COVID-19 severity correlates with airway epithelium-immune cell interactions identified by single-cell analysis.

To investigate the immune response and mechanisms associated with severe coronavirus disease 2019 (COVID-19), we performed single-cell RNA sequencing on nasopharyngeal and bronchial samples from 19 clinically well-characterized patients with moderate or critical disease and from five healthy controls. We identified airway epithelial cell types and states vulnerable to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In patients with COVID-19, epithelial cells showed an average three-fold increase in expression of the SARS-CoV-2 entry receptor ACE2, which correlated with interferon signals by immune cells. Compared to moderate cases, critical cases exhibited stronger interactions between epithelial and immune cells, as indicated by ligand-receptor expression profiles, and activated immune cells, including inflammatory macrophages expressing CCL2, CCL3, CCL20, CXCL1, CXCL3, CXCL10, IL8, IL1B and TNF. The transcriptional differences in critical cases compared to moderate cases likely contribute to clinical observations of heightened inflammatory tissue damage, lung injury and respiratory failure. Our data suggest that pharmacologic inhibition of the CCR1 and/or CCR5 pathways might suppress immune hyperactivation in critical COVID-19.

SARS-CoV-2 receptor ACE2 and TMPRSS2 are primarily expressed in bronchial transient secretory cells.

The SARS-CoV-2 pandemic affecting the human respiratory system severely challenges public health and urgently demands for increasing our understanding of COVID-19 pathogenesis, especially host factors facilitating virus infection and replication. SARS-CoV-2 was reported to enter cells via binding to ACE2, followed by its priming by TMPRSS2. Here, we investigate ACE2 and TMPRSS2 expression levels and their distribution across cell types in lung tissue (twelve donors, 39,778 cells) and in cells derived from subsegmental bronchial branches (four donors, 17,521 cells) by single nuclei and single cell RNA sequencing, respectively. While TMPRSS2 is strongly expressed in both tissues, in the subsegmental bronchial branches ACE2 is predominantly expressed in a transient secretory cell type. Interestingly, these transiently differentiating cells show an enrichment for pathways related to RHO GTPase function and viral processes suggesting increased vulnerability for SARS-CoV-2 infection. Our data provide a rich resource for future investigations of COVID-19 infection and pathogenesis.

Chromatin-sensitive cryptic promoters putatively drive expression of alternative protein isoforms in yeast.

Cryptic transcription is widespread and generates a heterogeneous group of RNA molecules of unknown function. To improve our understanding of cryptic transcription, we investigated their transcription start site (TSS) usage, chromatin organization, and posttranscriptional consequences in Saccharomyces cerevisiae We show that TSSs of chromatin-sensitive internal cryptic transcripts retain comparable features of canonical TSSs in terms of DNA sequence, directionality, and chromatin accessibility. We define the 5' and 3' boundaries of cryptic transcripts and show that, contrary to RNA degradation-sensitive ones, they often overlap with the end of the gene, thereby using the canonical polyadenylation site, and associate to polyribosomes. We show that chromatin-sensitive cryptic transcripts can be recognized by ribosomes and may produce truncated polypeptides from downstream, in-frame start codons. Finally, we confirm the presence of the predicted polypeptides by reanalyzing N-terminal proteomic data sets. Our work suggests that a fraction of chromatin-sensitive internal cryptic promoters initiates the transcription of alternative truncated mRNA isoforms. The expression of these chromatin-sensitive isoforms is conserved from yeast to human, expanding the functional consequences of cryptic transcription and proteome complexity.

Human haematopoietic stem cell lineage commitment is a continuous process.

Blood formation is believed to occur through stepwise progression of haematopoietic stem cells (HSCs) following a tree-like hierarchy of oligo-, bi- and unipotent progenitors. However, this model is based on the analysis of predefined flow-sorted cell populations. Here we integrated flow cytometric, transcriptomic and functional data at single-cell resolution to quantitatively map early differentiation of human HSCs towards lineage commitment. During homeostasis, individual HSCs gradually acquire lineage biases along multiple directions without passing through discrete hierarchically organized progenitor populations. Instead, unilineage-restricted cells emerge directly from a 'continuum of low-primed undifferentiated haematopoietic stem and progenitor cells' (CLOUD-HSPCs). Distinct gene expression modules operate in a combinatorial manner to control stemness, early lineage priming and the subsequent progression into all major branches of haematopoiesis. These data reveal a continuous landscape of human steady-state haematopoiesis downstream of HSCs and provide a basis for the understanding of haematopoietic malignancies.

Chd1 chromatin remodelers maintain nucleosome organization and repress cryptic transcription.

Chromatin organization is essential for defining transcription units and maintaining genomic integrity in eukaryotes. In this study, we found that deletion of the Schizosaccharomyces pombe Chd1 chromatin remodelers, hrp1 and hrp3, causes strong, genome-wide accumulation of antisense transcripts. Nucleosome mapping revealed a specific role for Chd1 remodelers in the positioning of nucleosomes in gene coding regions. Other mutations associated with enhanced cryptic transcription activity, such as set2Δ, alp13Δ and FACT complex subunit pob3Δ, did not, or only mildly, affect nucleosome positioning. These data indicate several mechanisms in the repression of cryptic promoter activity in eukaryotic cells.

Mesomelic dysplasia Kantaputra type is associated with duplications of the HOXD locus on chromosome 2q.

Mesomelic dysplasia Kantaputra type (MDK) is characterized by marked mesomelic shortening of the upper and lower limbs originally described in a Thai family. To identify the cause of MDK, we performed array CGH and identified two microduplications on chromosome 2 (2q31.1-q31.2) encompassing ∼481 and 507 kb, separated by a segment of normal copy number. The more centromeric duplication encompasses the entire HOXD cluster, as well as the neighboring genes EVX2 and MTX2. The breakpoints of the duplication localize to the same region as the previously identified inversion of the mouse mutant ulnaless (Ul), which has a similar phenotype as MDK. We propose that MDK is caused by duplications that modify the topography of the locus and as such result in deregulation of HOXD gene expression.

Deletion and point mutations of PTHLH cause brachydactyly type E.

Autosomal-dominant brachydactyly type E (BDE) is a congenital limb malformation characterized by small hands and feet predominantly as a result of shortened metacarpals and metatarsals. In a large pedigree with BDE, short stature, and learning disabilities, we detected a microdeletion of approximately 900 kb encompassing PTHLH, the gene coding for parathyroid hormone related protein (PTHRP). PTHRP is known to regulate the balance between chondrocyte proliferation and the onset of hypertrophic differentiation during endochondral bone development. Inactivation of Pthrp in mice results in short-limbed dwarfism because of premature differentiation of chondrocyte. On the basis of our initial finding, we tested further individuals with BDE and short stature for mutations in PTHLH. We identified two missense (L44P and L60P), a nonstop (X178WextX( *)54), and a nonsense (K120X) mutation. The missense mutation L60P was tested in chicken micromass culture with the replication-competent avian sarcoma leukosis virus retroviral expression system and was shown to result in a loss of function. Thus, loss-of-function mutations in PTHLH cause BDE with short stature.