Estrogen receptor-related receptor (Esrra) induces ribosomal protein Rplp1-mediated adaptive hepatic translation during prolonged starvation.

Protein translation is an energy-intensive ribosome-driven process that is reduced during nutrient scarcity to conserve cellular resources. During prolonged starvation, cells selectively translate specific proteins to enhance their survival (adaptive translation); however, this process is poorly understood. Accordingly, we analyzed protein translation and mRNA transcription by multiple methods in vitro and in vivo to investigate adaptive hepatic translation during starvation. While acute starvation suppressed protein translation in general, proteomic analysis showed that prolonged starvation selectively induced translation of lysosome and autolysosome proteins. Significantly, the expression of the orphan nuclear receptor, estrogen-related receptor alpha (Esrra) increased during prolonged starvation and served as a master regulator of this adaptive translation by transcriptionally stimulating 60S acidic ribosomal protein P1 (Rplp1) gene expression. Overexpression or siRNA knockdown of Esrra expression in vitro or in vivo led to parallel changes in Rplp1 gene expression, lysosome/autophagy protein translation, and autophagy. Remarkably, we have found that Esrra had dual functions by not only regulating transcription but also controling adaptive translation via the Esrra/Rplp1/lysosome/autophagy pathway during prolonged starvation.

Viral proteases activate the CARD8 inflammasome in the human cardiovascular system.

Nucleotide-binding oligomerization domain (NBD), leucine-rich repeat (LRR) containing protein family (NLRs) are intracellular pattern recognition receptors that mediate innate immunity against infections. The endothelium is the first line of defense against blood-borne pathogens, but it is unclear which NLRs control endothelial cell (EC) intrinsic immunity. Here, we demonstrate that human ECs simultaneously activate NLRP1 and CARD8 inflammasomes in response to DPP8/9 inhibitor Val-boro-Pro (VbP). Enterovirus Coxsackie virus B3 (CVB3)-the most common cause of viral myocarditis-predominantly activates CARD8 in ECs in a manner that requires viral 2A and 3C protease cleavage at CARD8 p.G38 and proteasome function. Genetic deletion of CARD8 in ECs and human embryonic stem cell-derived cardiomyocytes (HCMs) attenuates CVB3-induced pyroptosis, inflammation, and viral propagation. Furthermore, using a stratified endothelial-cardiomyocyte co-culture system, we demonstrate that deleting CARD8 in ECs reduces CVB3 infection of the underlying cardiomyocytes. Our study uncovers the unique role of CARD8 inflammasome in endothelium-intrinsic anti-viral immunity.

ZAKα-driven ribotoxic stress response activates the human NLRP1 inflammasome.

Human NLRP1 (NACHT, LRR, and PYD domain-containing protein 1) is an innate immune sensor predominantly expressed in the skin and airway epithelium. Here, we report that human NLRP1 senses the ultraviolet B (UVB)- and toxin-induced ribotoxic stress response (RSR). Biochemically, RSR leads to the direct hyperphosphorylation of a human-specific disordered linker region of NLRP1 (NLRP1DR) by MAP3K20/ZAKα kinase and its downstream effector, p38. Mutating a single ZAKα phosphorylation site in NLRP1DR abrogates UVB- and ribotoxin-driven pyroptosis in human keratinocytes. Moreover, fusing NLRP1DR to CARD8, which is insensitive to RSR by itself, creates a minimal inflammasome sensor for UVB and ribotoxins. These results provide insight into UVB sensing by human skin keratinocytes, identify several ribotoxins as NLRP1 agonists, and establish inflammasome-driven pyroptosis as an integral component of the RSR.

PLK1 inhibition selectively induces apoptosis in ARID1A deficient cells through uncoupling of oxygen consumption from ATP production.

Inhibitors of the mitotic kinase PLK1 yield objective responses in a subset of refractory cancers. However, PLK1 overexpression in cancer does not correlate with drug sensitivity, and the clinical development of PLK1 inhibitors has been hampered by the lack of patient selection marker. Using a high-throughput chemical screen, we discovered that cells deficient for the tumor suppressor ARID1A are highly sensitive to PLK1 inhibition. Interestingly this sensitivity was unrelated to canonical functions of PLK1 in mediating G2/M cell cycle transition. Instead, a whole-genome CRISPR screen revealed PLK1 inhibitor sensitivity in ARID1A deficient cells to be dependent on the mitochondrial translation machinery. We find that ARID1A knock-out (KO) cells have an unusual mitochondrial phenotype with aberrant biogenesis, increased oxygen consumption/expression of oxidative phosphorylation genes, but without increased ATP production. Using expansion microscopy and biochemical fractionation, we see that a subset of PLK1 localizes to the mitochondria in interphase cells. Inhibition of PLK1 in ARID1A KO cells further uncouples oxygen consumption from ATP production, with subsequent membrane depolarization and apoptosis. Knockdown of specific subunits of the mitochondrial ribosome reverses PLK1-inhibitor induced apoptosis in ARID1A deficient cells, confirming specificity of the phenotype. Together, these findings highlight a novel interphase role for PLK1 in maintaining mitochondrial fitness under metabolic stress, and a strategy for therapeutic use of PLK1 inhibitors. To translate these findings, we describe a quantitative microscopy assay for assessment of ARID1A protein loss, which could offer a novel patient selection strategy for the clinical development of PLK1 inhibitors in cancer.

Coding and non-coding roles of MOCCI (C15ORF48) coordinate to regulate host inflammation and immunity.

Mito-SEPs are small open reading frame-encoded peptides that localize to the mitochondria to regulate metabolism. Motivated by an intriguing negative association between mito-SEPs and inflammation, here we screen for mito-SEPs that modify inflammatory outcomes and report a mito-SEP named "Modulator of cytochrome C oxidase during Inflammation" (MOCCI) that is upregulated during inflammation and infection to promote host-protective resolution. MOCCI, a paralog of the NDUFA4 subunit of cytochrome C oxidase (Complex IV), replaces NDUFA4 in Complex IV during inflammation to lower mitochondrial membrane potential and reduce ROS production, leading to cyto-protection and dampened immune response. The MOCCI transcript also generates miR-147b, which targets the NDUFA4 mRNA with similar immune dampening effects as MOCCI, but simultaneously enhances RIG-I/MDA-5-mediated viral immunity. Our work uncovers a dual-component pleiotropic regulation of host inflammation and immunity by MOCCI (C15ORF48) for safeguarding the host during infection and inflammation.

sORF-Encoded MicroPeptides: New players in inflammation, metabolism, and precision medicine.

Significant technological advances have enabled the discovery and identification of a new class of molecules, micropeptides or small ORF encoded peptides (SEPs) within non-coding RNAs (ncRNAs). As ncRNAs are well known to be transcriptionally silent, the discovery of SEPs implies that many ncRNAs are misannotated or play both coding and non-coding functions. SEPs have reportedly diverse regulatory roles in embryogenesis, myogenesis, inflammation, diseases, and cancer. SEPs appearing in different subcellular compartments show distinct functions. In this review, we summarized the functions of SEPs that have been characterized thus far. As SEPs are amenable to therapeutic development as biologics, understanding their underlying functions will provide novel targets for the treatment of inflammatory or metabolic disorders.

Dominant-negative NFKBIA mutation promotes IL-1ß production causing hepatic disease with severe immunodeficiency.

Although IKK-ß has previously been shown as a negative regulator of IL-1ß secretion in mice, this role has not been proven in humans. Genetic studies of NF-κB signaling in humans with inherited diseases of the immune system have not demonstrated the relevance of the NF-κB pathway in suppressing IL-1ß expression. Here, we report an infant with a clinical pathology comprising neutrophil-mediated autoinflammation and recurrent bacterial infections. Whole-exome sequencing revealed a de novo heterozygous missense mutation of NFKBIA, resulting in a L34P IκBα variant that severely repressed NF-κB activation and downstream cytokine production. Paradoxically, IL-1ß secretion was elevated in the patient's stimulated leukocytes, in her induced pluripotent stem cell-derived macrophages, and in murine bone marrow-derived macrophages containing the L34P mutation. The patient's hypersecretion of IL-1ß correlated with activated neutrophilia and liver fibrosis with neutrophil accumulation. Hematopoietic stem cell transplantation reversed neutrophilia, restored a resting state in neutrophils, and normalized IL-1ß release from stimulated leukocytes. Additional therapeutic blockade of IL-1 ameliorated liver damage, while decreasing neutrophil activation and associated IL-1ß secretion. Our studies reveal a previously unrecognized role of human IκBα as an essential regulator of canonical NF-κB signaling in the prevention of neutrophil-dependent autoinflammatory diseases. These findings also highlight the therapeutic potential of IL-1 inhibitors in treating complications arising from systemic NF-κB inhibition.

Mitochondrial peptide BRAWNIN is essential for vertebrate respiratory complex III assembly.

The emergence of small open reading frame (sORF)-encoded peptides (SEPs) is rapidly expanding the known proteome at the lower end of the size distribution. Here, we show that the mitochondrial proteome, particularly the respiratory chain, is enriched for small proteins. Using a prediction and validation pipeline for SEPs, we report the discovery of 16 endogenous nuclear encoded, mitochondrial-localized SEPs (mito-SEPs). Through functional prediction, proteomics, metabolomics and metabolic flux modeling, we demonstrate that BRAWNIN, a 71 a.a. peptide encoded by C12orf73, is essential for respiratory chain complex III (CIII) assembly. In human cells, BRAWNIN is induced by the energy-sensing AMPK pathway, and its depletion impairs mitochondrial ATP production. In zebrafish, Brawnin deletion causes complete CIII loss, resulting in severe growth retardation, lactic acidosis and early death. Our findings demonstrate that BRAWNIN is essential for vertebrate oxidative phosphorylation. We propose that mito-SEPs are an untapped resource for essential regulators of oxidative metabolism.

Alternative Progenitor Cells Compensate to Rebuild the Coronary Vasculature in Elabela- and Apj-Deficient Hearts.

Organogenesis during embryonic development occurs through the differentiation of progenitor cells. This process is extraordinarily accurate, but the mechanisms ensuring high fidelity are poorly understood. Coronary vessels of the mouse heart derive from at least two progenitor pools, the sinus venosus and endocardium. We find that the ELABELA (ELA)-APJ signaling axis is only required for sinus venosus-derived progenitors. Because they do not depend on ELA-APJ, endocardial progenitors are able to expand and compensate for faulty sinus venosus development in Apj mutants, leading to normal adult heart function. An upregulation of endocardial SOX17 accompanied compensation in Apj mutants, which was also seen in Ccbe1 knockouts, indicating that the endocardium is activated in multiple cases where sinus venosus angiogenesis is stunted. Our data demonstrate that by diversifying their responsivity to growth cues, distinct coronary progenitor pools are able to compensate for each other during coronary development, thereby providing robustness to organ development.

ELABELA deficiency promotes preeclampsia and cardiovascular malformations in mice.

Preeclampsia (PE) is a gestational hypertensive syndrome affecting between 5 and 8% of all pregnancies. Although PE is the leading cause of fetal and maternal morbidity and mortality, its molecular etiology is still unclear. Here, we show that ELABELA (ELA), an endogenous ligand of the apelin receptor (APLNR, or APJ), is a circulating hormone secreted by the placenta. Elabela but not Apelin knockout pregnant mice exhibit PE-like symptoms, including proteinuria and elevated blood pressure due to defective placental angiogenesis. In mice, infusion of exogenous ELA normalizes hypertension, proteinuria, and birth weight. ELA, which is abundant in human placentas, increases the invasiveness of trophoblast-like cells, suggesting that it enhances placental development to prevent PE. The ELA-APLNR signaling axis may offer a new paradigm for the treatment of common pregnancy-related complications, including PE.

ELABELA-APJ axis protects from pressure overload heart failure and angiotensin II-induced cardiac damage.

AIMS: Elabela/Toddler/Apela (ELA) has been identified as a novel endogenous peptide ligand for APJ/Apelin receptor/Aplnr. ELA plays a crucial role in early cardiac development of zebrafish as well as in maintenance of self-renewal of human embryonic stem cells. Apelin was the first identified APJ ligand, and exerts positive inotropic heart effects and regulates the renin-angiotensin system. The aim of this study was to investigate the biological effects of ELA in the cardiovascular system. METHODS AND RESULTS: Continuous infusion of ELA peptide significantly suppressed pressure overload-induced cardiac hypertrophy, fibrosis and impaired contractility in mice. ELA treatment reduced mRNA expression levels of genes associated with heart failure and fibrosis. The cardioprotective effects of ELA were diminished in APJ knockout mice, indicating that APJ is the key receptor for ELA in the adult heart. Mechanistically, ELA downregulated angiotensin-converting enzyme (ACE) expression in the stressed hearts, whereas it showed little effects on angiotensin-converting enzyme 2 (ACE2) expression, which are distinct from the effects of Apelin. FoxM1 transcription factor, which induces ACE expression in the stressed hearts, was downregulated by ELA but not by Apelin. ELA antagonized angiotensin II-induced hypertension, cardiac hypertrophy, and fibrosis in mice. CONCLUSION: The ELA-APJ axis protects from pressure overload-induced heart failure possibly via suppression of ACE expression and pathogenic angiotensin II signalling. The different effects of ELA and Apelin on the expression of ACE and ACE2 implicate fine-tuned mechanisms for a ligand-induced APJ activation and downstream signalling.

ELABELA Is an Endogenous Growth Factor that Sustains hESC Self-Renewal via the PI3K/AKT Pathway.

ELABELA (ELA) is a peptide hormone required for heart development that signals via the Apelin Receptor (APLNR, APJ). ELA is also abundantly secreted by human embryonic stem cells (hESCs), which do not express APLNR. Here we show that ELA signals in a paracrine fashion in hESCs to maintain self-renewal. ELA inhibition by CRISPR/Cas9-mediated deletion, shRNA, or neutralizing antibodies causes reduced hESC growth, cell death, and loss of pluripotency. Global phosphoproteomic and transcriptomic analyses of ELA-pulsed hESCs show that it activates PI3K/AKT/mTORC1 signaling required for cell survival. ELA promotes hESC cell-cycle progression and protein translation and blocks stress-induced apoptosis. INSULIN and ELA have partially overlapping functions in hESC medium, but only ELA can potentiate the TGFß pathway to prime hESCs toward the endoderm lineage. We propose that ELA, acting through an alternate cell-surface receptor, is an endogenous secreted growth factor in human embryos and hESCs that promotes growth and pluripotency.

Essential role of BRG, the ATPase subunit of BAF chromatin remodeling complexes, in leukemia maintenance.

In mammals, combinatorial assembly of alternative families of subunits confers functional specificity to adenosine triphosphate (ATP)-dependent SWI/SNF-like Brg/Brm-associated factor (BAF) chromatin remodeling complexes by creating distinct polymorphic surfaces for interaction with regulatory elements and DNA-binding factors. Although redundant in terms of biochemical activity, the core ATPase subunits, BRG/SMARCA4 and BRM/SMARCA2, are functionally distinct and may contribute to complex specificity. Here we show using quantitative proteomics that BAF complexes expressed in leukemia are specifically assembled around the BRG ATPase. Moreover, using a mouse model of acute myeloid leukemia, we demonstrate that BRG is essential for leukemia maintenance, as leukemic cells lacking BRG rapidly undergo cell-cycle arrest and apoptosis. Most importantly, we show that BRG is dispensable for the maintenance of immunophenotypic long-term repopulating hematopoietic stem cells, suggesting that adroit targeting of BRG in leukemia may have potent and specific therapeutic effects.

C5orf42 is the major gene responsible for OFD syndrome type VI.

Oral-facial-digital syndrome type VI (OFD VI) is a recessive ciliopathy defined by two diagnostic criteria: molar tooth sign (MTS) and one or more of the following: (1) tongue hamartoma (s) and/or additional frenula and/or upper lip notch; (2) mesoaxial polydactyly of one or more hands or feet; (3) hypothalamic hamartoma. Because of the MTS, OFD VI belongs to the "Joubert syndrome related disorders". Its genetic aetiology remains largely unknown although mutations in the TMEM216 gene, responsible for Joubert (JBS2) and Meckel-Gruber (MKS2) syndromes, have been reported in two OFD VI patients. To explore the molecular cause(s) of OFD VI syndrome, we used an exome sequencing strategy in six unrelated families followed by Sanger sequencing. We identified a total of 14 novel mutations in the C5orf42 gene in 9/11 families with positive OFD VI diagnostic criteria including a severe fetal case with microphthalmia, cerebellar hypoplasia, corpus callosum agenesis, polydactyly and skeletal dysplasia. C5orf42 mutations have already been reported in Joubert syndrome confirming that OFD VI and JBS are allelic disorders, thus enhancing our knowledge of the complex, highly heterogeneous nature of ciliopathies.

ELABELA: a hormone essential for heart development signals via the apelin receptor.

We report here the discovery and characterization of a gene, ELABELA (ELA), encoding a conserved hormone of 32 amino acids. Present in human embryonic stem cells, ELA is expressed at the onset of zebrafish zygotic transcription and is ubiquitous in the naive ectodermal cells of the embryo. Using zinc-finger-nuclease-mediated gene inactivation in zebrafish, we created an allelic series of ela mutants. ela null embryos have impaired endoderm differentiation potential marked by reduced gata5 and sox17 expression. Loss of Ela causes embryos to develop with a rudimentary heart or no heart at all, surprisingly phenocopying the loss of the apelin receptor (aplnr), which we show serves as Ela's cognate G protein-coupled receptor. Our results reveal the existence of a peptide hormone, ELA, which, together with APLNR, forms an essential signaling axis for early cardiovascular development.

Proteomic and bioinformatic analysis of mammalian SWI/SNF complexes identifies extensive roles in human malignancy.

Subunits of mammalian SWI/SNF (mSWI/SNF or BAF) complexes have recently been implicated as tumor suppressors in human malignancies. To understand the full extent of their involvement, we conducted a proteomic analysis of endogenous mSWI/SNF complexes, which identified several new dedicated, stable subunits not found in yeast SWI/SNF complexes, including BCL7A, BCL7B and BCL7C, BCL11A and BCL11B, BRD9 and SS18. Incorporating these new members, we determined mSWI/SNF subunit mutation frequency in exome and whole-genome sequencing studies of primary human tumors. Notably, mSWI/SNF subunits are mutated in 19.6% of all human tumors reported in 44 studies. Our analysis suggests that specific subunits protect against cancer in specific tissues. In addition, mutations affecting more than one subunit, defined here as compound heterozygosity, are prevalent in certain cancers. Our studies demonstrate that mSWI/SNF is the most frequently mutated chromatin-regulatory complex (CRC) in human cancer, exhibiting a broad mutation pattern, similar to that of TP53. Thus, proper functioning of polymorphic BAF complexes may constitute a major mechanism of tumor suppression.

esBAF facilitates pluripotency by conditioning the genome for LIF/STAT3 signalling and by regulating polycomb function.

Signalling by the cytokine LIF and its downstream transcription factor, STAT3, prevents differentiation of pluripotent embryonic stem cells (ESCs). This contrasts with most cell types where STAT3 signalling induces differentiation. We find that STAT3 binding across the pluripotent genome is dependent on Brg1, the ATPase subunit of a specialized chromatin remodelling complex (esBAF) found in ESCs. Brg1 is required to establish chromatin accessibility at STAT3 binding targets, preparing these sites to respond to LIF signalling. Brg1 deletion leads to rapid polycomb (PcG) binding and H3K27me3-mediated silencing of many Brg1-activated targets genome wide, including the target genes of the LIF signalling pathway. Hence, one crucial role of Brg1 in ESCs involves its ability to potentiate LIF signalling by opposing PcG. Contrary to expectations, Brg1 also facilitates PcG function at classical PcG targets, including all four Hox loci, reinforcing their repression in ESCs. Therefore, esBAF does not simply antagonize PcG. Rather, the two chromatin regulators act both antagonistically and synergistically with the common goal of supporting pluripotency.

Comprehensive methylome map of lineage commitment from haematopoietic progenitors.

Epigenetic modifications must underlie lineage-specific differentiation as terminally differentiated cells express tissue-specific genes, but their DNA sequence is unchanged. Haematopoiesis provides a well-defined model to study epigenetic modifications during cell-fate decisions, as multipotent progenitors (MPPs) differentiate into progressively restricted myeloid or lymphoid progenitors. Although DNA methylation is critical for myeloid versus lymphoid differentiation, as demonstrated by the myeloerythroid bias in Dnmt1 hypomorphs, a comprehensive DNA methylation map of haematopoietic progenitors, or of any multipotent/oligopotent lineage, does not exist. Here we examined 4.6 million CpG sites throughout the genome for MPPs, common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and thymocyte progenitors (DN1, DN2, DN3). Marked epigenetic plasticity accompanied both lymphoid and myeloid restriction. Myeloid commitment involved less global DNA methylation than lymphoid commitment, supported functionally by myeloid skewing of progenitors following treatment with a DNA methyltransferase inhibitor. Differential DNA methylation correlated with gene expression more strongly at CpG island shores than CpG islands. Many examples of genes and pathways not previously known to be involved in choice between lymphoid/myeloid differentiation have been identified, such as Arl4c and Jdp2. Several transcription factors, including Meis1, were methylated and silenced during differentiation, indicating a role in maintaining an undifferentiated state. Additionally, epigenetic modification of modifiers of the epigenome seems to be important in haematopoietic differentiation. Our results directly demonstrate that modulation of DNA methylation occurs during lineage-specific differentiation and defines a comprehensive map of the methylation and transcriptional changes that accompany myeloid versus lymphoid fate decisions.

An embryonic stem cell chromatin remodeling complex, esBAF, is an essential component of the core pluripotency transcriptional network.

Distinctive SWI/SNF-like ATP-dependent chromatin remodeling esBAF complexes are indispensable for the maintenance and pluripotency of mouse embryonic stem (ES) cells [Ho L, et al. (2009) Proc Natl Acad Sci USA 10.1073/pnas.0812889106]. To understand the mechanism underlying the roles of these complexes in ES cells, we performed high-resolution genome-wide mapping of the core ATPase subunit, Brg, using ChIP-Seq technology. We find that esBAF, as represented by Brg, binds to genes encoding components of the core ES transcriptional circuitry, including Polycomb group proteins. esBAF colocalizes extensively with transcription factors Oct4, Sox2 and Nanog genome-wide, and shows distinct functional interactions with Oct4 and Sox2 at its target genes. Surprisingly, no significant colocalization of esBAF with PRC2 complexes, represented by Suz12, is observed. Lastly, esBAF colocalizes with Stat3 and Smad1 genome-wide, consistent with a direct and critical role in LIF and BMP signaling for maintaining self-renewal. Taken together, our studies indicate that esBAF is an essential component of the core pluripotency transcriptional network, and might also be a critical component of the LIF and BMP signaling pathways essential for maintenance of self-renewal and pluripotency.