Jade1 and the HBO1 complex are spatial-selective cofactors of Oct4.

Oct4 is a master regulator of pluripotency. Potential Oct4 interactors have been cataloged extensively but the manner and significance of these interactions are incompletely defined. Like other POU domain proteins, Oct4 is capable of binding to DNA in multiple configurations, however the relationship between these configurations and cofactor recruitment (and hence transcription output) are unknown.

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.

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 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.

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.

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.

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.

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.

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.

Quantitative Assessment of Histone H2B Monoubiquitination in Yeast Using Immunoblotting.

Studies in and 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.

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.

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.

Physico-chemical characterization study of coffee husk for feasibility assessment in fluidized bed gasification process.

The biomasses like post-harvest agricultural residues are usually disposed of as landfills and used as cattle fodder and manure. Large quantities of such wastes are often set to open firing. The open firing of such waste biomasses leads to particulate matter emission and air pollution.

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.

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.

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

Unlabelled: 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.

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 IDH1 requires a wild-type allele to produce (D)-2-hydroxyglutarate for epigenetic reprogramming, loss of IDH1 heterozygosity is associated with glioma progression in an IDH1-wildtype-like phenotype.

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 -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.

Establishment and Maintenance of Chromatin Architecture Are Promoted Independently of Transcription by the Histone Chaperone FACT and H3-K56 Acetylation in .

FACT (FAcilitates Chromatin Transcription/Transactions) is a histone chaperone that can destabilize or assemble nucleosomes. Acetylation of histone H3-K56 weakens a histone-DNA contact that is central to FACT activity, suggesting that this modification could affect FACT functions. We tested this by asking how mutations of H3-K56 and FACT affect nucleosome reorganization activity , and chromatin integrity and transcript output Mimics of unacetylated or permanently acetylated H3-K56 had different effects on FACT activity as expected, but the same mutations had surprisingly similar effects on global transcript levels.

HDAC1,2 inhibition and doxorubicin impair Mre11-dependent DNA repair and DISC to override BCR-ABL1-driven DSB repair in Philadelphia chromosome-positive B-cell precursor acute lymphoblastic leukemia.

Philadelphia chromosome-positive (Ph+) B-cell precursor acute lymphoblastic leukemia (ALL) expressing BCR-ABL1 oncoprotein is a major subclass of ALL with poor prognosis. BCR-ABL1-expressing leukemic cells are highly dependent on double-strand break (DSB) repair signals for their survival. Here we report that a first-in-class HDAC1,2 selective inhibitor and doxorubicin (a hyper-CVAD chemotherapy regimen component) impair DSB repair networks in Ph+ B-cell precursor ALL cells using common as well as distinct mechanisms.

Counteracting H3K4 methylation modulators Set1 and Jhd2 co-regulate chromatin dynamics and gene transcription.

Histone H3K4 methylation is connected to gene transcription from yeast to humans, but its mechanistic roles in transcription and chromatin dynamics remain poorly understood. We investigated the functions for Set1 and Jhd2, the sole H3K4 methyltransferase and H3K4 demethylase, respectively, in S. cerevisiae.

Next generation sequencing analysis of lung cancer datasets: A functional genomics perspective.

Cigarette smoking leads to serious epidemics in humans, creating torsion of infection in epithelial cells lining the respiratory tracts. Several researchers in the recent past have theorized that the next generation sequencing (NGS), especially transcriptome sequencing has enhanced understanding lung cancers and other epithelial epidemics. Conversely, pathogenesis specific to lung cancer with respect to molecular fraction of genomic ribonucleic acid has some mutant effect in various populations like smokers with lung cancer, healthy never smokers and vice versa.

SUMOylation Regulates Growth Factor Independence 1 in Transcriptional Control and Hematopoiesis.

Cell fate specification requires precise coordination of transcription factors and their regulators to achieve fidelity and flexibility in lineage allocation. The transcriptional repressor growth factor independence 1 (GFI1) is comprised of conserved Snail/Slug/Gfi1 (SNAG) and zinc finger motifs separated by a linker region poorly conserved with GFI1B, its closest homolog. Moreover, GFI1 and GFI1B coordinate distinct developmental fates in hematopoiesis, suggesting that their functional differences may derive from structures within their linkers.

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.

Annotation and curation of uncharacterized proteins- challenges.

Hypothetical proteins (HPs) are the proteins predicted to be expressed from an open reading frame, making a substantial fraction of proteomes in both prokaryotes and eukaryotes. Genome projects have led to the identification of many therapeutic targets, the putative function of the protein, and their interactions. In this review we enlist various methods linking annotation to structural and functional prediction of HPs that assist in the discovery of new structures and functions serving as markers and pharmacological targets for drug designing, discovery, and screening.