A parathyroid hormone/salt-inducible kinase signaling axis controls renal vitamin D activation and organismal calcium homeostasis.

The renal actions of parathyroid hormone (PTH) promote 1,25-vitamin D generation; however, the signaling mechanisms that control PTH-dependent vitamin D activation remain unknown. Here, we demonstrated that salt-inducible kinases (SIKs) orchestrated renal 1,25-vitamin D production downstream of PTH signaling. PTH inhibited SIK cellular activity by cAMP-dependent PKA phosphorylation. Whole-tissue and single-cell transcriptomics demonstrated that both PTH and pharmacologic SIK inhibitors regulated a vitamin D gene module in the proximal tubule. SIK inhibitors increased 1,25-vitamin D production and renal Cyp27b1 mRNA expression in mice and in human embryonic stem cell-derived kidney organoids. Global- and kidney-specific Sik2/Sik3 mutant mice showed Cyp27b1 upregulation, elevated serum 1,25-vitamin D, and PTH-independent hypercalcemia. The SIK substrate CRTC2 showed PTH and SIK inhibitor-inducible binding to key Cyp27b1 regulatory enhancers in the kidney, which were also required for SIK inhibitors to increase Cyp27b1 in vivo. Finally, in a podocyte injury model of chronic kidney disease-mineral bone disorder (CKD-MBD), SIK inhibitor treatment stimulated renal Cyp27b1 expression and 1,25-vitamin D production. Together, these results demonstrated a PTH/SIK/CRTC signaling axis in the kidney that controls Cyp27b1 expression and 1,25-vitamin D synthesis. These findings indicate that SIK inhibitors might be helpful for stimulation of 1,25-vitamin D production in CKD-MBD.

tiRNA signaling via stress-regulated vesicle transfer in the hematopoietic niche.

Extracellular vesicles (EVs) transfer complex biologic material between cells. However, the role of this process in vivo is poorly defined. Here, we demonstrate that osteoblastic cells in the bone marrow (BM) niche elaborate extracellular vesicles that are taken up by hematopoietic progenitor cells in vivo. Genotoxic or infectious stress rapidly increased stromal-derived extracellular vesicle transfer to granulocyte-monocyte progenitors. The extracellular vesicles contained processed tRNAs (tiRNAs) known to modulate protein translation. 5'-ti-Pro-CGG-1 was preferentially abundant in osteoblast-derived extracellular vesicles and, when transferred to granulocyte-monocyte progenitors, increased protein translation, cell proliferation, and myeloid differentiation. Upregulating EV transfer improved hematopoietic recovery from genotoxic injury and survival from fungal sepsis. Therefore, EV-mediated tiRNA transfer provides a stress-modulated signaling axis in the BM niche distinct from conventional cytokine-driven stress responses.

Inducible histone K-to-M mutations are dynamic tools to probe the physiological role of site-specific histone methylation in vitro and in vivo.

Development and differentiation are associated with profound changes to histone modifications, yet their in vivo function remains incompletely understood. Here, we generated mouse models expressing inducible histone H3 lysine-to-methionine (K-to-M) mutants, which globally inhibit methylation at specific sites. Mice expressing H3K36M developed severe anaemia with arrested erythropoiesis, a marked haematopoietic stem cell defect, and rapid lethality. By contrast, mice expressing H3K9M survived up to a year and showed expansion of multipotent progenitors, aberrant lymphopoiesis and thrombocytosis. Additionally, some H3K9M mice succumbed to aggressive T cell leukaemia/lymphoma, while H3K36M mice exhibited differentiation defects in testis and intestine. Mechanistically, induction of either mutant reduced corresponding histone trimethylation patterns genome-wide and altered chromatin accessibility as well as gene expression landscapes. Strikingly, discontinuation of transgene expression largely restored differentiation programmes. Our work shows that individual chromatin modifications are required at several specific stages of differentiation and introduces powerful tools to interrogate their roles in vivo.

The RNA Helicase DDX6 Controls Cellular Plasticity by Modulating P-Body Homeostasis.

Post-transcriptional mechanisms have the potential to influence complex changes in gene expression, yet their role in cell fate transitions remains largely unexplored. Here, we show that suppression of the RNA helicase DDX6 endows human and mouse primed embryonic stem cells (ESCs) with a differentiation-resistant, "hyper-pluripotent" state, which readily reprograms to a naive state resembling the preimplantation embryo. We further demonstrate that DDX6 plays a key role in adult progenitors where it controls the balance between self-renewal and differentiation in a context-dependent manner. Mechanistically, DDX6 mediates the translational suppression of target mRNAs in P-bodies. Upon loss of DDX6 activity, P-bodies dissolve and release mRNAs encoding fate-instructive transcription and chromatin factors that re-enter the ribosome pool. Increased translation of these targets impacts cell fate by rewiring the enhancer, heterochromatin, and DNA methylation landscapes of undifferentiated cell types. Collectively, our data establish a link between P-body homeostasis, chromatin organization, and stem cell potency.

TREM2 Acts Downstream of CD33 in Modulating Microglial Pathology in Alzheimer's Disease.

The microglial receptors CD33 and TREM2 have been associated with risk for Alzheimer's disease (AD). Here, we investigated crosstalk between CD33 and TREM2. We showed that knockout of CD33 attenuated amyloid beta (Aß) pathology and improved cognition in 5xFAD mice, both of which were abrogated by additional TREM2 knockout. Knocking out TREM2 in 5xFAD mice exacerbated Aß pathology and neurodegeneration but reduced Iba1+ cell numbers, all of which could not be rescued by additional CD33 knockout. RNA-seq profiling of microglia revealed that genes related to phagocytosis and signaling (IL-6, IL-8, acute phase response) are upregulated in 5xFAD;CD33-/- and downregulated in 5xFAD;TREM2-/- mice. Differential gene expression in 5xFAD;CD33-/- microglia depended on the presence of TREM2, suggesting TREM2 acts downstream of CD33. Crosstalk between CD33 and TREM2 includes regulation of the IL-1ß/IL-1RN axis and a gene set in the "receptor activity chemokine" cluster. Our results should facilitate AD therapeutics targeting these receptors.

Human gut derived-organoids provide model to study gluten response and effects of microbiota-derived molecules in celiac disease.

Celiac disease (CD) is an immune-mediated disorder triggered by gluten exposure. The contribution of the adaptive immune response to CD pathogenesis has been extensively studied, but the absence of valid experimental models has hampered our understanding of the early steps leading to loss of gluten tolerance. Using intestinal organoids developed from duodenal biopsies from both non-celiac (NC) and celiac (CD) patients, we explored the contribution of gut epithelium to CD pathogenesis and the role of microbiota-derived molecules in modulating the epithelium's response to gluten. When compared to NC, RNA sequencing of CD organoids revealed significantly altered expression of genes associated with gut barrier, innate immune response, and stem cell functions. Monolayers derived from CD organoids exposed to gliadin showed increased intestinal permeability and enhanced secretion of pro-inflammatory cytokines compared to NC controls. Microbiota-derived bioproducts butyrate, lactate, and polysaccharide A improved barrier function and reduced gliadin-induced cytokine secretion. We concluded that: (1) patient-derived organoids faithfully express established and newly identified molecular signatures characteristic of CD. (2) microbiota-derived bioproducts can be used to modulate the epithelial response to gluten. Finally, we validated the use of patient-derived organoids monolayers as a novel tool for the study of CD.

Exploration of CTCF post-translation modifications uncovers Serine-224 phosphorylation by PLK1 at pericentric regions during the G2/M transition.

The zinc finger CCCTC-binding protein (CTCF) carries out many functions in the cell. Although previous studies sought to explain CTCF multivalency based on sequence composition of binding sites, few examined how CTCF post-translational modification (PTM) could contribute to function. Here, we performed CTCF mass spectrometry, identified a novel phosphorylation site at Serine 224 (Ser224-P), and demonstrate that phosphorylation is carried out by Polo-like kinase 1 (PLK1). CTCF Ser224-P is chromatin-associated, mapping to at least a subset of known CTCF sites. CTCF Ser224-P accumulates during the G2/M transition of the cell cycle and is enriched at pericentric regions. The phospho-obviation mutant, S224A, appeared normal. However, the phospho-mimic mutant, S224E, is detrimental to mouse embryonic stem cell colonies. While ploidy and chromatin architecture appear unaffected, S224E mutants differentially express hundreds of genes, including p53 and p21. We have thus identified a new CTCF PTM and provided evidence of biological function.

The surveillance of pre-mRNA splicing is an early step in C. elegans RNAi of endogenous genes.

RNAi pathways detect and silence foreign nucleic acids such as viruses as well as endogenous genes in many species. The phylogenetic profile across eukaryotes of proteins that mediate key steps in RNAi is correlated with the profiles of multiple mRNA splicing proteins and with intron number, suggesting that RNAi may surveil mRNA splicing to detect the divergent or absent introns of viruses. Here we examine the role of mRNA splicing in Caenorhabditis elegans RNAi. We found that viable null mutations in U1 and U2 small nuclear ribonucleic protein (snRNP)-specific splicing factor genes cause defects in RNAi. The U1A ortholog rnp-2 is required for normal ERGO-1 Argonaute class 26G siRNA biogenesis, trans-splicing of the eri-6/7 transcript, and targeting of poorly conserved gene transcripts by WAGO Argonaute class 22G siRNAs. We found that gene transcripts engaged by the siRNA-generating machinery are poorly conserved, possess few introns, and often have introns that are divergent from introns with strong consensus splicing sites found in highly conserved genes. We present biochemical evidence that RNAi targeted transcripts are tightly bound to spliceosomes. These findings suggest multiple layers of regulation by the spliceosome at early steps of small RNA-mediated gene silencing.

Direct Reprogramming of Mouse Fibroblasts into Functional Skeletal Muscle Progenitors.

Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell markers including Pax7 and Myf5 and give rise to dystrophin-expressing myofibers upon transplantation in vivo. Notably, a subset of transplanted iMPCs maintain Pax7 expression and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from Pax7+ cells and require Pax7 itself for maintenance. Finally, we show that myogenic progenitor cell lines can be established from muscle tissue following small-molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/progenitor-like cells from multiple cell types.

Genetic Intersection of Tsix and Hedgehog Signaling during the Initiation of X-Chromosome Inactivation.

X-chromosome inactivation (XCI) silences one X chromosome in the female mammal and is essential to peri-implantation development. XCI is thought to be cell autonomous, with all factors required being produced within each cell. Nevertheless, external cues may exist. Here, we search for such developmental signals by combining bioinformatic, biochemical, and genetic approaches. Using ex vivo and in vivo models, we identify the Hedgehog (HH) paracrine system as a candidate signaling cascade. HH signaling keeps XCI in check in pluripotent cells and is transduced by GLI transcription factors to binding sites in Tsix, the antisense repressor of XCI. GLI potentiates Tsix expression and impedes XCI. In vivo, mutating Indian Hedgehog results in a sex ratio bias against females, and the female lethality is rescued by a second-site mutation in Tsix. These data demonstrate a genetic and functional intersection between HH and XCI and support a role for intercellular signaling during XCI.

Polycomb Repressive Complex 2 Methylates Elongin A to Regulate Transcription.

Polycomb repressive complex 2 (PRC2-EZH2) methylates histone H3 at lysine 27 (H3K27) and is required to maintain gene repression during development. Misregulation of PRC2 is linked to a range of neoplastic malignancies, which is believed to involve methylation of H3K27. However, the full spectrum of non-histone substrates of PRC2 that might also contribute to PRC2 function is not known. We characterized the target recognition specificity of the PRC2 active site and used the resultant data to screen for uncharacterized potential targets. The RNA polymerase II (Pol II) transcription elongation factor, Elongin A (EloA), is methylated by PRC2 in vivo. Mutation of the methylated EloA residue decreased repression of a subset of PRC2 target genes as measured by both steady-state and nascent RNA levels and perturbed embryonic stem cell differentiation. We propose that PRC2 modulates transcription of a subset of low expression target genes in part via methylation of EloA.

Phf8 loss confers resistance to depression-like and anxiety-like behaviors in mice.

PHF8 is a histone demethylase with specificity for repressive modifications. While mutations of PHF8 have been associated with cognitive defects and cleft lip/palate, its role in mammalian development and physiology remains unexplored. Here, we have generated a Phf8 knockout allele in mice to examine the consequences of Phf8 loss for development and behaviour. Phf8 deficient mice neither display obvious developmental defects nor signs of cognitive impairment. However, we report a striking resiliency to stress-induced anxiety- and depression-like behaviour on loss of Phf8. We further observe misregulation of serotonin signalling within the prefrontal cortex of Phf8 deficient mice and identify the serotonin receptors Htr1a and Htr2a as direct targets of PHF8. Our results clarify the functional role of Phf8 in mammalian development and behaviour and establish a direct link between Phf8 expression and serotonin signalling, identifying this histone demethylase as a potential target for the treatment of anxiety and depression.

Shank is a dose-dependent regulator of Cav1 calcium current and CREB target expression.

Shank is a post-synaptic scaffolding protein that has many binding partners. Shank mutations and copy number variations (CNVs) are linked to several psychiatric disorders, and to synaptic and behavioral defects in mice. It is not known which Shank binding partners are responsible for these defects. Here we show that the C. elegans SHN-1/Shank binds L-type calcium channels and that increased and decreased shn-1 gene dosage alter L-channel current and activity-induced expression of a CRH-1/CREB transcriptional target (gem-4 Copine), which parallels the effects of human Shank copy number variations (CNVs) on Autism spectrum disorders and schizophrenia. These results suggest that an important function of Shank proteins is to regulate L-channel current and activity induced gene expression.

HELZ2 Is an IFN Effector Mediating Suppression of Dengue Virus.

Flaviviral infections including dengue virus are an increasing clinical problem worldwide. Dengue infection triggers host production of the type 1 IFN, IFN alpha, one of the strongest and broadest acting antivirals known. However, dengue virus subverts host IFN signaling at early steps of IFN signal transduction. This subversion allows unbridled viral replication which subsequently triggers ongoing production of IFN which, again, is subverted. Identification of downstream IFN antiviral effectors will provide targets which could be activated to restore broad acting antiviral activity, stopping the signal to produce endogenous IFN at toxic levels. To this end, we performed a targeted functional genomic screen for IFN antiviral effector genes (IEGs), identifying 56 IEGs required for antiviral effects of IFN against fully infectious dengue virus. Dengue IEGs were enriched for genes encoding nuclear receptor interacting proteins, including HELZ2, MAP2K4, SLC27A2, HSP90AA1, and HSP90AB1. We focused on HELZ2 (Helicase With Zinc Finger 2), an IFN stimulated gene and IEG which encodes a promiscuous nuclear factor coactivator that exists in two isoforms. The two unique HELZ2 isoforms are both IFN responsive, contain ISRE elements, and gene products increase in the nucleus upon IFN stimulation. Chromatin immunoprecipitation-sequencing revealed that the HELZ2 complex interacts with triglyceride-regulator LMF1. Mass spectrometry revealed that HELZ2 knockdown cells are depleted of triglyceride subsets. We thus sought to determine whether HELZ2 interacts with a nuclear receptor known to regulate immune response and lipid metabolism, AHR, and identified HELZ2:AHR interactions via co-immunoprecipitation, found that AHR is a dengue IEG, and that an AHR ligand, FICZ, exhibits anti-dengue activity. Primary bone marrow derived macrophages from HELZ2 knockout mice, compared to wild type controls, exhibit enhanced dengue infectivity. Overall, these findings reveal that IFN antiviral response is mediated by HELZ2 transcriptional upregulation, enrichment of HELZ2 protein levels in the nucleus, and activation of a transcriptional program that appears to modulate intracellular lipid state. IEGs identified in this study may serve as both (1) potential targets for host directed antiviral design, downstream of the common flaviviral subversion point, as well as (2) possible biomarkers, whose variation, natural, or iatrogenic, could affect host response to viral infections.

T-Helper 17 Cell Cytokine Responses in Lyme Disease Correlate With Borrelia burgdorferi Antibodies During Early Infection and With Autoantibodies Late in the Illness in Patients With Antibiotic-Refractory Lyme Arthritis.

BACKGROUND: Control of Lyme disease is attributed predominantly to innate and adaptive T-helper 1 cell (TH1) immune responses, whereas the role of T-helper 17 cell (TH17) responses is less clear. Here we characterized these inflammatory responses in patients with erythema migrans (EM) or Lyme arthritis (LA) to elucidate their role early and late in the infection. METHODS: Levels of 21 cytokines and chemokines, representative of innate, TH1, and TH17 immune responses, were assessed by Luminex in acute and convalescent sera from 91 EM patients, in serum and synovial fluid from 141 LA patients, and in serum from 57 healthy subjects. Antibodies to Borrelia burgdorferi or autoantigens were measured by enzyme-linked immunosorbent assay. RESULTS: Compared with healthy subjects, EM patients had significantly higher levels of innate, TH1, and TH17-associated mediators (P ≤ .05) in serum. In these patients, the levels of inflammatory mediators, particularly TH17-associated cytokines, correlated directly with B. burgdorferi immunoglobulin G antibodies (P ≤ .02), suggesting a beneficial role for these responses in control of early infection. Late in the disease, in patients with LA, innate and TH1-associated mediators were often >10-fold higher in synovial fluid than serum. In contrast, the levels of TH17-associated mediators were more variable, but correlated strongly with autoantibodies to endothelial cell growth factor, matrix metalloproteinase 10, and apolipoprotein B-100 in joints of patients with antibiotic-refractory LA, implying a shift in TH17 responses toward an autoimmune phenotype. CONCLUSIONS: Patients with Lyme disease often develop pronounced TH17 immune responses that may help control early infection. However, late in the disease, excessive TH17 responses may be disadvantageous by contributing to autoimmune responses associated with antibiotic-refractory LA.

Tribbles ortholog NIPI-3 and bZIP transcription factor CEBP-1 regulate a Caenorhabditis elegans intestinal immune surveillance pathway.

BACKGROUND: Many pathogens secrete toxins that target key host processes resulting in the activation of immune pathways. The secreted Pseudomonas aeruginosa toxin Exotoxin A (ToxA) disrupts intestinal protein synthesis, which triggers the induction of a subset of P. aeruginosa-response genes in the nematode Caenorhabditis elegans. RESULTS: We show here that one ToxA-induced C. elegans gene, the Tribbles pseudokinase ortholog nipi-3, is essential for host survival following exposure to P. aeruginosa or ToxA. We find that NIPI-3 mediates the post-developmental expression of intestinal immune genes and proteins and primarily functions in parallel to known immune pathways, including p38 MAPK signaling. Through mutagenesis screening, we identify mutants of the bZIP C/EBP transcription factor cebp-1 that suppress the hypersusceptibility defects of nipi-3 mutants. CONCLUSIONS: NIPI-3 is a negative regulator of CEBP-1, which in turn negatively regulates protective immune mechanisms. This pathway represents a previously unknown innate immune signaling pathway in intestinal epithelial cells that is involved in the surveillance of cellular homeostasis. Because NIPI-3 and CEBP-1 are also essential for C. elegans development, NIPI-3 is analogous to other key innate immune signaling molecules such as the Toll receptors in Drosophila that have an independent role during development.

Single-cell imaging of normal and malignant cell engraftment into optically clear prkdc-null SCID zebrafish.

Cell transplantation into immunodeficient mice has revolutionized our understanding of regeneration, stem cell self-renewal, and cancer; yet models for direct imaging of engrafted cells has been limited. Here, we characterize zebrafish with mutations in recombination activating gene 2 (rag2), DNA-dependent protein kinase (prkdc), and janus kinase 3 (jak3). Histology, RNA sequencing, and single-cell transcriptional profiling of blood showed that rag2 hypomorphic mutant zebrafish lack T cells, whereas prkdc deficiency results in loss of mature T and B cells and jak3 in T and putative Natural Killer cells. Although all mutant lines engraft fluorescently labeled normal and malignant cells, only the prkdc mutant fish reproduced as homozygotes and also survived injury after cell transplantation. Engraftment into optically clear casper, prkdc-mutant zebrafish facilitated dynamic live cell imaging of muscle regeneration, repopulation of muscle stem cells within their endogenous niche, and muscle fiber fusion at single-cell resolution. Serial imaging approaches also uncovered stochasticity in fluorescently labeled leukemia regrowth after competitive cell transplantation into prkdc mutant fish, providing refined models to assess clonal dominance and progression in the zebrafish. Our experiments provide an optimized and facile transplantation model, the casper, prkdc mutant zebrafish, for efficient engraftment and direct visualization of fluorescently labeled normal and malignant cells at single-cell resolution.

SIKs control osteocyte responses to parathyroid hormone.

Parathyroid hormone (PTH) activates receptors on osteocytes to orchestrate bone formation and resorption. Here we show that PTH inhibition of SOST (sclerostin), a WNT antagonist, requires HDAC4 and HDAC5, whereas PTH stimulation of RANKL, a stimulator of bone resorption, requires CRTC2. Salt inducible kinases (SIKs) control subcellular localization of HDAC4/5 and CRTC2. PTH regulates both HDAC4/5 and CRTC2 localization via phosphorylation and inhibition of SIK2. Like PTH, new small molecule SIK inhibitors cause decreased phosphorylation and increased nuclear translocation of HDAC4/5 and CRTC2. SIK inhibition mimics many of the effects of PTH in osteocytes as assessed by RNA-seq in cultured osteocytes and following in vivo administration. Once daily treatment with the small molecule SIK inhibitor YKL-05-099 increases bone formation and bone mass. Therefore, a major arm of PTH signalling in osteocytes involves SIK inhibition, and small molecule SIK inhibitors may be applied therapeutically to mimic skeletal effects of PTH.

T Cell Immune Deficiency in zap70 Mutant Zebrafish.

ZAP70 [zeta-chain (TCR)-associated protein kinase, 70-kDa], is required for T cell activation. ZAP70 deficiencies in humans and null mutations in mice lead to severe combined immune deficiency. Here, we describe a zap70 loss-of-function mutation in zebrafish (zap70 y442 ) that was created using transcription activator-like effector nucleases (TALENs). In contrast to what has been reported for morphant zebrafish, zap70 y442 homozygous mutant zebrafish displayed normal development of blood and lymphatic vasculature. Hematopoietic cell development was also largely unaffected in mutant larvae. However, mutant fish had reduced lck:GFP + thymic T cells by 5 days postfertilization that persisted into adult stages. Morphological analysis, RNA sequencing, and single-cell gene expression profiling of whole kidney marrow cells of adult fish revealed complete loss of mature T cells in zap70 y442 mutant animals. T cell immune deficiency was confirmed through transplantation of unmatched normal and malignant donor cells into zap70 y442 mutant zebrafish, with T cell loss being sufficient for robust allogeneic cell engraftment. zap70 mutant zebrafish show remarkable conservation of immune cell dysfunction as found in mice and humans and will serve as a valuable model to study zap70 immune deficiency.