Biology subjects

Katayama, S.

Publications and source records attributed to Katayama, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Placental cis-eQTL analysis identifies preeclampsia-dependent genetic regulation at promoters

Background: Preeclampsia (PE) is a hypertensive pregnancy disorder with contributions from both maternal and fetal genetic factors, but how both genomes regulate placental transcription remains incompletely understood. Results: We analyzed placental 5'-end RNA sequencing data from 59 pregnancies in the FINNPEC cohort and performed separate maternal and fetal cis-eQTL analyses using gene-level expression and promoter-level expression defined as Transcript Far 5' Ends (TFE). Genotype-by-PE interaction models were used to identify genetic effects that differ according to PE status. No gene-level interaction eQTLs remained significant after multiple-testing correction, although suggestive signals included LILRB2 in the maternal and LPCAT3 in the fetal analyses. In contrast, we identified two significant syntenic promoter-level interaction eQTLs: rs12504642-TFE61820 at the FHDC1 locus in the maternal analysis and rs1557150-TFE9032 at the PPIAP31 locus in the fetal analysis. Conclusion: These findings suggest that PE-dependent genetic interaction effects may be detectable at specific transcript promoter regions even when they are not captured at annotated gene levels.

genetics↗

bioRxiv2026

Structure and genomic organization of the human DUX4 homologue bovine DUXC

BackgroundDUXC is a multi-copy transcription factor gene found within a long tandem repeat locus in several Laurasiatherians. It is suggested to be functionally similar to human DUX4 because of its shared C-terminal domain and its close phylogenetic relationship to DUX4. DUX family genes are transiently expressed in preimplantation embryos of placental mammals. However, early embryo-derived cDNA proof for DUXC, which is needed for its further functional characterization, has not been reported so far. ResultsOur study provides a full-length sequence of DUXC mRNA, derived from the 8-cell stage in vitro fertilization (IVF) bovine embryos, containing double homeobox and 9aa transactivation domain (9aaTAD)-encoding sequences. Identified DUXC sequence uncovered a first exon that was not previously annotated. We showed that DUXC mRNA levels are independent of the embryonic transcription at the 2-, 4-, and 8-cell stage, whereas its decline, observed from the 8-cell stage onwards, is minor embryonic genome activation (EGA)-dependent. We also investigated the genomic organisation of the DUXC array in eight different cattle breed assemblies, revealing polymorphic internal repeats flanked by an incomplete distal unit at the telomeric end and a much shorter unit at the proximal end of the DUXC array. Despite the presence of a putative polyadenylation signal downstream the distal unit, we presented evidence for the expression of internal but not distal DUXC in early bovine IVF embryos. ConclusionsDUXC is a potential bovine EGA inducer, supported by its expression at peak levels at pre-EGA stages followed by a decrease with a dependency on minor EGA.

developmental biology↗

bioRxiv2026

Fast and Slow Gene Expression Changes in Blood Following Acute Social Stress

Social stress is a risk factor for psychiatric disorders and also influences immune function. While it is known that acute social stress impacts the number of immune cells in circulation, the temporal dynamics of stress induced immune-related transcriptional changes in human blood remain unclear. To investigate changes in gene expression, we exposed 26 adults to the Trier Social Stress Test (TSST), and collected blood at baseline, as well as 5, 30, 60 and 90 min after stress. Whole-blood gene expression was profiled using a 5 targeted RNA-sequencing method (STRT). Differential expression was analyzed using linear and cubic models. We observed a total of 54 differentially expressed genes following stress. Fast responses, with a transient peak immediately following stress, were enriched for cytotoxic T cell, NK cell and dendritic cell functions (e.g., GZMB, GNLY, CCL4 and GZMA) and paralleled lymphocyte count changes. In contrast, gradual, linear responses without any evident peak were enriched for neutrophil related genes (e.g., FPR2, PLAUR, CXCR2, AQP9, and QPCT) and did not mirror neutrophil counts, indicating cell intrinsic transcriptional changes. From pathway and transcription factor enrichment analyses, IL-12 family mediated signaling is inferred as a central mechanism linking stress to immune gene regulation. Our results show that acute psychosocial stress induces both fast and slower changes in gene expression in different immune cell populations. The involvement of the IL-12-STAT4 axis and genes such as PLAUR and FPR2 suggests molecular mechanisms through which stress-related immune activation may contribute to vulnerability for anxiety and depressive disorders.

genetics↗

bioRxiv2025

Hic2 fast-tracks iPS cell generation by suppressing KLF4-dependent epidermal detour

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) is the most established cellular conversion by exogenous master transcription factors (TFs). A deeper understanding of this yet inefficient process is critical to extending our capability to control cellular identity for medical applications. Here we report 14 genes essential for efficient iPSC generation, but dispensable for self-renewal. Of those, overexpression of Hic2, a transcriptional suppressor highly expressed in PSCs, enhances iPSC generation [~]10-fold. This is achieved through a more direct transition towards pluripotency, bypassing an intermediate state with KLF4-dependent transient epidermal gene expression during iPSC generation. Mechanistically, HIC2 co-occupies these KLF4 targets and directly inhibits their expression. Our work demonstrates that master TFs necessary for cellular conversions can also activate obstructive genes during cellular reprogramming. We propose that identifying transcriptional suppressors against such side effects, like Hic2, can be a powerful strategy to achieve more efficient TF-mediated cell conversions.

developmental biology↗

bioRxiv2024

Oxygen level alters energy metabolism in bovine preimplantation embryos

STUDY QUESTIONWhat is the effect of different oxygen (O2) levels on the transcriptomic profile of bovine embryos during the in vitro culture? SUMMARY ANSWEREmbryos grown in hypoxia (6% O2) from zygotes until the blastocyst stage had the highest blastocyst formation rate, whereas normoxia (20% O2) delayed transcriptomic reprogramming and embryonic genome activation, and induced changes in energy metabolism gene expression. WHAT IS KNOWN ALREADYMammalian preimplantation embryo development is a complex sequence of events where, within a week, the zygote is reprogrammed to totipotency and subsequently diverges to embryonic and extraembryonic cell lineages for post-implantation embryo development. This period of development is sensitive to oxygen levels that can affect various cellular processes. STUDY DESIGN, SIZE, DURATIONIn this study, we used triplicate bovine embryos as a model for human embryogenesis to compare the influence of O2 levels on preimplantation embryonic development by culturing embryos either in normoxic (20% O2) or physiological hypoxic (6% O2) conditions, or sequential hypoxia until 16-cell stage and then switching to ultrahypoxic culture (2% O2). PARTICIPANTS/MATERIALS, SETTING, METHODSAs the readout for varied O2 effects, we performed RNA sequencing using 5 targeted STRT-N method on single embryos. We compared zygotes, 4-, 8-, 16-cell and blastocyst stage embryos grown in either normoxic or hypoxic condition, adding ultrahypoxia for blastocyst stage embryos as the third condition. MAIN RESULTS AND THE ROLE OF CHANCEWe found that the initial cleavage rate was not affected by O2 levels but there was a clear difference in blastocyst formation rate. In hypoxia, 36% of embryos reached blastocyst stage while in normoxia the blastocyst formation rate was 13%. In final ultrahypoxia condition only 4.6% of embryos reached blastocyst stage. Transcriptomic profiles showed that normoxic conditions slowed down oocyte transcript degradation and embryonic genome activation. Key metabolic enzyme genes were also altered between hypoxic and normoxic conditions at the blastocyst stage. Both hypoxic and ultrahypoxic conditions induced energy production by upregulating genes involved in glycolysis and lipid metabolism typical to in vivo embryos. In contrast, normoxic conditions failed to upregulate glycolysis genes and only depended on primitive oxidative phosphorylation metabolism. We conclude that constant hypoxia culture of in vitro embryos provided the highest blastocyst formation rate and appropriate energy metabolism. Normoxia altered the energy metabolism and decreased the blastocyst formation rate. Even though ultrahypoxia at blastocyst stage resulted in a drop of blastocyst formation, the transcriptional profile of surviving embryos was normal. LARGE SCALE DATAThe raw data (BCL files) are available at Zenodo: XXXXX FASTQ files generated are available in the EMBLs European Bioinformatics Institute (EMBL-EBI)-BioStudies with accession number X-XXXX. LIMITATIONS, REASONS FOR CAUTIONThe limitation of this study is the use of bovine as an animal model instead of human embryos. Due to this, the direct translation of the results to human should be taken with caution. WIDER IMPLICATIONS OF THE FINDINGSThis study supports previous literature on hypoxic culture conditions being the most suitable for in vitro embryo culture. In addition, we provide new insights on why embryos grown in normoxia do not have the same success rate as embryos grown in hypoxia. We did not observe any benefits of lowering the oxygen levels to 2%, calling for caution of switching to this culture system. STUDY FUNDING/COMPETING INTEREST(S)This project has received funding from the European Unions Horizon 2020 Research and Innovation Programme under the Marie Sklodowska-Curie grant agreement No. 813707.; Work in the JK laboratory is supported by Jane and Aatos Erkko Foundation, Sigrid Juselius Foundation, Liv och Halsa (Finland), Swedish Brain Foundation and Swedish Research Council. The study was also supported by the Estonian Research Council (grant no. PRG1076) and the Horizon Europe NESTOR project (grant no. 101120075). What this means for patients?During assisted reproduction treatment (ART) oocytes are fertilized in vitro by sperm to form an embryo. The embryos are then exposed to environmental effects during the 5 days of culture before implantation. Embryo culture takes place inside the incubator, where different oxygen levels can be used. In this study, we compared 3 different oxygen concentrations for embryo culture: atmospheric or normoxia (20%), physiologic or hypoxia (6%) and combined low and ultra-low concentration with initial culture at 6% oxygen until day 3 followed by 2% (ultrahypoxia) until day 5 of culture. We conducted this study because many ART clinics use different oxygen concentrations for embryo culture, and we aimed to provide insight on how these different concentrations impact embryo development by monitoring gene expression. As using human embryos for this type of experiments is not commonly approved, we used bovine embryos whose early development resembles that of humans. We found that culturing embryos for 5 days at constant low oxygen concentration (hypoxia) gives the highest number of embryos that reach the blastocyst stage (day 5) and have expected gene expression that provides the physiological path of embryo development. Culturing embryos at atmospheric oxygen concentration showed that only 13% of fertilised eggs reached the blastocyst stage at day 5, while culturing at the final ultra-low oxygen concentration had the lowest percentage of matured embryos, and we did not observe any benefits that would justify the use of such a culture system. We suggest that in vitro culturing of embryos at constant 5-6% oxygen gives the best results.

developmental biology↗

bioRxiv2024

Methanol chemoreceptor MtpA- and flagellin protein FliC-dependent methylotaxis determines the spaciotemporal colonization of PPFM in the phyllosphere

Pink-pigmented facultative methylotrophs (PPFMs) capable of growth on methanol are dominant and versatile phyllosphere bacteria that provide positive effects on plant growth through symbiosis. However, the spatiotemporal behavior of PPFMs on plant surfaces and its molecular basis are unknown. Here we show that Methylobacterium sp. strain OR01 inoculated onto red perilla seeds colonized across the entire plant surface in the phyllosphere concomitant with the plant growth. FliC flagellin proteins required for motility were necessary for distributed colonization on plant leaves, but not for colonization at the leaf periphery. Methanol-sensing chemoreceptor MtpA-dependent chemotaxis (methylotaxis; chemotaxis toward methanol) facilitated the bacterial movement from the peripheral to the inner part of leaves and further entry into the stomatal cavity, indicating that methanol functions as a volatile messenger attracting PPFMs to particular leaf locations possibly to support with photosynthesis and to afford protection from pathogenic invasion.

microbiology↗

bioRxiv2024

Impacts of Cell Ranger versions on Chromium gene expression data

In droplet-based single cell gene expression data, cell barcode processing by Cell Ranger (CR) is a standard pipeline. But no systematic evaluation of the impact of CR version on single cell gene expression data has been conducted. To comprehensively evaluate the impact of CR version, we considered six molecular quality criteria, quantified gene expression, and performed downstream analysis for 12 single-cell datasets. Each dataset was processed by 15 versions of CR. We demonstrated that different versions of CR yield different numbers of cell barcodes with significant variation in detected UMIs, features, molecular qualities and average gene expression of protein-coding and lncRNA for the same dataset. Our analysis finds distinction between two diverse categories of cell barcodes: common barcodes unmasked by all versions of CR, and specific barcodes only unmasked/masked by some versions. Surprisingly, we observed variation in molecular read-out between common cell barcodes when called by different versions of CR. The specific barcodes yield skewed gene body coverage and form distinct clusters. The choice of CR version affects scores for quality, average gene expression, clustering results, and top cluster marker genes of the dataset.

bioinformatics↗

bioRxiv2023

Genistein enhances NAD+ biosynthesis by binding to Prohibitin 1 and upregulating nicotinamide phosphoribosyltransferase in adipocytes

Decreased NAD+ levels in adipocytes cause adipose-tissue dysfunction, leading to systemic glucose and lipid metabolism failure. Therefore, developing small molecules and nutraceuticals that can increase NAD+ levels in adipocytes is necessary. Genistein, a nutraceutical derived from soybeans, has various physiological activities and improves glucose and lipid metabolism. In this study, we aimed to unravel the effects of genistein on the intracellular NAD+ levels in adipocytes and the underlying molecular mechanisms. We showed that genistein enhanced NAD+ biosynthesis by increasing the expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in NAD+ biosynthesis. A pull-down assay using genistein-immobilized beads identified prohibitin 1 (PHB1) as a target protein of genistein. The knockdown of PHB1 suppressed the genistein-induced increase in NAMPT expression and NAD+ levels in adipocytes. Genistein-bound PHB1 contributed to the stabilization of the transcription factor CCAAT/enhancer-binding protein {beta} through activation of extracellular signal-regulated kinase, resulting in increased NAMPT expression at the transcriptional level. Genistein induced dephosphorylation of peroxisome proliferator-activated receptor at serine 273 and increased the insulin-sensitizing adipokine, adiponectin, in adipocytes, whereas the knockdown of NAMPT and PHB1 abolished these genistein-mediated effects. Our results proved the potential efficacy of nutraceuticals in promoting NAD+ levels and restoring metabolic function in adipocytes. Furthermore, we identified PHB1, localized to the plasma membrane, as a candidate target protein for increased expression of NAMPT in adipocytes. Overall, these findings will assist in developing NAD+ boosting strategies to alleviate the metabolic dysfunctions in adipose tissues. Significance StatementIncreasing NAD+ levels is an important preventive strategy for maintaining metabolic function. Here, we showed that genistein, a nutraceutical, which increases NAD+ levels in adipocytes, increased NAD+ biosynthesis by upregulating nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in the NAD+ biosynthesis pathway. Our findings also showed that genistein increased NAMPT expression by binding to prohibitin 1 in the plasma membrane. Genistein-induced increase in NAD+ levels promoted adiponectin expression, an insulin-sensitizing adipokine, in adipocytes. This study provides evidence that nutraceuticals, such as genistein, are effective in enhancing NAD+ biosynthesis in adipocytes and that PHB1 is a candidate target protein for increased expression of NAMPT to maintain metabolic functions in adipose tissues.

biochemistry↗

bioRxiv2022

B1 SINE-binding ZFP266 impedes reprogramming through suppression of chromatin opening by pioneering factors

Induced pluripotent stem cell reprogramming is inherently inefficient and understanding the molecular mechanisms underlying this inefficiency holds the key to successfully control cellular identity. Here, we report 16 novel reprogramming roadblock genes identified by CRISPR/Cas9-mediated genome-wide knockout (KO) screening. Of these, depletion of the predicted KRAB zinc finger protein (KRAB-ZFP) Zfp266 strongly and consistently enhanced iPSC generation in several iPSC reprogramming settings, emerging as the most robust roadblock. Further analyses revealed that ZFP266 binds Short Interspersed Nuclear Elements (SINEs) adjacent to binding sites of pioneering factors, OCT4 (POU5F1), SOX2 and KLF4, and impedes chromatin opening. Replacing the KRAB co-suppressor with a co-activator domain converted ZFP266 from a reprogramming inhibitor to a potent reprogramming facilitator. This work proposes SINE-KRAB-ZFP interaction to be a critical regulator of chromatin accessibility at enhancers for efficient cellular identity changes and also serves as a resource to further illuminate molecular mechanisms hindering reprogramming.

cell biology↗

bioRxiv2021

Transient DUX4 expression induces blastomere-like expression program that is marked by SLC34A2

DUX4 has recently been recognized as a key regulator in human embryonic genome activation (EGA). The exact role of DUX4 in human embryo is still elusive, partly due to the cytotoxicity of persistent DUX4 expression in cellular models. We report here that a transient DUX4 expression in human embryonic stem cells (hESCs) retains cell viability while inducing an EGA-like expression program in a subpopulation of the cells. These cells showed resemblance to 8-cell stage blastomeres and were thus named induced blastomere-like (iBM) cells. Trajectory inference from the single-cell RNA-seq data suggested that the expression profile of these cells progressed in a manner similar to the morula to blastocyst transition in human embryo. Finally, viable iBM cells could be enriched using an antibody against NaPi2b (SLC34A2), paving the way for further experimental approaches. The iBM cells can become a powerful tool to model transcriptional dynamics and regulation during early human embryogenesis.

cell biology↗

bioRxiv2020

Ectopic histone clipping in the mouse model of progressive myoclonus epilepsy

We establish cystatin B (CSTB) as a regulator of histone H3 tail clipping in murine neural progenitor cells (NPCs) and provide evidence suggesting that epigenetic dysregulation contributes to the early pathogenesis in brain disorders associated with deficient CSTB function. We show that NPCs undergo regulated cleavage of the N-terminal tail of histone H3 at threonine 22 (H3T22) transiently upon induction of differentiation. CSTB-deficient NPCs present premature activation of H3T22 clipping during self-renewal mediated by increased activity of cathepsins L and B. During differentiation, the proportion of immature committed neurons undergoing H3T22 clipping is significantly higher in CSTB-deficient than in wild-type NPCs, with no observable decline within 12 days post-differentiation. CSTB-deficient NPCs exhibit significant transcriptional changes highlighting altered expression of nuclear-encoded mitochondrial genes. These changes are associated with significantly impaired respiratory capacity of differentiating NPCs devoid of CSTB. Our data expand the mechanistic understanding of diseases associated with CSTB deficiency.

pathology↗

bioRxiv2020

Branched-Chain Amino Acid Metabolism is Regulated by ERRα and is Further Impaired by Glucose Loading in Type 2 Diabetes

Aims/hypothesisIncreased levels of branched-chain amino acids (BCAAs) are associated with type 2 diabetes pathogenesis. However, most metabolomic studies are limited to an analysis of plasma metabolites under fasting conditions, rather than the dynamic shift in response to a metabolic challenge. Moreover, metabolomic profiles of peripheral tissues involved in glucose homeostasis are scarce and the transcriptomic regulation of genes involved in BCAA catabolism is partially unknown. This study aimed to identify differences in circulating and skeletal muscle BCAA levels in response to an OGTT in individuals with normal glucose tolerance (NGT) or type 2 diabetes. Additionally, transcription factors involved in the regulation of the BCAA gene set were identified. MethodsPlasma and vastus lateralis muscle biopsies were obtained from individuals with NGT or type 2 diabetes before and after an OGTT. Plasma and quadriceps muscles were harvested from skeletal muscle-specific PGC-1 knockout and transgenic mice. BCAA-related metabolites and genes were assessed by LC-MS/MS and RT-PCR, respectively. Small interfering RNA and adenovirus-mediated overexpression techniques were used in primary human skeletal muscle cells to study the role of PGC-1A and ESRRA in the expression of the BCAA gene set. Radiolabeled leucine was used to analyze the impact of ERR knockdown on leucine oxidation. ResultsImpairments in BCAA catabolism in people with type 2 diabetes under fasting conditions were exacerbated after a glucose load. Branched-chain keto acids were reduced 37-56% after an OGTT in the NGT group, whereas no changes were detected in individuals with T2D. These changes were concomitant with a stronger correlation with glucose homeostasis biomarkers and downregulated expression of BCAT2, BCKDH complex subunits and 69% of downstream BCAA-related genes in skeletal muscle. In primary human myotubes overexpressing PGC-1, 61% of the analyzed BCAA genes were upregulated, while 67% were downregulated in the quadriceps of skeletal muscle-specific PGC-1 knockout mice. ESRRA (encoding estrogen-related receptor , ERR) silencing completely abrogated the PGC-1-induced upregulation of BCAA-related genes in primary human myotubes. Conclusions/interpretationMetabolic inflexibility in type 2 diabetes impacts BCAA homeostasis and attenuates the decrease of circulating and skeletal muscle BCAA-related metabolites after a glucose challenge. Transcriptional regulation of BCAA genes in primary human myotubes via a PGC-1 is ERR-dependent. Research in contextO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LICirculating levels of BCAA are elevated in type 2 diabetes. C_LIO_LIPGC-1 is involved in the transcription of the BCAA gene set. C_LI What is the key question?O_LIDoes metabolic inflexibility associated with type 2 diabetes encompass BCAA homeostasis and PGC-1 mediated transcription of the BCAA gene set? C_LI What are the new findings?O_LIBCAA homeostasis is further compromised by a glucose challenge in type 2 diabetes. C_LIO_LIAn OGTT reveals coordinated regulation between BCAA metabolites, blood glucose, and HbA1c levels. C_LIO_LIERR is essential for PGC-1-mediated BCAA gene expression in primary human myotubes. C_LI How might this impact on clinical practice in the foreseeable future?O_LIAn OGTT can be used to underscore impairments in BCAA metabolism. These findings suggest that interventions targeting the PGC-1/ ERR axis may improve BCAA homeostasis. C_LI

physiology↗

bioRxiv2020

Differentiation of ciliated human midbrain-derived LUHMES neurons

Many human cell types are ciliated, including neural progenitors and differentiated neurons. Ciliopathies are characterized by defective cilia and comprise various disease states, including brain phenotypes, where the underlying biological pathways are largely unknown. Our understanding of neuronal cilia is rudimentary and an easy-to-maintain, ciliated human neuronal cell model is missing. LUHMES is a ciliated neuronal cell line derived from human fetal mesencephalon. LUHMES cells can easily be maintained and differentiated into mature, functional neurons within one week. They have a single primary cilium as proliferating progenitor cells and as post-mitotic, differentiating neurons. These developmental stages are completely separable within one day of culture condition change. The Sonic Hedgehog (SHH) signaling pathway is active in differentiating LUHMES neurons. RNA-seq time course analyses reveal molecular pathways and gene-regulatory networks critical for ciliogenesis and axon outgrowth at the interface between progenitor cell proliferation, polarization and neuronal differentiation. Gene expression dynamics of cultured LUHMES neurons faithfully mimic the corresponding in vivo dynamics of human fetal midbrain. In LUHMES, neuronal cilia biology can be investigated along a complete timeline: from proliferation through differentiation to mature neurons. Summary StatementWith LUHMES, a ciliated human neuronal cell model, the underlying "neurobiology" of cilia and ciliopathies can be investigated along a complete time line: from proliferation through differentiation to mature neurons.

neuroscience↗

bioRxiv2019

Quality assessment of single-cell RNA sequencing data by coverage skewness analysis

Analysis and interpretation of single-cell RNA-sequencing (scRNA-seq) experiments are compromised by the presence of poor quality cells. For meaningful analyses, such poor quality cells should be excluded to avoid biases and large variation. However, no clear guidelines exist. We introduce SkewC, a novel quality-assessment method to identify poor quality single-cells in scRNA-seq experiments. The method is based on the assessment of gene coverage for each single cell and its skewness as a quality measure. To validate the method, we investigated the impact of poor quality cells on downstream analyses and compared biological differences between typical and poor quality cells. Moreover, we measured the ratio of intergenic expression, suggesting genomic contamination, and foreign organism contamination of single-cell samples. SkewC is tested in 37,993 single-cells generated by 15 scRNA-seq protocols. We envision SkewC as an indispensable QC method to be incorporated into scRNA-seq experiment to preclude the possibility of scRNA-seq data misinterpretation.

genomics↗

bioRxiv2019

Single cell map of the human ovarian cortex.

The human ovary orchestrates sex hormone production and undergoes monthly structural changes to release mature oocytes. The outer lining of the ovary (cortex) has a key role in defining fertility in women as it harbors the ovarian reserve. It has been postulated that putative oogonial stem cells exist in the ovarian cortex and that these can be captured by DDX4 antibody isolation. We analysed on a single cell level the transcriptome and cell surface antigen profiles of over 24,000 cells from high quality ovarian cortex samples from 21 patients. Our single cell mapping reveals transcriptional profiles of six main cell types; oocytes, granulosa cells, immune cells, endothelial cells, perivascular cells, and stromal cells. Cells captured by DDX4 antibody are perivascular cells, not oogonial stem cells. Our data does not support the existence of germline stem cells in adult human ovaries thereby reinforcing the dogma of a limited ovarian reserve.

cell biology↗

bioRxiv2019

DUX4 regulates oocyte to embryo transition in human

During the human oocyte-to-embryo transition, the fertilized oocyte undergoes final maturation and the embryo genome is gradually activated during the first three cell divisions. How this transition is coordinated in humans is largely unknown. We show that the double homeodomain transcription factor DUX4 contributes to this transition. DUX4 knockdown in human zygotes caused insufficient transcriptome reprogramming as observed three days after fertilization. Induced DUX4 expression in human embryonic stem cells activated transcription of thousands of newly identified bi-directional transcripts, including putative enhancers for embryonic genome activation genes such as LEUTX. DUX4 protein interacted with transcriptional modifiers that are known to couple enhancers and promoters. Taken together, our results reveal that DUX4 is a pioneer regulating oocyte-to-embryo transition in human through activation of intergenic genome, especially enhancers, and hence setting the stage for early human embryo development.

developmental biology↗

bioRxiv2019

Dynamics and Topology of Human Transcribed Cis-regulatory Elements

Promoters and enhancers are key cis-regulatory elements, but how they orchestrate to generate cell-type-specific transcriptomes remains elusive. We developed a simple and robust approach to globally determine 5-ends of nascent RNAs (NET-CAGE) in diverse cells and tissues, thereby sensitively detecting unstable transcripts including enhancer-derived RNAs. We studied RNA synthesis and degradation at the transcription start site level, uncovering the impact of differential promoter usage on transcript stability. We quantified transcription from cis-regulatory elements without influence of RNA turnover, and identified enhancer-promoter pairs which were simultaneously activated upon cellular stimulation. By integrating NET-CAGE data with chromatin interaction maps, we revealed that cis-regulatory elements are topologically connected according to their cell-type specificity. We discovered new enhancers with high sensitivity, and delineated primary locations of transcription within super-enhancers. Our collection of NET-CAGE data from human and mouse significantly expanded the FANTOM5 catalogue of transcribed enhancers, with broad applicability to biomedical research. (148 words)

genomics↗

bioRxiv2019

Complement in human pre-implantation embryos: attack and defense

It is essential for early human life that immunological responses to developing embryos are tightly regulated. An imbalance in the activation and regulation of the human complement system occurs in pregnancy complications, such as pre-eclampsia and recurrent miscarriage. We hereby present the first full analysis of the expression and deposition of complement molecules in human pre-implantation embryos. Thus far, immunological imbalance has been considered in stages of pregnancy following implantation. We here show that complement activation and deposition takes place on developing human embryos already at the pre-implantation stage. Using confocal microscopy, we observed deposition of activation products such as C1q, C3 and C5 on healthy developing embryos, which highlights the need for strict complement regulation. The early embryos express the complement membrane inhibitors CD46, CD55 and CD59 and bind the soluble regulators C4bp and factor H. These findings show that complement targets human embryos, and indicate potential adverse pregnancy outcomes, if regulation of activation fails. In addition, single-cell RNA sequencing of embryos at oocyte, zygote, 4-cell and 8-cell stages showed expression of complement genes, e.g. C1s, C2, C3, C5, factor B and factor D. This shows that the embryonic cells themselves have the capacity to express C3 and C5, which may become activated and function as mediators of cellular signaling. The specific local embryonic expression of complement components, regulators, and deposition of activation products on the surface of embryos suggests that complement has immunoregulatory functions and may impact cellular homeostasis and differentiation at the earliest stage of human life.\n\nStatement of significanceWhile canonical functions of the complement system relate to pathogen-defence, it is known to drive certain immune pathologies. The work here described shows, for the first time, expression and localization of a full range of complement molecules in human pre-implantation embryos. We demonstrate complement attack against early embryos, and show presence of embryonic defence mechanisms. Furthermore, we reveal early embryonic production of complement activators, suggesting non-canonical roles in cell signalling and development. Our findings thus reveal a fundamental role for complement at the earliest stages of human embryogenesis. Our data opens up for future studies into the role of complement, both in relation to infertility and pregnancy complications, as well as basic cellular processes during early human development.

developmental biology↗