive effect on most Sertoli cell specific genes, which suggested that Sertoli cells underwent a cellular rearrangement. We propose that gonadotropin-dependent increases in and expression drove Sertoli cell proliferation and germ cell self-renewal and supported the transition of gonocytes to Ad spermatogonia, independent of inhibins.
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http://dx.doi.org/10.1186/s12610-018-0067-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5806254PMC
February 2018

The Dynamics of mRNA Turnover Revealed by Single-Molecule Imaging in Single Cells.

Mol Cell 2017 Nov 19;68(3):615-625.e9. Epub 2017 Oct 19.

Friedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland. Electronic address:

RNA degradation plays a fundamental role in regulating gene expression. In order to characterize the spatiotemporal dynamics of RNA turnover in single cells, we developed a fluorescent biosensor based on dual-color, single-molecule RNA imaging that allows intact transcripts to be distinguished from stabilized degradation intermediates. Using this method, we measured mRNA decay in single cells and found that individual degradation events occur independently within the cytosol and are not enriched within processing bodies. We show that slicing of an mRNA targeted for endonucleolytic cleavage by the RNA-induced silencing complex can be observed in real time in living cells. This methodology provides a framework for investigating the entire life history of individual mRNAs from birth to death in single cells.
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http://dx.doi.org/10.1016/j.molcel.2017.09.030DOI Listing
November 2017

DMRTC2, PAX7, BRACHYURY/T and TERT Are Implicated in Male Germ Cell Development Following Curative Hormone Treatment for Cryptorchidism-Induced Infertility.

Genes (Basel) 2017 Oct 11;8(10). Epub 2017 Oct 11.

Cryptorchidism Research Institute, Kindermedizinisches Zentrum Liestal, 4410 Liestal, Switzerland.

Defective mini-puberty results in insufficient testosterone secretion that impairs the differentiation of gonocytes into dark-type (Ad) spermatogonia. The differentiation of gonocytes into Ad spermatogonia can be induced by administration of the gonadotropin-releasing hormone agonist, GnRHa (Buserelin, INN)). Nothing is known about the mechanism that underlies successful GnRHa treatment in the germ cells. Using RNA-sequencing of testicular biopsies, we recently examined RNA profiles of testes with and without GnRHa treatment. Here, we focused on the expression patterns of known gene markers for gonocytes and spermatogonia, and found that , , BRACHYURY/, and were associated with defective mini-puberty and were responsive to GnRHa. These results indicate novel testosterone-dependent genes and provide valuable insight into the transcriptional response to both defective mini-puberty and curative GnRHa treatment, which prevents infertility in man with one or both undescended (cryptorchid) testes.
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http://dx.doi.org/10.3390/genes8100267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5664117PMC
October 2017

Genes Involved in Long-Term Memory Are Expressed in Testis of Cryptorchid Boys and Respond to GnRHa Treatment.

Cytogenet Genome Res 2017 13;152(1):9-15. Epub 2017 Jul 13.

Cryptorchidism Research Institute, Kindermedizinisches Zentrum Liestal, Liestal, Switzerland.

It has been known for many years that boys with unilateral or bilateral undescended testis (cryptorchidism) tend to have a low IQ, and those who belong to the high infertility risk (HIR) group perform less well at school than low infertility risk (LIR) patients. However, the molecular biological processes underlying this phenomenon are not understood. In this study, we report the outcome of testicular RNA profiling for genes involved in long-term memory formation. We analyzed the histology and the transcriptome of testicular biopsies from bilateral HIR cryptorchid boys, comparing those who received GnRHa treatment for 6 months after the first surgery with those who did not receive GnRHa before the second surgery. We found that GnRHa treatment alters the testicular mRNA levels of neuronal genes that are involved in long-term memory and testosterone synthesis. These data highlight a possible molecular link between cryptorchidism, impaired mini-puberty, and diminished cognitive functions. Our results are consistent with the hypothesis that hypogonadotropic hypogonadism in cryptorchid boys with altered mini-puberty may affect neuronal genes important for memory and learning, which could help explaining the negative correlation between cryptorchidism and intellectual abilities.
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http://dx.doi.org/10.1159/000477522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5637305PMC
September 2017

MicroRNAs-143 and -145 induce epithelial to mesenchymal transition and modulate the expression of junction proteins.

Cell Death Differ 2017 10 23;24(10):1750-1760. Epub 2017 Jun 23.

Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Turin, Turin, Italy.

Transforming growth factor (TGF)-β is one of the major inducers of epithelial to mesenchymal transition (EMT), a crucial program that has a critical role in promoting carcinoma's metastasis formation. MicroRNAs-143 and -145, which are both TGF-β direct transcriptional targets, are essential for the differentiation of vascular smooth muscle cells (VSMC) during embryogenesis, a TGF-β-dependent process reminiscent of EMT. Their role in adult tissues is however less well defined and even ambiguous, as their expression was correlated both positively and negatively with tumor progression. Here we show that high expression of both miRs-143 and -145 in mouse mammary tumor cells expressing constitutively active STAT3 (S3C) is involved in mediating their disrupted cell-cell junctions. Additionally, miR-143 appears to have a unique role in tumorigenesis by enhancing cell migration in vitro and extravasation in vivo while impairing anchorage-independent growth, which may explain the contradictory reports about its role in tumors. Accordingly, we demonstrate that overexpression of either miRNA in the non-transformed mammary epithelial NMuMG cells leads to upregulation of EMT markers and of several endogenous TGF-β targets, downmodulation of a number of junction proteins and increased motility, correlating with enhanced basal and TGF-β-induced SMAD-mediated transcription. Moreover, pervasive transcriptome perturbation consistent with the described phenotype was observed. In particular, the expression of several transcription factors involved in the mitogenic responses, of MAPK family members and, importantly, of several tight junction proteins and the SMAD co-repressor TGIF was significantly reduced. Our results provide important mechanistic insight into the non-redundant role of miRs-143 and -145 in EMT-related processes in both transformed and non-transformed cells, and suggest that their expression must be finely coordinated to warrant optimal migration/invasion while not interfering with cell growth.
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http://dx.doi.org/10.1038/cdd.2017.103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5596419PMC
October 2017

pSILAC mass spectrometry reveals ZFP91 as IMiD-dependent substrate of the CRL4 ubiquitin ligase.

Nat Commun 2017 05 22;8:15398. Epub 2017 May 22.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA.

Thalidomide and its derivatives lenalidomide and pomalidomide (IMiDs) are effective treatments of haematologic malignancies. It was shown that IMiDs impart gain-of-function properties to the CUL4-RBX1-DDB1-CRBN (CRL4) ubiquitin ligase that enable binding, ubiquitination and degradation of key therapeutic targets such as IKZF1, IKZF3 and CSNK1A1. While these substrates have been implicated as efficacy targets in multiple myeloma (MM) and 5q deletion associated myelodysplastic syndrome (del(5q)-MDS), other targets likely exist. Using a pulse-chase SILAC mass spectrometry-based proteomics approach, we demonstrate that lenalidomide induces the ubiquitination and degradation of ZFP91. We establish ZFP91 as a bona fide IMiD-dependent CRL4 substrate and further show that ZFP91 harbours a zinc finger (ZnF) motif, related to the IKZF1/3 ZnF, critical for IMiD-dependent CRBN binding. These findings demonstrate that single time point pulse-chase SILAC mass spectrometry-based proteomics (pSILAC MS) is a sensitive approach for target identification of small molecules inducing selective protein degradation.
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http://dx.doi.org/10.1038/ncomms15398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5458144PMC
May 2017

GnRHa Treatment of Cryptorchid Boys Affects Genes Involved in Hormonal Control of the HPG Axis and Fertility.

Sex Dev 2017 16;11(3):126-136. Epub 2017 May 16.

Cryptorchidism Research Institute, Kindermedizinisches Zentrum Liestal, Liestal, Switzerland.

The gonadotropin-releasing hormone agonist (GnRHa; Buserelin) rescues fertility during adulthood in the majority of high infertility risk cryptorchid boys presenting with defective mini-puberty. However, the molecular events governing this effect are not understood. We report the outcome of an RNA profiling analysis of testicular biopsies from 4 operated patients who were treated with GnRHa for 6 months versus 3 operated controls who were not treated. GnRHa induces a significant transcriptional response, including protein-coding genes involved in pituitary development, the hypothalamic-pituitary-gonadal axis, and testosterone synthesis. Furthermore, we observed an increased abundance of long noncoding RNAs (lncRNAs) participating in epigenetic processes, including AIRN, FENDRR, XIST, and HOTAIR. These data are consistent with the hypothesis that hypogonadotropic hypogonadism in boys with altered mini-puberty is the consequence of a profoundly altered gene expression program involving protein-coding genes and lncRNAs. Our results point to molecular mechanisms that underlie the ability of GnRHa to rescue fertility.
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http://dx.doi.org/10.1159/000471937DOI Listing
April 2018

Gene bivalency at Polycomb domains regulates cranial neural crest positional identity.

Science 2017 03;355(6332)

Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4051 Basel, Switzerland.

The cranial neural crest cells are multipotent cells that provide head skeletogenic mesenchyme and are crucial for craniofacial patterning. We analyzed the chromatin landscapes of mouse cranial neural crest subpopulations in vivo. Early postmigratory subpopulations contributing to distinct mouse craniofacial structures displayed similar chromatin accessibility patterns yet differed transcriptionally. Accessible promoters and enhancers of differentially silenced genes carried H3K27me3/H3K4me2 bivalent chromatin marks embedded in large -dependent Polycomb domains, indicating transcriptional poising. These postmigratory bivalent chromatin regions were already present in premigratory progenitors. At Polycomb domains, H3K27me3 antagonized H3K4me2 deposition, which was restricted to accessible sites. Thus, bivalent Polycomb domains provide a chromatin template for the regulation of cranial neural crest cell positional identity in vivo, contributing insights into the epigenetic regulation of face morphogenesis.
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http://dx.doi.org/10.1126/science.aal2913DOI Listing
March 2017

The Hippo kinases LATS1 and 2 control human breast cell fate via crosstalk with ERα.

Nature 2017 01 9;541(7638):541-545. Epub 2017 Jan 9.

Friedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland.

Cell fate perturbations underlie many human diseases, including breast cancer. Unfortunately, the mechanisms by which breast cell fate are regulated are largely unknown. The mammary gland epithelium consists of differentiated luminal epithelial and basal myoepithelial cells, as well as undifferentiated stem cells and more restricted progenitors. Breast cancer originates from this epithelium, but the molecular mechanisms that underlie breast epithelial hierarchy remain ill-defined. Here, we use a high-content confocal image-based short hairpin RNA screen to identify tumour suppressors that regulate breast cell fate in primary human breast epithelial cells. We show that ablation of the large tumour suppressor kinases (LATS) 1 and 2 (refs 5, 6), which are part of the Hippo pathway, promotes the luminal phenotype and increases the number of bipotent and luminal progenitors, the proposed cells-of-origin of most human breast cancers. Mechanistically, we have identified a direct interaction between Hippo and oestrogen receptor-α (ERα) signalling. In the presence of LATS, ERα was targeted for ubiquitination and Ddb1-cullin4-associated-factor 1 (DCAF1)-dependent proteasomal degradation. Absence of LATS stabilized ERα and the Hippo effectors YAP and TAZ (hereafter YAP/TAZ), which together control breast cell fate through intrinsic and paracrine mechanisms. Our findings reveal a non-canonical (that is, YAP/TAZ-independent) effect of LATS in the regulation of human breast cell fate.
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http://dx.doi.org/10.1038/nature20829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726477PMC
January 2017

XRN2 Autoregulation and Control of Polycistronic Gene Expresssion in Caenorhabditis elegans.

PLoS Genet 2016 09 15;12(9):e1006313. Epub 2016 Sep 15.

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

XRN2 is a conserved 5'→3' exoribonuclease that complexes with proteins that contain XRN2-binding domains (XTBDs). In Caenorhabditis elegans (C. elegans), the XTBD-protein PAXT-1 stabilizes XRN2 to retain its activity. XRN2 activity is also promoted by 3'(2'),5'-bisphosphate nucleotidase 1 (BPNT1) through hydrolysis of an endogenous XRN inhibitor 3'-phosphoadenosine-5'-phosphate (PAP). Here, we find through unbiased screening that loss of bpnt-1 function suppresses lethality caused by paxt-1 deletion. This unexpected finding is explained by XRN2 autoregulation, which occurs through repression of a cryptic promoter activity and destabilization of the xrn-2 transcript. De-repression appears to be triggered such that more robust XRN2 perturbation, by elimination of both PAXT-1 and BPNT1, is less detrimental to worm viability than absence of PAXT-1 alone. Indeed, we find that two distinct XRN2 repression mechanisms are alleviated at different thresholds of XRN2 inactivation. Like more than 15% of C. elegans genes, xrn-2 occurs in an operon, and we identify additional operons under its control, consistent with a broader function of XRN2 in polycistronic gene regulation. Regulation occurs through intercistronic regions that link genes in an operon, but a part of the mechanisms may allow XRN2 to operate on monocistronic genes in organisms lacking operons.
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http://dx.doi.org/10.1371/journal.pgen.1006313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5025045PMC
September 2016
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