Extensive Recovery of Embryonic Enhancer and Gene Memory Stored in Hypomethylated Enhancer DNA.

Developing and adult tissues use different cis-regulatory elements. Although DNA at some decommissioned embryonic enhancers is hypomethylated in adult cells, it is unknown whether this putative epigenetic memory is complete and recoverable. We find that, in adult mouse cells, hypomethylated CpG dinucleotides preserve a nearly complete archive of tissue-specific developmental enhancers. Sites that carry the active histone mark H3K4me1, and are therefore considered "primed," are mainly cis elements that act late in organogenesis. In contrast, sites decommissioned early in development retain hypomethylated DNA as a singular property. In adult intestinal and blood cells, sustained absence of polycomb repressive complex 2 indirectly reactivates most-and only-hypomethylated developmental enhancers. Embryonic and fetal transcriptional programs re-emerge as a result, in reverse chronology to cis element inactivation during development. Thus, hypomethylated DNA in adult cells preserves a "fossil record" of tissue-specific developmental enhancers, stably marking decommissioned sites and enabling recovery of this epigenetic memory.

Quercetin inhibits the proliferation of multiple myeloma cells by upregulating PTPRR expression.

Multiple myeloma (MM) is an incurable disease characterized by malignant plasma cell clonal expansion in the bone marrow; therefore, inhibiting the proliferation of plasma cells is an important approach to overcome the progression of MM. Quercetin (Que) is a promising flavonoid with broad-spectrum anti-tumor activity against various cancers, including MM; however, the underlying mechanism is not yet understood. The present study aimed to reveal the gene expression profile of Que-treated MM cells and clarify its potential mechanism. The 30% inhibitory concentration (IC30) of Que against MM cells was calculated, and the proliferation rate was significantly reduced after Que treatment. Next, 495 dysregulated genes were identified via RNA sequencing in Que-treated MM cells. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses indicated that the dysregulated genes were enriched in various apoptosis-related GO terms and amino acid metabolism-related pathways. qPCR validation showed that protein tyrosine phosphatase receptor-type R (PTPRR) had the highest verified log2 FC (abs) among the top 15 dysregulated genes. Overexpression of PTPRR increased the sensitivity of MM cells against Que, significantly inhibiting their proliferation and colony formation ability; silencing of PTPRR showed the opposite results. Furthermore, bioinformatics analyses and PPI network construction of PTPRR indicated that dephosphorylation of ERK might be the potential pathway for the PTPRR-induced inhibition of MM cell proliferation. In summary, our study identified the gene expression profile in Que-treated MM cells and demonstrated that the upregulation of PTPRR was one of the important mechanisms for the Que-induced inhibition of MM cell proliferation.

Functional epigenetics approach identifies BRM/SMARCA2 as a critical synthetic lethal target in BRG1-deficient cancers.

Defects in epigenetic regulation play a fundamental role in the development of cancer, and epigenetic regulators have recently emerged as promising therapeutic candidates. We therefore set out to systematically interrogate epigenetic cancer dependencies by screening an epigenome-focused deep-coverage design shRNA (DECODER) library across 58 cancer cell lines. This screen identified BRM/SMARCA2, a DNA-dependent ATPase of the mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complex, as being essential for the growth of tumor cells that harbor loss of function mutations in BRG1/SMARCA4. Depletion of BRM in BRG1-deficient cancer cells leads to a cell cycle arrest, induction of senescence, and increased levels of global H3K9me3. We further demonstrate the selective dependency of BRG1-mutant tumors on BRM in vivo. Genetic alterations of the mSWI/SNF chromatin remodeling complexes are the most frequent among chromatin regulators in cancers, with BRG1/SMARCA4 mutations occurring in ∼10-15% of lung adenocarcinomas. Our findings position BRM as an attractive therapeutic target for BRG1 mutated cancers. Because BRG1 and BRM function as mutually exclusive catalytic subunits of the mSWI/SNF complex, we propose that such synthetic lethality may be explained by paralog insufficiency, in which loss of one family member unveils critical dependence on paralogous subunits. This concept of "cancer-selective paralog dependency" may provide a more general strategy for targeting other tumor suppressor lesions/complexes with paralogous subunits.

Sugar-Protein Connectivity Impacts on the Immunogenicity of Site-Selective Salmonella O-Antigen Glycoconjugate Vaccines.

A series of glycoconjugates with defined connectivity were synthesized to investigate the impact of coupling Salmonella typhimurium O-antigen to different amino acids of CRM197 protein carrier. In particular, two novel methods for site-selective glycan conjugation were developed to obtain conjugates with single attachment site on the protein, based on chemical modification of a disulfide bond and pH-controlled transglutaminase-catalyzed modification of lysine, respectively. Importantly, conjugation at the C186-201 bond resulted in significantly higher anti O-antigen bactericidal antibody titers than coupling to K37/39, and in comparable titers to conjugates bearing a larger number of saccharides. This study demonstrates that the conjugation site plays a role in determining the immunogenicity in mice and one single attachment point may be sufficient to induce high levels of bactericidal antibodies.

Absolute quantification of histone PTM marks by MRM-based LC-MS/MS.

The N-terminal tails of core histones harbor the sites of numerous post-translational modifications (PTMs) with important roles in the regulation of chromatin structure and function. Profiling histone PTM marks provides data that help understand the epigenetics events in cells and their connections with cancer and other diseases. Our previous study demonstrated that specific derivatization of histone peptides by NHS propionate significantly improved their chromatographic performance on reversed phase columns for LC/MS analysis. As a step forward, we recently developed a multiple reaction monitoring (MRM) based LC-MS/MS method to analyze 42 targeted histone peptides. By using stable isotopic labeled peptides as internal standards that are spiked into the reconstituted solutions, this method allows to measure absolute concentration of the tryptic peptides of H3 histone proteins extracted from cancer cell lines. The method was thoroughly validated for the accuracy and reproducibility through analyzing recombinant histone proteins and cellular samples. The linear dynamic range of the MRM assays was achieved in 3 orders of magnitude from 1 nM to 1 µM for all targeted peptides. Excellent intrabatch and interbatch reproducibility (<15% CV) was obtained. This method has been used to study translocated NSD2 (a histone lysine methyltransferase that catalyzes the histone lysine 36 methylation) function with its overexpression in KMS11 multiple myeloma cells. From the results we have successfully quantitated both individual and combinatorial histone marks in parental and NSD2 selective knockout KMS11 cells.

Defined conjugation of glycans to the lysines of CRM197 guided by their reactivity mapping.

Systematic characterisation of the reactivity of the lysine moieties in CRM197 towards N-hydroxysuccinimide linkers bearing alkynes or azides is described. This involves two-step conjugation of various glycans to CRM197 by click chemistry in a well-defined manner. By semiquantitative LC-MS/MS analysis of proteolytic digests of the conjugates formed, the reactivity of lysine residues in the protein was mapped and ranked. Computational analysis of the solvent accessibility of each lysine residue (based on the CRM197 crystal structure) established a correlation between reactivity and surface exposure. By this approach, conjugation involving lysine residues (normally a random process) can be controlled. It enables the preparation of lysine-mediated glycoconjugates with improved batch-to-batch reproducibility, thereby producing neo-glycoconjugates with more-consistent biological activity.

Tyrosine-directed conjugation of large glycans to proteins via copper-free click chemistry.

We have demonstrated that the insertion of alkyne-containing bifunctional linkers into the tyrosine residues of the carrier protein, followed by the copper mediated azide-alkyne [3 + 2] cycloaddition of carbohydrates, is a robust approach for the preparation of glycoconjugates with defined glycans, carrier, and connectivity. Conjugation of Group B Streptococcus (GBS) capsular polysaccharides to streptococcal pilus protein could extend the vaccine coverage to a variety of strains. Application of our protocol to these large charged polysaccharides occurred at low yields. Herein we developed a tyrosine-directed conjugation approach based on the copper-free click chemistry of sugars modified with cyclooctynes, which enables efficient condensation of synthetic carbohydrates. Most importantly, this strategy was demonstrated to be more effective than the corresponding copper catalyzed reaction for the insertion of GBS onto the tyrosine residues of GBS pilus proteins, previously selected as vaccine antigens through the so-called reverse vaccinology. Integrity of protein epitopes in the modified proteins was ascertained by competitive ELISA, and conjugation of polysaccharide to protein was confirmed by SDS page electrophoresis and immunoblot assays. The amount of conjugated polysaccharide was estimated by high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). The described technology is particularly suitable for proteins used with the dual role of vaccine antigen and carrier for the carbohydrate haptens.

Theoretical and experimental studies of polarization fluctuations over atmospheric turbulent channels for wireless optical communication systems.

In wireless optical communications (WOC), polarization multiplexing systems and coherent polarization systems have excellent performance and wide applications, while its state of polarization affected by atmospheric turbulence is not clearly understood. This paper focuses on the polarization fluctuations caused by atmospheric turbulence in a WOC link. Firstly, the relationship between the polarization fluctuations and the index of refraction structure parameter is introduced and the distribution of received polarization angle is obtained through theoretical derivations. Then, turbulent conditions are adjusted and measured elaborately in a wide range of scintillation indexes (SI). As a result, the root-mean-square (RMS) variation and probability distribution function (PDF) of polarization angle conforms closely to that of theoretical model.

Specific and efficient N-propionylation of histones with propionic acid N-hydroxysuccinimide ester for histone marks characterization by LC-MS.

Histones participate in epigenetic regulation via a variety of dynamic posttranslational modifications (PTMs) on them. Mass spectrometry (MS) has become a powerful tool to investigate histone PTMs. With the bottom-up mass spectrometry approach, chemical derivatization of histones with propionic anhydride or deuterated acetic anhydride followed by trypsin digestion was widely used to block the hydrophilic lysine residues and generate compatible peptides for LC-MS analysis. However, certain severe side reactions (such as acylation on tyrosine or serine) caused by acid anhydrides will lead to a number of analytical issues such as reducing results accuracy and impairing the reproducibility and sensitivity of MS analysis. As an alternative approach, we report a novel derivatization method that utilizes N-hydroxysuccinimide ester to specifically and efficiently derivatize both free and monomethylated amine groups in histones. A competitive inhibiting strategy was implemented in our method to effectively prevent the side reactions. We demonstrated that our method can achieve excellent specificity and efficiency for histones derivatization in a reproducible manner. Using this derivatization method, we succeeded to quantitatively profile the histone PTMs in KMS11 cell line with selective knock out of translocated NSD2 allele (TKO) and the original parental KMS11 cell lines (PAR) (NSD2, a histone methyltransferase that catalyzes the histone H3 K36 methylation), which revealed a significant crosstalk between H3 protein K27 methylation and adjacent K36 methylation.

Ratio of hemoglobin to mean corpuscular volume: A new index for discriminating between iron deficiency anemia and thalassemia trait.

BACKGROUND: Iron deficiency anemia (IDA) and thalassemia trait (TT) are the most common microcytic and hypochromic anemias. Differentiation between mild TT and early IDA is still a clinical challenge. AIM: To develop and validate a new index for discriminating between IDA and TT. METHODS: Blood count data from 126 patients, consisting of 43 TT patients and 83 IDA patients, was retrospectively analyzed to develop a new index formula. This formula was further validated in another 61 patients, consisting of 48 TT patients and 13 IDA patients. RESULTS: The new index is the ratio of hemoglobin to mean corpuscular volume. Its sensitivity, specificity, accuracy, Youden's Index, area under the receiver operating characteristic curve, and Kappa coefficient in discriminating between IDA and TT were 93.5%, 78.4%, 83.3%, 0.72, 0.97, and 0.65, respectively. CONCLUSION: This new index has good diagnostic performance in discriminating between mild TT and early IDA. It requires only two results of complete blood count, which can be a very desirable feature in under-resourced scenarios.

The novel HLA-DPB1*02:02:07 allele identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-DPB1*02:02:07 differs from HLA-DPB1*02:02:01:01 by one nucleotide in exon 2.

The histone H3K79 methyltransferase Dot1L is essential for mammalian development and heterochromatin structure.

Dot1 is an evolutionarily conserved histone methyltransferase specific for lysine 79 of histone H3 (H3K79). In Saccharomyces cerevisiae, Dot1-mediated H3K79 methylation is associated with telomere silencing, meiotic checkpoint control, and DNA damage response. The biological function of H3K79 methylation in mammals, however, remains poorly understood. Using gene targeting, we generated mice deficient for Dot1L, the murine Dot1 homologue. Dot1L-deficient embryos show multiple developmental abnormalities, including growth impairment, angiogenesis defects in the yolk sac, and cardiac dilation, and die between 9.5 and 10.5 days post coitum. To gain insights into the cellular function of Dot1L, we derived embryonic stem (ES) cells from Dot1L mutant blastocysts. Dot1L-deficient ES cells show global loss of H3K79 methylation as well as reduced levels of heterochromatic marks (H3K9 di-methylation and H4K20 tri-methylation) at centromeres and telomeres. These changes are accompanied by aneuploidy, telomere elongation, and proliferation defects. Taken together, these results indicate that Dot1L and H3K79 methylation play important roles in heterochromatin formation and in embryonic development.

Acetylation of sox2 induces its nuclear export in embryonic stem cells.

Embryonic stem (ES) cells require a coordinated network of transcription factors to maintain pluripotency or trigger lineage specific differentiation. Central to these processes are the proteins Oct4, Nanog, and Sox2. Although the transcriptional targets of these factors have been extensively studied, very little is known about how the proteins themselves are regulated, especially at the post-translational level. Post-translational modifications are well documented to have broad effects on protein stability, activity, and cellular distribution. Here, we identify a key lysine residue in the nuclear export signal of Sox2 that is acetylated, and demonstrate that blocking acetylation at this site retains Sox2 in the nucleus and sustains expression of its target genes under hyperacetylation or differentiation conditions. Mimicking acetylation at this site promotes association of Sox2 with the nuclear export machinery. In addition, increased cellular acetylation leads to reduction in Sox2 levels by ubiquitination and proteasomal degradation, thus abrogating its ability to drive transcription of its target genes. Acetylation-mediated nuclear export may be a commonly used regulatory mechanism for many Sox family members, as this lysine is conserved across species and in orthologous proteins.

HDAC4 Controls Muscle Homeostasis through Deacetylation of Myosin Heavy Chain, PGC-1α, and Hsc70.

HDAC4, a class IIa histone deacetylase, is upregulated in skeletal muscle in response to denervation-induced atrophy. When HDAC4 is deleted postnatally, mice are partially protected from denervation. Despite the name "histone" deacetylase, HDAC4 demonstrably deacetylates cytosolic and non-histone nuclear proteins. We developed potent and selective class IIa HDAC inhibitors. Using these tools and genetic knockdown, we identified three previously unidentified substrates of HDAC4: myosin heavy chain, peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1α), and heat shock cognate 71 kDa protein (Hsc70). HDAC4 inhibition almost completely prevented denervation-induced loss of myosin heavy chain isoforms and blocked the action of their E3 ligase, MuRF1. PGC-1α directly interacts with class IIa HDACs; selective inhibitors increased PGC-1α protein in muscles. Hsc70 deacetylation by HDAC4 affects its chaperone activity. Through these endogenous HDAC4 substrates, we identified several muscle metabolic pathways that are regulated by class IIa HDACs, opening up new therapeutic options to treat skeletal muscle disorders and potentially other disease where these specific pathways are affected.

Dissimilarity in the reductive unfolding pathways of two ribonuclease homologues.

Using DTT(red) as the reducing agent, the kinetics of the reductive unfolding of onconase, a frog ribonuclease, has been examined. An intermediate containing three disulfides, Ir, that is formed rapidly in the reductive pathway, is more resistant to further reduction than the parent molecule, indicating that the remaining disulfides in onconase are less accessible to DTT(red). Disulfide-bond mapping of Ir indicated that it is a single species lacking the (30-75) disulfide bond. The reductive unfolding pattern of onconase is consistent with an analysis of the exposed surface area of the cysteine sulfur atoms in the (30-75) disulfide bond, which reveals that these atoms are about four- and sevenfold, respectively, more exposed than those in the next two maximally exposed disulfides. By contrast, in the reductive unfolding of the homologue, RNase A, there are two intermediates, arising from the reduction of the (40-95) and (65-72) disulfide bonds, which takes place in parallel, and on a much longer time-scale, compared to the initial reduction of onconase; this behavior is consistent with the almost equally exposed surface areas of the cysteine sulfur atoms that form the (40-95) and (65-72) disulfide bonds in RNase A and the fourfold more exposed cysteine sulfur atoms of the (30-75) disulfide bond in onconase. Analysis and in silico mutation of the residues around the (40-95) disulfide bond in RNase A, which is analogous to the (30-75) disulfide bond of onconase, reveal that the side-chain of tyrosine 92 of RNase A, a highly conserved residue among mammalian pancreatic ribonucleases, lies atop the (40-95) disulfide bond, resulting in a shielding of the corresponding sulfur atoms from the solvent; such burial of the (30-75) sulfur atoms is absent from onconase, due to the replacement of Tyr92 by Arg73, which is situated away from the (30-75) disulfide bond and into the solvent, resulting in the large exposed surface-area of the cysteine sulfur atoms forming this bond. Removal of Tyr92 from RNase A resulted in the relatively rapid reduction of the mutant to form a single intermediate (des [40-95] Y92A), i.e. it resulted in an onconase-like reductive unfolding behavior. The reduction of the P93A mutant of RNase A proceeds through a single intermediate, the des [40-95] P93A species, as in onconase. Although mutation of Pro93 to Ala does not increase the exposed surface area of the (40-95) cysteine sulfur atoms, structural analysis of the mutant reveals that there is greater flexibility in the (40-95) disulfide bond compared to the (65-72) disulfide bond that may make the (40-95) disulfide bond much easier to expose, consistent with the reductive unfolding pathway and kinetics of P93A. Mutation of Tyr92 to Phe92 in RNase A has no effect on its reductive unfolding pathway, suggesting that the hydrogen bond between the hydroxyl group of Tyr92 and the carbonyl group of Lys37 has no impact on the local unfolding free energy required to expose the (40-95) disulfide bond. Thus, these data shed light on the differences between the reductive unfolding pathways of the two homologous proteins and provide a structural basis for the origin of this difference.

Simultaneous kinetic characterization of multiple protein forms by top down mass spectrometry.

Top down mass spectrometry, using a Fourier transform instrument, has unique capabilities for biomolecule kinetic studies, in that the concentration of large molecules in a reaction mixture can be monitored simultaneously from its mass spectrum produced by electrospray ionization. This is demonstrated with enzyme modifications occurring in the biosynthesis of the thiazole moiety of thiamin phosphate. The formation rate of ThiS-thiocarboxylate from ThiS was determined from the relative abundance of the corresponding m/z 10162 and 10146 isotopic peak clusters for all the observable charge states in the mass spectra measured at different reaction times. Even without measuring standard ionization efficiencies, the rate and precision of 0.018 +/- 0.004 min(-1) agree well with the 0.027 +/- 0.003 min(-1) obtained with a radiochemical assay, which requires a separate derivatization step. To illustrate the simultaneous characterization of the reaction kinetics of a native enzyme and its mutant, the imine formation rate of ThiG and its substrate DXP was compared between the native protein (M(r) = 26803.9) and its E98A (M(r) = 26745.9) or D182A (M(r) = 26759.9) mutant in the same reaction mixture. The kinetic data show clearly that neither the E98 nor the D182 residues participate in the imine formation. The high resolution and MS/MS capabilities of FTMS should make possible the extension of this kinetics approach to far more complicated systems, such as simultaneous monitoring of 24 native, intermediate, and reduced forms in the reductive unfolding of a mixture of ribonuclease A and the five isoforms of ribonuclease B. Stable intermediates with different SS bonding (same molecular weight) can be differentiated by MS/MS, while molecular ions differing by only 2 Da are distinguished clearly by synthesizing isotopically depleted proteins.

The biosynthesis of the thiazole phosphate moiety of thiamin: the sulfur transfer mediated by the sulfur carrier protein ThiS.

Thiamin-pyrophosphate is an essential cofactor in all living systems. The biosynthesis of both the thiazole and the pyrimidine moieties of this cofactor involves new biosynthetic chemistry. Thiazole-phosphate synthase (ThiG) catalyses the formation of the thiazole moiety of thiamin-pyrophosphate from 1-deoxy-D-xylulose-5-phosphate (DXP), dehydroglycine and the sulfur carrier protein (ThiS), modified on its carboxy terminus as a thiocarboxylate (ThiS-thiocarboxylate). Thiazole biosynthesis is initiated by the formation of a ThiG/DXP imine, which then tautomerizes to an amino-ketone. In this paper we study the sulfur transfer from ThiS-thiocarboxylate to this amino-ketone and trap a new thioenolate intermediate. Surprisingly, thiazole formation results in the replacement of the ThiS-thiocarboxylate sulfur with an oxygen from DXP and not from the buffer, as shown by electrospray ionization Fourier transform mass spectrometry (ESI-FTMS) using (18)O labeling of the 13C-, 15N-depleted protein. These observations further clarify the mechanism of the complex thiazole biosynthesis in bacteria.

Simultaneous characterization of the reductive unfolding pathways of RNase B isoforms by top-down mass spectrometry.

A novel method for characterization of the simultaneous reductive unfolding pathways of five isoforms of bovine pancreatic ribonuclease B (RNase B) is demonstrated. The results indicate that each isoform unfolds reductively through two three-disulfide-containing structured intermediates before proceeding to the fully reduced form, as in the reductive unfolding pathways of the A variant lacking the carbohydrate chain. The rates of reduction of bovine pancreatic ribonuclease A (RNase A) and RNase B and the formation and consumption of their reductive intermediates are identical, indicating that the unfolding events necessary to expose disulfide bonds for reduction are not affected by the oligosaccharide. The method utilizes top-down mass spectrometry and a naturally occurring tag on the protein, viz. the carbohydrate moiety, to obtain unfolding information of an ensemble of protein isoforms and is a generally applicable methodological advance for conducting folding studies on mixtures of different proteins.

RAF inhibitors activate the MAPK pathway by relieving inhibitory autophosphorylation.

ATP competitive inhibitors of the BRAF(V600E) oncogene paradoxically activate downstream signaling in cells bearing wild-type BRAF (BRAF(WT)). In this study, we investigate the biochemical mechanism of wild-type RAF (RAF(WT)) activation by multiple catalytic inhibitors using kinetic analysis of purified BRAF(V600E) and RAF(WT) enzymes. We show that activation of RAF(WT) is ATP dependent and directly linked to RAF kinase activity. These data support a mechanism involving inhibitory autophosphorylation of RAF's phosphate-binding loop that, when disrupted either through pharmacologic or genetic alterations, results in activation of RAF and the mitogen-activated protein kinase (MAPK) pathway. This mechanism accounts not only for compound-mediated activation of the MAPK pathway in BRAF(WT) cells but also offers a biochemical mechanism for BRAF oncogenesis.

A systematic screen for CDK4/6 substrates links FOXM1 phosphorylation to senescence suppression in cancer cells.

Cyclin D-dependent kinases (CDK4 and CDK6) are positive regulators of cell cycle entry and they are overactive in the majority of human cancers. However, it is currently not completely understood by which cellular mechanisms CDK4/6 promote tumorigenesis, largely due to the limited number of identified substrates. Here we performed a systematic screen for substrates of cyclin D1-CDK4 and cyclin D3-CDK6. We identified the Forkhead Box M1 (FOXM1) transcription factor as a common critical phosphorylation target. CDK4/6 stabilize and activate FOXM1, thereby maintain expression of G1/S phase genes, suppress the levels of reactive oxygen species (ROS), and protect cancer cells from senescence. Melanoma cells, unlike melanocytes, are highly reliant on CDK4/6-mediated senescence suppression, which makes them particularly susceptible to CDK4/6 inhibition.