Oct4 redox sensitivity potentiates reprogramming and differentiation.

The transcription factor Oct4/Pou5f1 is a component of the regulatory circuitry governing pluripotency and is widely used to induce pluripotency from somatic cells. Here we used domain swapping and mutagenesis to study Oct4's reprogramming ability, identifying a redox-sensitive DNA binding domain, cysteine residue (Cys48), as a key determinant of reprogramming and differentiation. Oct4 Cys48 sensitizes the protein to oxidative inhibition of DNA binding activity and promotes oxidation-mediated protein ubiquitylation. Pou5f1 C48S point mutation has little effect on undifferentiated embryonic stem cells (ESCs) but upon retinoic acid (RA) treatment causes retention of Oct4 expression, deregulated gene expression, and aberrant differentiation. Pou5f1 C48S ESCs also form less differentiated teratomas and contribute poorly to adult somatic tissues. Finally, we describe Pou5f1 C48S (Janky) mice, which in the homozygous condition are severely developmentally restricted after E4.5. Rare animals bypassing this restriction appear normal at birth but are sterile. Collectively, these findings uncover a novel Oct4 redox mechanism involved in both entry into and exit from pluripotency.

Structure and functional determinants of Rad6-Bre1 subunits in the histone H2B ubiquitin-conjugating complex.

The conserved complex of the Rad6 E2 ubiquitin-conjugating enzyme and the Bre1 E3 ubiquitin ligase catalyzes histone H2B monoubiquitination (H2Bub1), which regulates chromatin dynamics during transcription and other nuclear processes. Here, we report a crystal structure of Rad6 and the non-RING domain N-terminal region of Bre1, which shows an asymmetric homodimer of Bre1 contacting a conserved loop on the Rad6 'backside'. This contact is distant from the Rad6 catalytic site and is the location of mutations that impair telomeric silencing in yeast. Mutational analyses validated the importance of this contact for the Rad6-Bre1 interaction, chromatin-binding dynamics, H2Bub1 formation and gene expression. Moreover, the non-RING N-terminal region of Bre1 is sufficient to confer nucleosome binding ability to Rad6 in vitro. Interestingly, Rad6 P43L protein, an interaction interface mutant and equivalent to a cancer mutation in the human homolog, bound Bre1 5-fold more tightly than native Rad6 in vitro, but showed reduced chromatin association of Bre1 and reduced levels of H2Bub1 in vivo. These surprising observations imply conformational transitions of the Rad6-Bre1 complex during its chromatin-associated functional cycle, and reveal the differential effects of specific disease-relevant mutations on the chromatin-bound and unbound states. Overall, our study provides structural insights into Rad6-Bre1 interaction through a novel interface that is important for their biochemical and biological responses.

A toolbox for class I HDACs reveals isoform specific roles in gene regulation and protein acetylation.

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimics class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.

Mutations of Rad6 E2 ubiquitin-conjugating enzymes at alanine-126 in helix-3 affect ubiquitination activity and decrease enzyme stability.

Rad6, an E2 ubiquitin-conjugating enzyme conserved from yeast to humans, functions in transcription, genome maintenance, and proteostasis. The contributions of many conserved secondary structures of Rad6 and its human homologs UBE2A and UBE2B to their biological functions are not understood. A mutant RAD6 allele with a missense substitution at alanine-126 (A126) of helix-3 that causes defects in telomeric gene silencing, DNA repair, and protein degradation was reported over 2 decades ago. Here, using a combination of genetics, biochemical, biophysical, and computational approaches, we discovered that helix-3 A126 mutations compromise the ability of Rad6 to ubiquitinate target proteins without disrupting interactions with partner E3 ubiquitin-ligases that are required for their various biological functions in vivo. Explaining the defective in vitro or in vivo ubiquitination activities, molecular dynamics simulations and NMR showed that helix-3 A126 mutations cause local disorder of the catalytic pocket of Rad6 in addition to disorganizing the global structure of the protein to decrease its stability in vivo. We also show that helix-3 A126 mutations deform the structures of UBE2A and UBE2B, the human Rad6 homologs, and compromise the in vitro ubiquitination activity and folding of UBE2B. Providing insights into their ubiquitination defects, we determined helix-3 A126 mutations impair the initial ubiquitin charging and the final discharging steps during substrate ubiquitination by Rad6. In summary, our studies reveal that the conserved helix-3 is a crucial structural constituent that controls the organization of catalytic pockets, enzymatic activities, and biological functions of the Rad6-family E2 ubiquitin-conjugating enzymes.

Rapid purification of rabbit immunoglobulins using a single-step, negative-selection chromatography.

Custom polyclonal antibodies raised in rabbits are routinely used in immunoblotting and other protein analysis techniques. Custom rabbit polyclonal antisera are generally purified using immunoaffinity or Protein A-affinity chromatography; however, these methods require harsh elution conditions that can compromise the antigen binding efficacy. We evaluated the utility of Melon™ Gel chromatography for purification of IgG from crude rabbit serum. We show that Melon Gel-purified rabbit IgGs are active and perform well in immunoblotting. In summary, the Melon Gel method is a rapid, one-step, negative-selection approach that can be employed in either preparative or small-scale format to purify IgG from crude rabbit serum without the need for denaturing eluent.

The histone H4 basic patch regulates SAGA-mediated H2B deubiquitination and histone acetylation.

Histone H2B monoubiquitylation (H2Bub1) has central functions in multiple DNA-templated processes, including gene transcription, DNA repair, and replication. H2Bub1 also is required for the trans-histone regulation of H3K4 and H3K79 methylation. Although previous studies have elucidated the basic mechanisms that establish and remove H2Bub1, we have only an incomplete understanding of how H2Bub1 is regulated. We report here that the histone H4 basic patch regulates H2Bub1. Yeast cells with arginine-to-alanine mutations in the H4 basic patch (H42RA) exhibited a significant loss of global H2Bub1. H42RA mutant yeast strains also displayed chemotoxin sensitivities similar to, but less severe than, strains containing a complete loss of H2Bub1. We found that the H4 basic patch regulates H2Bub1 levels independently of interactions with chromatin remodelers and separately from its regulation of H3K79 methylation. To measure H2B ubiquitylation and deubiquitination kinetics in vivo, we used a rapid and reversible optogenetic tool, the light-inducible nuclear exporter, to control the subcellular location of the H2Bub1 E3 ligase, Bre1. The ability of Bre1 to ubiquitylate H2B was unaffected in the H42RA mutant. In contrast, H2Bub1 deubiquitination by SAGA-associated Ubp8, but not by Ubp10, increased in the H42RA mutant. Consistent with a function for the H4 basic patch in regulating SAGA deubiquitinase activity, we also detected increased SAGA-mediated histone acetylation in H4 basic patch mutants. Our findings uncover that the H4 basic patch has a regulatory function in SAGA-mediated histone modifications.

Interaction of the Jhd2 Histone H3 Lys-4 Demethylase with Chromatin Is Controlled by Histone H2A Surfaces and Restricted by H2B Ubiquitination.

Histone H3 lysine 4 (H3K4) methylation is a dynamic modification. In budding yeast, H3K4 methylation is catalyzed by the Set1-COMPASS methyltransferase complex and is removed by Jhd2, a JMJC domain family demethylase. The catalytic JmjC and JmjN domains of Jhd2 have the ability to remove all three degrees (mono-, di-, and tri-) of H3K4 methylation. Jhd2 also contains a plant homeodomain (PHD) finger required for its chromatin association and H3K4 demethylase functions. The Jhd2 PHD finger associates with chromatin independent of H3K4 methylation and the H3 N-terminal tail. Therefore, how Jhd2 associates with chromatin to perform H3K4 demethylation has remained unknown. We report a novel interaction between the Jhd2 PHD finger and histone H2A. Two residues in H2A (Phe-26 and Glu-57) serve as a binding site for Jhd2 in vitro and mediate its chromatin association and H3K4 demethylase functions in vivo. Using RNA sequencing, we have identified the functional target genes for Jhd2 and the H2A Phe-26 and Glu-57 residues. We demonstrate that H2A Phe-26 and Glu-57 residues control chromatin association and H3K4 demethylase functions of Jhd2 during positive or negative regulation of transcription at target genes. Importantly, we show that H2B Lys-123 ubiquitination blocks Jhd2 from accessing its binding site on chromatin, and thereby, we have uncovered a second mechanism by which H2B ubiquitination contributes to the trans-histone regulation of H3K4 methylation. Overall, our study provides novel insights into the chromatin binding dynamics and H3K4 demethylase functions of Jhd2.

Oct4 redox sensitivity potentiates reprogramming and differentiation.

The transcription factor Oct4/Pou5f1 is a component of the regulatory circuitry governing pluripotency and is widely used to induce pluripotency from somatic cells. Here we use domain swapping and mutagenesis to study Oct4s reprogramming ability, identifying a redox-sensitive DNA binding domain cysteine residue (Cys48) as a key determinant of reprogramming and differentiation. Oct4 Cys48 sensitizes the protein to oxidative inhibition of DNA binding activity and promotes oxidation-mediated protein ubiquitylation. Pou5f1C48S point mutation has little effect on undifferentiated embryonic stem cells (ESCs), but upon retinoic acid (RA) treatment causes retention of Oct4 expression, deregulated gene expression and aberrant differentiation. Pou5f1C48S ESCs also form less differentiated teratomas and contribute poorly to adult somatic tissues. Finally, we describe Pou5f1C48S (Janky) mice, which in the homozygous condition are severely developmentally restricted after E4.5. Rare animals bypassing this restriction appear normal at birth but are sterile. Collectively, these findings uncover a novel Oct4 redox mechanism involved in both entry into and exit from pluripotency.

Structure-function analysis of histone H2B and PCNA ubiquitination dynamics using deubiquitinase-deficient strains.

Post-translational covalent conjugation of ubiquitin onto proteins or ubiquitination is important in nearly all cellular processes. Steady-state ubiquitination of individual proteins in vivo is maintained by two countering enzymatic activities: conjugation of ubiquitin by E1, E2 and E3 enzymes and removal by deubiquitinases. Here, we deleted one or more genes encoding deubiquitinases in yeast and evaluated the requirements for ubiquitin conjugation onto a target protein. Our proof-of-principle studies demonstrate that absence of relevant deubiquitinase(s) provides a facile and versatile method that can be used to study the nuances of ubiquitin conjugation and deubiquitination of target proteins in vivo . We verified our method using mutants lacking the deubiquitinases Ubp8 and/or Ubp10 that remove ubiquitin from histone H2B or PCNA. Our studies reveal that the C-terminal coiled-domain of the adapter protein Lge1 and the C-terminal acidic tail of Rad6 E2 contribute to monoubiquitination of histone H2BK123, whereas the distal acidic residues of helix-4 of Rad6, but not the acidic tail, is required for monoubiquitination of PCNA. Further, charged substitution at alanine-120 in the H2B C-terminal helix adversely affected histone H2BK123 monoubiquitination by inhibiting Rad6-Bre1-mediated ubiquitin conjugation and by promoting Ubp8/Ubp10-mediated deubiquitination. In summary, absence of yeast deubiquitinases UBP8 and/or UBP10 allows uncovering the regulation of and requirements for ubiquitin addition and removal from their physiological substrates such as histone H2B or PCNA in vivo .

Structure-function analysis of histone H2B and PCNA ubiquitination dynamics using deubiquitinase-deficient strains.

Post-translational covalent conjugation of ubiquitin onto proteins or ubiquitination is important in nearly all cellular processes. Steady-state ubiquitination of individual proteins in vivo is maintained by two countering enzymatic activities: conjugation of ubiquitin by E1, E2 and E3 enzymes and removal by deubiquitinases. Here, we deleted one or more genes encoding deubiquitinases in yeast and evaluated the requirements for ubiquitin conjugation onto a target protein. Our proof-of-principle studies demonstrate that absence of relevant deubiquitinase(s) provides a facile and versatile method that can be used to study the nuances of ubiquitin conjugation and deubiquitination of target proteins in vivo. We verified our method using mutants lacking the deubiquitinases Ubp8 and/or Ubp10 that remove ubiquitin from histone H2B or PCNA. Our studies reveal that the C-terminal coiled-domain of the adapter protein Lge1 and the C-terminal acidic tail of Rad6 E2 contribute to monoubiquitination of histone H2BK123, whereas the distal acidic residues of helix-4 of Rad6, but not the acidic tail, is required for monoubiquitination of PCNA. Further, charged substitution at alanine-120 in the H2B C-terminal helix adversely affected histone H2BK123 monoubiquitination by inhibiting Rad6-Bre1-mediated ubiquitin conjugation and by promoting Ubp8/Ubp10-mediated deubiquitination. In summary, absence of yeast deubiquitinases UBP8 and/or UBP10 allows uncovering the regulation of and requirements for ubiquitin addition and removal from their physiological substrates such as histone H2B or PCNA in vivo.

Oct1 cooperates with the Smad family of transcription factors to promote mesodermal lineage specification.

The transition between pluripotent and tissue-specific states is a key aspect of development. Understanding the pathways driving these transitions will facilitate the engineering of properly differentiated cells for experimental and therapeutic uses. Here, we showed that during mesoderm differentiation, the transcription factor Oct1 activated developmental lineage-appropriate genes that were silent in pluripotent cells. Using mouse embryonic stem cells (ESCs) with an inducible knockout of Oct1, we showed that Oct1 deficiency resulted in poor induction of mesoderm-specific genes, leading to impaired mesodermal and terminal muscle differentiation. Oct1-deficient cells exhibited poor temporal coordination of the induction of lineage-specific genes and showed inappropriate developmental lineage branching, resulting in poorly differentiated cell states retaining epithelial characteristics. In ESCs, Oct1 localized with the pluripotency factor Oct4 at mesoderm-associated genes and remained bound to those loci during differentiation after the dissociation of Oct4. Binding events for Oct1 overlapped with those for the histone lysine demethylase Utx, and an interaction between Oct1 and Utx suggested that these two proteins cooperate to activate gene expression. The specificity of the ubiquitous Oct1 for the induction of mesodermal genes could be partially explained by the frequent coexistence of Smad and Oct binding sites at mesoderm-specific genes and the cooperative stimulation of mesodermal gene transcription by Oct1 and Smad3. Together, these results identify Oct1 as a key mediator of mesoderm lineage-specific gene induction.

Regulation of histone H2A and H2B deubiquitination and Xenopus development by USP12 and USP46.

Post-translational histone modifications play important roles in regulating gene expression programs, which in turn determine cell fate and lineage commitment during development. One such modification is histone ubiquitination, which primarily targets histone H2A and H2B. Although ubiquitination of H2A and H2B has been generally linked to gene silencing and gene activation, respectively, the functions of histone ubiquitination during eukaryote development are not well understood. Here, we identified USP12 and USP46 as histone H2A and H2B deubiquitinases that regulate Xenopus development. USP12 and USP46 prefer nucleosomal substrates and deubiquitinate both histone H2A and H2B in vitro and in vivo. WDR48, a WD40 repeat-containing protein, interacts with USP12 and USP46 and is required for the histone deubiquitination activity. Overexpression of either gene leads to gastrulation defects without affecting mesodermal cell fate, whereas knockdown of USP12 in Xenopus embryos results in reduction of a subset of mesodermal genes at gastrula stages. Immunohistochemical staining and chromatin immunoprecipitation assays revealed that USP12 regulates histone deubiquitination in the mesoderm and at specific gene promoters during Xenopus development. Taken together, this study identifies USP12 and USP46 as histone deubiquitinases for H2A and H2B and reveals that USP12 regulates Xenopus development during gastrula stages.

Decoding the trans-histone crosstalk: methods to analyze H2B ubiquitination, H3 methylation and their regulatory factors.

Regulation of histone H3 lysine 4 and 79 methylation by histone H2B lysine 123 monoubiquitination is an evolutionarily conserved trans-histone crosstalk mechanism, which demonstrates a functional role for histone ubiquitination within the cell. The regulatory enzymes, factors and processes involved in the establishment and dynamic modulation of these modifications and their genome-wide distribution patterns have been determined in many model systems. Rapid progress in understanding this trans-histone crosstalk has been made using the standard experimental tools of chromatin biology in budding yeast (Saccharomyces cerevisiae), a highly tractable model organism. Here, we provide a set of modified and refined experimental procedures that can be used to gain further insights into the underlying mechanisms that govern this crosstalk in budding yeast. Importantly, the improved procedures and their underlying principles can also be applied to other model organisms. Methods presented here provide a rapid and efficient means to prepare enriched protein extracts to better preserve and assess the steady state levels of histones, non-histone proteins and their modifications. Improved chromatin immunoprecipitation and double immunoprecipitation protocols are provided to measure the occupancy and distribution of proteins and their modified forms at specific chromatin regions or loci. A quick and easy method to measure overall protein abundance and changes in protein-protein and protein-DNA interactions on native chromatin is also described.

Ubiquitination of histone H2B regulates chromatin dynamics by enhancing nucleosome stability.

The mechanism by which ubiquitination of histone H2B (H2Bub1) regulates H3-K4 and -K79 methylation and the histone H2A-H2B chaperone Spt16-mediated nucleosome dynamics during transcription is not fully understood. Upon investigating the effect of H2Bub1 on chromatin structure, we find that contrary to the supposed role for H2Bub1 in opening up chromatin, it is important for nucleosome stability. First, we show that H2Bub1 does not function as a "wedge" to non-specifically unfold chromatin, as replacement of ubiquitin with a bulkier SUMO molecule conjugated to the C-terminal helix of H2B cannot functionally support H3-K4 and -K79 methylation. Second, using a series of biochemical analyses, we demonstrate that nucleosome stability is reduced or enhanced, when the levels of H2Bub1 are abolished or increased, respectively. Besides transcription elongation, we show that H2Bub1 regulates initiation by stabilizing nucleosomes positioned over the promoters of repressed genes. Collectively, our study reveals an intrinsic difference in the property of chromatin assembled in the presence or absence of H2Bub1 and implicates the regulation of nucleosome stability as the mechanism by which H2Bub1 modulates nucleosome dynamics and histone methylation during transcription.

Quantitative Assessment of Histone H2B Monoubiquitination in Yeast Using Immunoblotting.

Studies in Saccharomyces cerevisiae and Schizosaccharomyces pombe have enhanced our understanding of the regulation and functions of histone H2B monoubiquitination (H2Bub1), a key epigenetic marker with important roles in transcription and other processes. The detection of H2Bub1 in yeasts using immunoblotting has been greatly facilitated by the commercial availability of antibodies against yeast histone H2B and the cross-reactivity of an antibody raised against monoubiquitinated human H2BK120. These antibodies have obviated the need to express epitope-tagged histone H2B to detect H2Bub1 in yeasts. Here, we provide a step-by-step protocol and best practices for the quantification of H2Bub1 in yeast systems, from cell extract preparation to immunoblotting using the commercially available antibodies. We demonstrate that the commercial antibodies can effectively and accurately detect H2Bub1 in S. cerevisiae and S. pombe. Further, we show that the C-terminal epitope-tagging of histone H2B alters the steady-state levels of H2Bub1 in yeast systems. We report a sectioned blot probing approach combined with the serial dilution of protein lysates and the use of reversibly stained proteins as loading controls that together provide a cost-effective and sensitive method for the quantitative evaluation of H2Bub1 in yeast.

The JmjN domain of Jhd2 is important for its protein stability, and the plant homeodomain (PHD) finger mediates its chromatin association independent of H3K4 methylation.

Histone lysine methylation is a dynamic process that plays an important role in regulating chromatin structure and gene expression. Recent studies have identified Jhd2, a JmjC domain-containing protein, as an H3K4-specific demethylase in budding yeast. However, important questions regarding the regulation and functions of Jhd2 remain unanswered. In this study, we show that Jhd2 has intrinsic activity to remove all three states of H3K4 methylation in vivo and can dynamically associate with chromatin to modulate H3K4 methylation levels on both active and repressed genes and at the telomeric regions. We found that the plant homeodomain (PHD) finger of Jhd2 is important for its chromatin association in vivo. However, this association is not dependent on H3K4 methylation and the H3 N-terminal tail, suggesting the presence of an alternative mechanism by which Jhd2 binds nucleosomes. We also provide evidence that the JmjN domain and its interaction with the JmjC catalytic domain are important for Jhd2 function and that Not4 (an E3 ligase) monitors the structural integrity of this interdomain interaction to maintain the overall protein levels of Jhd2. We show that the S451R mutation in human SMCX (a homolog of Jhd2), which has been linked to mental retardation, and the homologous T359R mutation in Jhd2 affect the protein stability of both of these proteins. Therefore, our findings provide a mechanistic explanation for the observed defects in patients harboring this SMCX mutant and suggest the presence of a conserved pathway involving Not4 that modulates the protein stability of both yeast Jhd2 and human SMCX.

Targeting DNA Repair and Chromatin Crosstalk in Cancer Therapy.

Aberrant DNA repair pathways that underlie developmental diseases and cancers are potential targets for therapeutic intervention. Targeting DNA repair signal effectors, modulators and checkpoint proteins, and utilizing the synthetic lethality phenomena has led to seminal discoveries. Efforts to efficiently translate the basic findings to the clinic are currently underway. Chromatin modulation is an integral part of DNA repair cascades and an emerging field of investigation. Here, we discuss some of the key advancements made in DNA repair-based therapeutics and what is known regarding crosstalk between chromatin and repair pathways during various cellular processes, with an emphasis on cancer.

Unification and extensive diversification of M/Orf3-related ion channel proteins in coronaviruses and other nidoviruses.

The coronavirus, Severe Acute Respiratory Syndrome (SARS)-CoV-2, responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic, has emphasized the need for a better understanding of the evolution of virus-host interactions. ORF3a in both SARS-CoV-1 and SARS-CoV-2 are ion channels (viroporins) implicated in virion assembly and membrane budding. Using sensitive profile-based homology detection methods, we unify the SARS-CoV ORF3a family with several families of viral proteins, including ORF5 from MERS-CoVs, proteins from beta-CoVs (ORF3c), alpha-CoVs (ORF3b), most importantly, the Matrix (M) proteins from CoVs, and more distant homologs from other nidoviruses. We present computational evidence that these viral families might utilize specific conserved polar residues to constitute an aqueous pore within the membrane-spanning region. We reconstruct an evolutionary history of these families and objectively establish the common origin of the M proteins of CoVs and Toroviruses. We also show that the divergent ORF3 clade (ORF3a/ORF3b/ORF3c/ORF5 families) represents a duplication stemming from the M protein in alpha- and beta-CoVs. By phyletic profiling of major structural components of primary nidoviruses, we present a hypothesis for their role in virion assembly of CoVs, ToroVs, and Arteriviruses. The unification of diverse M/ORF3 ion channel families in a wide range of nidoviruses, especially the typical M protein in CoVs, reveal a conserved, previously under-appreciated role of ion channels in virion assembly and membrane budding. We show that M and ORF3 are under different evolutionary pressures; in contrast to the slow evolution of M as core structural component, the ORF3 clade is under selection for diversification, which suggests it might act at the interface with host molecules and/or immune attack.

Unification of the M/ORF3-related proteins points to a diversified role for ion conductance in pathogenesis of coronaviruses and other nidoviruses.

The new coronavirus, SARS-CoV-2, responsible for the COVID-19 pandemic has emphasized the need for a better understanding of the evolution of virus-host conflicts. ORF3a in both SARS-CoV-1 and SARS-CoV-2 are ion channels (viroporins) and involved in virion assembly and membrane budding. Using sensitive profile-based homology detection methods, we unify the SARS-CoV ORF3a family with several families of viral proteins, including ORF5 from MERS-CoVs, proteins from beta-CoVs (ORF3c), alpha-CoVs (ORF3b), most importantly, the Matrix (M) proteins from CoVs, and more distant homologs from other nidoviruses. By sequence analysis and structural modeling, we show that these viral families utilize specific conserved polar residues to constitute an ion-conducting pore in the membrane. We reconstruct the evolutionary history of these families, objectively establish the common origin of the M proteins of CoVs and Toroviruses. We show that the divergent ORF3a/ORF3b/ORF5 families represent a duplication stemming from the M protein in alpha- and beta-CoVs. By phyletic profiling of major structural components of primary nidoviruses, we present a model for their role in virion assembly of CoVs, ToroVs and Arteriviruses. The unification of diverse M/ORF3 ion channel families in a wide range of nidoviruses, especially the typical M protein in CoVs, reveal a conserved, previously under-appreciated role of ion channels in virion assembly, membrane fusion and budding. We show that the M and ORF3 are under differential evolutionary pressures; in contrast to the slow evolution of M as core structural component, the CoV-ORF3 clade is under selection for diversification, which indicates it is likely at the interface with host molecules and/or immune attack. IMPORTANCE: Coronaviruses (CoVs) have become a major threat to human welfare as the causative agents of several severe infectious diseases, namely Severe Acute Respiratory Syndrome (SARS), Middle Eastern Respiratory Syndrome (MERS), and the recently emerging human coronavirus disease 2019 (COVID-19). The rapid spread, severity of these diseases, as well as the potential re-emergence of other CoV-associated diseases have imposed a strong need for a thorough understanding of function and evolution of these CoVs. By utilizing robust domain-centric computational strategies, we have established homologous relationships between many divergent families of CoV proteins, including SARS-CoV/SARS-CoV-2 ORF3a, MERS-CoV ORF5, proteins from both beta-CoVs (ORF3c) and alpha-CoVs (ORF3b), the typical CoV Matrix proteins, and many distant homologs from other nidoviruses. We present evidence that they are active ion channel proteins, and the Cov-specific ORF3 clade proteins are under selection for rapid diversification, suggesting they might have been involved in interfering host molecules and/or immune attack.

The neural stem-cell marker CD24 is specifically upregulated in IDH-mutant glioma.

BACKGROUND: Malignant gliomas have disproportionally high morbidity and mortality. Heterozygous mutations in the isocitrate dehydrogenase 1 (IDH1) gene are most common in glioma, resulting in predominantly arginine to histidine substitution at codon 132. Because IDH1R132H requires a wild-type allele to produce (D)-2-hydroxyglutarate for epigenetic reprogramming, loss of IDH1R132H heterozygosity is associated with glioma progression in an IDH1-wildtype-like phenotype. Although previous studies have reported that transgenic IDH1R132H induces the expression of nestin-a neural stem-cell marker, the underlying mechanism remains unclear. Furthermore, this finding seems at odds with better outcome of IDH1R132H glioma because of a negative association of nestin with overall survival. METHODS: Gene expression was compared between IDH1R132H-hemizygous and IDH1R132H-heterozygous glioma cells under adherent and spheroid growth conditions. The results were validated for (D)-2-hydroxyglutarate responsiveness by pharmacologic agents, associations with DNA methylation by bioinformatic analysis, and associations with overall survival. Bisulfite DNA sequencing, chromatin immunoprecipitation, and pharmacological approach were used. FINDINGS: Neural stem-cell marker genes, including CD44, NES, and PROM1, are generally downregulated in IDH-mutant gliomas and IDH1R132H-heterozygous spheroid growth compared respectively with IDH-wildtype gliomas and IDH1R132H-hemizygous spheroid growth, in agreement with their negative associations with patient outcome. In contrast, CD24 is specifically upregulated and apparently associated with better survival. CD24 and NES expression respond differentially to alteration of (D)-2-hydroxyglutarate levels. CD24 upregulation is associated with histone and DNA demethylation as opposed to hypermethylation in the downregulated genes. INTERPRETATION: The better outcome of IDH-mutant glioma is orchestrated exquisitely through epigenetic reprogramming that directs bidirectional expression of neural stem-cell marker genes.