DNA methylation at imprinting control regions (ICRs) is established in gametes in a sex-specific manner and has to be stably maintained during development and in somatic cells to ensure the correct monoallelic expression of imprinted genes. In addition to DNA methylation, the ICRs are marked by allele-specific histone modifications. Whether these marks are essential for maintenance of genomic imprinting is largely unclear. Here, we show that the histone H3 lysine 9 methylases G9a and GLP are required for stable maintenance of imprinted DNA methylation in embryonic stem cells; however, their catalytic activity and the G9a/GLP-dependent H3K9me2 mark are completely dispensable for imprinting maintenance despite the genome-wide loss of non-imprinted DNA methylation in H3K9me2-depleted cells. We provide additional evidence that the G9a/GLP complex protects imprinted DNA methylation by recruitment of de novo DNA methyltransferases, which antagonize TET dioxygenass-dependent erosion of DNA methylation at ICRs.

Nutrient transporters can be rapidly removed from the cell surface via substrate-stimulated endocytosis as a way to control nutrient influx, but the molecular underpinnings are not well understood. In this work, we focus on zinc-dependent endocytosis of human ZIP4 (hZIP4), a zinc transporter that is essential for dietary zinc uptake. Structure-guided mutagenesis and internalization assay reveal that hZIP4 per se acts as the exclusive zinc sensor, with the transport site's being responsible for zinc sensing. In an effort of seeking sorting signal, a scan of the longest cytosolic loop (L2) leads to identification of a conserved Leu-Gln-Leu motif that is essential for endocytosis. Partial proteolysis of purified hZIP4 demonstrates a structural coupling between the transport site and the L2 upon zinc binding, which supports a working model of how zinc ions at physiological concentration trigger a conformation-dependent endocytosis of the zinc transporter. This work provides a paradigm on post-translational regulation of nutrient transporters.

Morbidity and mortality in response to SARS-CoV-2 infection are significantly elevated in people of advanced age. To understand the underlying biology of this phenotype, we utilize the golden hamster model to compare how the innate and adaptive immune responses to SARS-CoV-2 infection differed between younger and older animals. We find that while both hamster cohorts showed similar virus kinetics in the lungs, the host response in older animals was dampened, with diminished tissue repair in the respiratory tract post-infection. Characterization of the adaptive immune response also revealed age-related differences, including fewer germinal center B cells in older hamsters, resulting in reduced potency of neutralizing antibodies. Moreover, older animals demonstrate elevated suppressor T cells and neutrophils in the respiratory tract, correlating with an increase in TGF-β and IL-17 induction. Together, these data support that diminished immunity is one of the underlying causes of age-related morbidity.

It is urgent to develop disease models to dissect mechanisms regulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we derive airway organoids from human pluripotent stem cells (hPSC-AOs). The hPSC-AOs, particularly ciliated-like cells, are permissive to SARS-CoV-2 infection. Using this platform, we perform a high content screen and identify GW6471, which blocks SARS-CoV-2 infection. GW6471 can also block infection of the B.1.351 SARS-CoV-2 variant. RNA sequencing (RNA-seq) analysis suggests that GW6471 blocks SARS-CoV-2 infection at least in part by inhibiting hypoxia inducible factor 1 subunit alpha (HIF1α), which is further validated by chemical inhibitor and genetic perturbation targeting HIF1α. Metabolic profiling identifies decreased rates of glycolysis upon GW6471 treatment, consistent with transcriptome profiling. Finally, xanthohumol, 5-(tetradecyloxy)-2-furoic acid, and ND-646, three compounds that suppress fatty acid biosynthesis, also block SARS-CoV-2 infection. Together, a high content screen coupled with transcriptome and metabolic profiling reveals a key role of the HIF1α-glycolysis axis in mediating SARS-CoV-2 infection of human airway epithelium.

Elevated circulating activity of adenosine deaminase 2 (ADA2) is associated with liver fibrosis in nonalcoholic fatty liver disease (NAFLD). In the liver of NAFLD patients, ADA2-positive portal macrophages are significantly associated with the degree of liver fibrosis. These liver macrophages are CD14- and CD16-positive and co-express chemokine receptors CCR2, CCR5, and CXCR3, indicating infiltrative monocyte origin. Human circulatory monocytes release ADA2 upon macrophage differentiation in vitro. When stimulated by recombinant human ADA2 (rhADA2), human monocyte-derived macrophages demonstrate upregulation of pro-inflammatory and pro-fibrotic genes, including PDGF-B, a key pro-fibrotic cytokine. This PDGF-B upregulation is reproduced by inosine, the enzymatic product of ADA2, but not adenosine, and is abolished by E359N, a loss-of-function mutation in ADA2. Finally, rhADA2 also stimulates PDGF-B production from Kupffer cells in primary human liver spheroids. Together, these data suggest that infiltrative monocytes promote fibrogenesis in NAFLD via ADA2-mediated autocrine/paracrine signaling culminating in enhanced PDGF-B production.

The phosphoinositide 3-kinase p110α is an essential mediator of insulin signaling and glucose homeostasis. We interrogated the human serine, threonine, and tyrosine kinome to search for novel regulators of p110α and found that the Hippo kinases phosphorylate p110α at T1061, which inhibits its activity. This inhibitory state corresponds to a conformational change of a membrane-binding domain on p110α, which impairs its ability to engage membranes. In human primary hepatocytes, cancer cell lines, and rodent tissues, activation of the Hippo kinases MST1/2 using forskolin or epinephrine is associated with phosphorylation of T1061 and inhibition of p110α, impairment of downstream insulin signaling, and suppression of glycolysis and glycogen synthesis. These changes are abrogated when MST1/2 are genetically deleted or inhibited with small molecules or if the T1061 is mutated to alanine. Our study defines an inhibitory pathway of PI3K signaling and a link between epinephrine and insulin signaling.

Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity phosphatases (DUSPs), the activities of which are tightly regulated during cell differentiation. Using knockdown screening and single-cell transcriptional analysis, we demonstrate that DUSP4 is the phosphatase that specifically inactivates p38 kinase to promote megakaryocyte (Mk) differentiation. Mechanistically, PRMT1-mediated methylation of DUSP4 triggers its ubiquitinylation by an E3 ligase HUWE1. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation in Mk cells. In the context of thrombocytopenia observed in myelodysplastic syndrome (MDS), we demonstrate that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis. These findings provide mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a strategy for treatment of thrombocytopenia associated with MDS.