Herpes simplex virus 1 (HSV-1) induces a profound host shut-off during lytic infection. The virion host shut-off (vhs) protein plays a key role in this process by efficiently cleaving host and viral mRNAs. Furthermore, the onset of viral DNA replication is accompanied by a rapid decline in host transcriptional activity. To dissect relative contributions of both mechanisms and elucidate gene-specific host transcriptional responses throughout the first 8h of lytic HSV-1 infection, we employed RNA-seq of total, newly transcribed (4sU-labelled) and chromatin-associated RNA in wild-type (WT) and Δvhs infection of primary human fibroblasts. Following virus entry, vhs activity rapidly plateaued at an elimination rate of around 30% of cellular mRNAs per hour until 8h p.i. In parallel, host transcriptional activity dropped to 10-20%. While the combined effects of both phenomena dominated infection-induced changes in total RNA, extensive gene-specific transcriptional regulation was observable in chromatin-associated RNA and was surprisingly concordant between WT and Δvhs infection. Both induced strong transcriptional up-regulation of a small subset of genes that were poorly expressed prior to infection but already primed by H3K4me3 histone marks at their promoters. Most interestingly, analysis of chromatin-associated RNA revealed vhs-nuclease-activity-dependent transcriptional down-regulation of at least 150 cellular genes, in particular of many integrin adhesome and extracellular matrix components. This was accompanied by a vhs-dependent reduction in protein levels by 8h p.i. for many of these genes. In summary, our study provides a comprehensive picture of the molecular mechanisms that govern cellular RNA metabolism during the first 8h of lytic HSV-1 infection. IMPORTANCE The HSV-1 virion host shut-off (vhs) protein efficiently cleaves both host and viral mRNAs in a translation-dependent manner. In this study, we model and quantify changes in vhs activity as well as virus-induced global loss of host transcriptional activity during productive HSV-1 infection. In general, HSV-1-induced alterations in total RNA levels were dominated by these two global effects. In contrast, chromatin-associated RNA depicted gene-specific transcriptional changes. This revealed highly concordant transcriptional changes in WT and Δvhs infection, confirmed DUX4 as a key transcriptional regulator in HSV-1 infection and depicted vhs-dependent, transcriptional down-regulation of the integrin adhesome and extracellular matrix components. The latter explained seemingly gene-specific effects previously attributed to vhs-mediated mRNA degradation and resulted in a concordant loss in protein levels by 8h p.i. for many of the respective genes.

The human adrenal gland is a complex endocrine tissue. Developmental studies on this tissue have been limited to animal models or human foetus. Here, we present a cell atlas analysis of the adult human normal adrenal gland, combining single-nuclei RNA sequencing and spatial transcriptome data to reconstruct adrenal gland development and tumourigenesis. We identified two populations of potential progenitor cells resident within the adrenal cortex: adrenocortical progenitors NR2F2+-ID1+ cells, located within and underneath the capsule, and medullary progenitors SYT1+-CHGA- cells, located in islets in the subcapsular region. Using pseudotime analyses, we provided evidence of the centripetal nature of adrenocortical cell development and of the essential role played by the Wnt/β-catenin pathway in the adrenocortical self-renewal. By comparing transcriptional profiles of cells of normal adrenal glands and adrenocortical adenomas we revealed a high heterogeneity with six adenoma-specific clusters. Overall, our results give insights into adrenal plasticity and mechanisms underlying adrenocortical tumourigenesis.

Blood vessels play a critical role in pancreatic islet health and function, yet current culture methods to generate islet organoids from human pluripotent stem cells (SC-islets) lack a vascular component. Here, we engineered 3D vascularized SC-islet organoids by assembling SC-islet cells, human primary endothelial cells (ECs) and fibroblasts both in a non-perfused model and a microfluidic device with perfused vessels. Vasculature improved stimulus-dependent Ca2+ influx into SC-β-cells, a hallmark of β-cell function that is blunted in non-vascularized SC-islets. We show that an islet-like basement membrane is formed by vasculature and contributes to the functional improvement of SC-β-cells. Furthermore, cell-cell communication networks based on scRNA-seq data predicted BMP2/4-BMPR2 signaling from ECs to SC-β-cells. Correspondingly, BMP4 augmented the SC-β-cell Ca2+ response and insulin secretion. These vascularized SC-islet models will enable further studies of crosstalk between β-cells and ECs and can serve as in vivo-mimicking platforms for disease modeling and therapeutic testing.

26Herpes simplex virus 1 (HSV-1) installs a profound host shut-off during lytic infection. 27 The virion host shut-off (vhs) protein plays a key role in this process by efficiently 28 cleaving both host and viral mRNAs in a translation-initiation-dependent manner. 29 Furthermore, the onset of viral DNA replication is accompanied by a rapid decline in 30 transcriptional activity of the host genome. Both mechanisms have tremendous impact 31 on the RNA expression profile of the infected cells. To dissect their relative 32 contributions and elucidate gene-specific host transcriptional responses throughout 33 the first 8h of lytic HSV-1 infection, we here employed RNA-seq of total, newly 34 transcribed (4sU-labelled) and chromatin-associated RNA in wild-type (WT) and vhs 35 infection of primary human fibroblasts. Following virus entry, vhs activity rapidly 36 plateaued at an elimination rate of around 30% of cellular mRNAs per hour until 8h p.i. 37In parallel, host transcriptional activity dropped down to 10-20%. While the combined 38 effects of both phenomena dominated infection-induced changes in total RNA, 39 extensive gene-specific transcriptional regulation was observable in chromatin-40 associated RNA. This was surprisingly concordant between WT HSV-1 and its vhs 41 mutant and at least in parts mediated by the embryonic transcription factor DUX4. 42 Furthermore, both WT and vhs infection induced strong transcriptional up-regulation 43 of a small subset of genes. Most of these were either poorly or not at all expressed 44 prior to infection but already primed by H3K4me3 histone marks at their promoters. 45 Most interestingly, analysis of chromatin-associated RNA revealed vhs-nuclease- 46 activity-dependent transcriptional down-regulation of at least 150 cellular genes, in 47 particular of many genes encoding integrin adhesome and extracellular matrix 48 components. This was accompanied by a vhs-dependent reduction in protein levels by 49 8h p.i. for many of these genes. In summary, our study provides a comprehensive 50 picture of the molecular mechanisms that govern cellular RNA metabolism during the 51 first 8h of lytic HSV-1 infection. 52 53 4 Author Summary 54The HSV-1 virion host shut-off (vhs) protein efficiently cleaves both host and viral 55 mRNAs in a translation-dependent manner. In this study, we model and quantify 56 changes in vhs activity as well as the virus-induced global loss of host transcriptional 57 activity during productive HSV-1 infection. In general, HSV-1-induced alterations in 58 total RNA levels were found to be predominantly shaped by these two global processes 59 rather than gene-specific regulation. In contrast, chromatin-associated RNA depicted 60 gene-specific transcriptional changes. This revealed highly concordant transcriptional 61 changes in WT and vhs infection, confirmed DUX4 as a key transcriptional regulator 62 in HSV-1 infection and depicted vhs-dependent, transcriptional down-regulation of the 63 integrin adhesome and extracellular matrix. The latt...

We applied the patch-seq technique to harvest transcripts from individual microglial cells from cortex, hippocampus and corpus callosum of acute brain slices from adult mice. After recording membrane currents with the patch-clamp technique, the cytoplasm was collected via the pipette and underwent adapted SMART-seq2 preparation with subsequent sequencing. On average, 4138 genes were detected in 113 cells from hippocampus, corpus callosum and cortex, including microglia markers such as Tmem119, P2ry12 and Siglec-H. Comparing our dataset to previously published single cell mRNA sequencing data from FACS-isolated microglia indicated that two clusters of cells were absent in our patch-seq dataset. Pathway analysis of marker genes in FACS-specific clusters revealed association with microglial activation and stress response. This indicates that under normal conditions microglia in situ lack transcripts associated with a stress-response, and that the microglia-isolation procedure by mechanical dissociation and FACS triggers the expression of genes related to activation and stress.

No abstract