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RNA has been shown to interact with various proteins to regulate chromatin dynamics and gene expression. However, it is unknown whether RNAs associate with epigenetic marks such as post-translational modifications of histones, including histone 4 lysine 20 trimethylation (H4K20me3) or trimethylated histone 3 lysine 4 (H3K4me3), to regulate chromatin and gene expression. Here, we used chromatin-associated RNA immunoprecipitation (CARIP) followed by next-generation sequencing (CARIP-Seq) to survey RNAs associated with H4K20me3- and H3K4me3-marked chromatin on a global scale in embryonic stem (ES) cells. We identified thousands of mRNAs and noncoding RNAs that associate with H4K20me3- and H3K4me3-marked chromatin. H4K20me3- and H3K4me3-interacting RNAs are involved in chromatin organization and modification and RNA processing, whereas H4K20me3-only RNAs are involved in cell motility and differentiation, and H3K4me3-only RNAs are involved in metabolic processes and RNA processing. Expressi ...

Elastin is an essential vertebrate protein responsible for the elasticity of force-bearing tissues such as those of the lungs, blood vessels, and skin. One of the key features required for the exceptional properties of this durable biopolymer is the extensive covalent cross-linking between domains of its monomer molecule tropoelastin. To date, elastin's exact molecular assembly and mechanical properties are poorly understood. Here, using bovine elastin, we investigated the different types of cross-links in mature elastin to gain insight into its structure. We purified and proteolytically cleaved elastin from a single tissue sample into soluble cross-linked and noncross-linked peptides that we studied by high-resolution MS. This analysis enabled the elucidation of cross-links and other elastin modifications. We found that the lysine residues within the tropoelastin sequence were simultaneously unmodified and involved in various types of cross-links with different other domains. The Lys? ...

Molecular chaperones often possess functional modules that are specialized in assisting the formation of specific structural elements, such as a disulfide bridges and peptidyl?prolyl bonds in cis form, in the client protein. A ribosome-associated molecular chaperone trigger factor (TF), which has a peptidyl?prolyl cis/trans isomerase (PPIase) domain, acts as a highly efficient catalyst in the folding process limited by peptidyl?prolyl isomerization. Herein we report a study on the mechanism through which TF recognizes the proline residue in the unfolded client protein during the cis/trans isomerization process. The solution structure of TF in complex with the client protein showed that TF recognizes the proline-aromatic motif located in the hydrophobic stretch of the unfolded client protein through its conserved hydrophobic cleft, which suggests that TF preferentially accelerates the isomerization of the peptidyl?prolyl bond that is eventually folded into the core of the protein in its ...

DNA polymerase ? (Pol ?) is essential for maintaining genomic integrity. During short-patch base excision repair (BER), Pol ? incorporates a nucleotide into a single-gapped DNA substrate. Pol ? may also function in long-patch BER, where the DNA substrate consists of larger gap sizes or 5?-modified downstream DNA. We have recently shown that Pol ? fills small gaps in DNA during microhomology-mediated end-joining as part of a process that increases genomic diversity. Our previous results with single-nucleotide gapped DNA show that Pol ? undergoes two pre-catalytic conformational changes upon binding to the correct nucleotide substrate. Here we use FRET to investigate nucleotide incorporation of Pol ? with various DNA substrates. The results show that increasing the gap size influences the fingers closing step by increasing its reverse rate. However, the 5?-phosphate group has a more significant effect. The absence of the 5?-phosphate decreases the DNA binding affinity of Pol ? and result ...

Gene targeting via homologous recombination can occasionally result in incomplete disruption of the targeted gene. Here, we show that a widely used Nur77-deficient transgenic mouse model expresses a truncated protein encoding for part of the N-terminal domain of nuclear receptor Nur77. This truncated Nur77 protein is absent in a newly developed Nur77-deficient mouse strain generated using Cre-Lox recombination. Comparison of these two mouse strains using immunohistochemistry, flow cytometry, and colony-forming assays shows that homologous recombination?derived Nur77-deficient mice, but not WT or Cre-Lox?derived Nur77-deficient mice, suffer from liver immune cell infiltrates, loss of splenic architecture, and increased numbers of bone marrow hematopoietic stem cells and splenic colony?forming cells with age. Mechanistically, we demonstrate that the truncated Nur77 N-terminal domain protein maintains the stability and activity of hypoxia-inducible factor (HIF)-1, a transcription factor k ...

Antibody-mediated blockade of cluster of differentiation 47 (CD47)?thrombospondin-1 (TSP-1) interactions blocks osteoclast formation in vitro and attenuates parathyroid hormone (PTH)-induced hypercalcemia in vivo in mice. Hypercalcemia in this model reflects increased bone resorption. TSP-1 has two cell-associated binding partners, CD47 and CD36. The roles of these two molecules in mediating the effects of TSP1 in osteoclasts are unclear. Osteoclast formation was attenuated but not absent when preosteoclasts isolated from CD47?/? mice were cocultured with WT osteoblasts. Suppressing CD36 in osteoclast progenitors also attenuated osteoclast formation. The hypercalcemic response to a PTH infusion was blunted in CD47?/?/CD36?/? (double knockout (DKO)) female mice but not CD47?/? mice or CD36?/? animals, supporting a role for both CD47 and CD36 in mediating this effect. Consistent with this, DKO osteoclasts had impaired resorptive activity when analyzed in vitro. Inhibition of nitric oxide ...

Although EcoR124 is one of the better-studied Type I restriction-modification enzymes, it still presents many challenges to detailed analyses because of its structural and functional complexity and missing structural information. In all available structures of its motor subunit HsdR, responsible for DNA translocation and cleavage, a large part of the HsdR C terminus remains unresolved. The crystal structure of the C terminus of HsdR, obtained with a crystallization chaperone in the form of pHluorin fusion and refined to 2.45 Å, revealed that this part of the protein forms an independent domain with its own hydrophobic core and displays a unique ?-helical fold. The full-length HsdR model, based on the WT structure and the C-terminal domain determined here, disclosed a proposed DNA-binding groove lined by positively charged residues. In vivo and in vitro assays with a C-terminal deletion mutant of HsdR supported the idea that this domain is involved in complex assembly and DNA binding. ...

Environmental exposure and cellular metabolism can give rise to DNA alkylation, which can occur on the nitrogen and oxygen atoms of nucleobases, as well as on the phosphate backbone. Although O6-alkyl-2?-deoxyguanosine (O6-alkyl-dG) lesions are known to be associated with cancer, not much is known about how the alkyl group structures in these lesions affect their repair and replicative bypass in vivo or how translesion synthesis DNA polymerases influence the latter process. To answer these questions, here we synthesized oligodeoxyribonucleotides harboring seven O6-alkyl-dG lesions, with the alkyl group being Me, Et, nPr, iPr, nBu, iBu, or sBu, and examined the impact of these lesions on DNA replication in Escherichia coli cells. We found that replication past all the O6-alkyl-dG lesions was highly efficient and that SOS-induced DNA polymerases play redundant roles in bypassing these lesions. Moreover, these lesions directed exclusively the G ? A mutation, the frequency of which increas ...

N-Formylation of the Met-tRNAMet by the nuclearly encoded mitochondrial methionyl-tRNA formyltransferase (MTFMT) has been found to be a key determinant of protein synthesis initiation in mitochondria. In humans, mutations in the MTFMT gene result in Leigh syndrome, a progressive and severe neurometabolic disorder. However, the absolute requirement of formylation of Met-tRNAMet for protein synthesis in mammalian mitochondria is still debated. Here, we generated a Mtfmt-KO mouse fibroblast cell line and demonstrated that N-formylation of the first methionine via fMet-tRNAMet by MTFMT is not an absolute requirement for initiation of protein synthesis. However, it differentially affected the efficiency of synthesis of mtDNA-coded polypeptides. Lack of methionine N-formylation did not compromise the stability of these individual subunits but had a marked effect on the assembly and stability of the OXPHOS complexes I and IV and on their supercomplexes. In summary, N-formylation is not essent ...

Protein minimization is an attractive approach for designing vaccines against rapidly evolving pathogens such as human immunodeficiency virus, type 1 (HIV-1), because it can help in focusing the immune response toward conserved conformational epitopes present on complex targets. The outer domain (OD) of HIV-1 gp120 contains epitopes for a large number of neutralizing antibodies and therefore is a primary target for structure-based vaccine design. We have previously designed a bacterially expressed outer-domain immunogen (ODEC) that bound CD4-binding site (CD4bs) ligands with 3?12 ?m affinity and elicited a modest neutralizing antibody response in rabbits. In this study, we have optimized ODEC using consensus sequence design, cyclic permutation, and structure-guided mutations to generate a number of variants with improved yields, biophysical properties, stabilities, and affinities (KD of 10?50 nm) for various CD4bs targeting broadly neutralizing antibodies, including the germline-revert ...