Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

University of Nottingham

Activation of signalling by fibroblast growth factor receptor leads to phosphorylation of the signalling attenuator human Sprouty 2 (hSpry2) on residue Y55. This event requires the presence of the signalling adaptor fibroblast growth factor receptor substrate 2 (FRS2). The phosphorylation of hSpry2 is therefore mediated by an intermediate kinase. Using a SRC family kinase-specific inhibitor and mutant cells, we show that hSpry2 is a direct substrate for SRC family kinases, including SRC itself. Activation of SRC via fibroblast growth factor signalling is dependent upon FRS2 and fibroblast growth factor receptor kinase activity. SRC forms a complex with hSpry2 and this interaction is enhanced by hSpry2 phosphorylation. Phosphorylation of hSpry2 is required for hSpry2 to inhibit activation of the extracellular signal-regulated kinase pathway. These results show that recruitment of SRC to FRS2 leads to activation of signal attenuation pathways.

FRS2-dependent SRC activation is required for fibroblast growth factor receptor-induced phosphorylation of Sprouty and suppression of ERK activity.

Within the mammalian genome, there are many multimember gene families that encode membrane proteins with extracellular leucine rich repeats which are thought to act as cell adhesion or signalling molecules. We previously showed that the members of the NLRR gene family are expressed in a developmentally restricted manner in the mouse with NLRR-1 being expressed in the developing myotome. The FLRT gene family shows a similar genomic layout and predicted protein secondary structure to the NLRRs so we analysed expression of the three FLRT genes during mouse development. FLRTs are glycosylated membrane proteins expressed at the cell surface which localise in a homophilic manner to cell-cell contacts expressing the focal adhesion marker vinculin. Each member of the FLRT family has a distinct, highly regulated expression pattern, as was seen for the NLRR family. FLRT3 has a provocative expression pattern during somite development being expressed in regions of the somite where muscle precursor cells migrate from the dermomyotome and move into the myotome, and later in myotomal precursors destined to migrate towards their final destination, for example, those that form the ventral body wall. FLRT3 is also expressed at the midbrain/hindbrain boundary and in the apical ectodermal ridge, regions where FGF signalling is known to be important, suggesting that the role for FLRT3 in FGF signalling identified in Xenopus is conserved in mammals. FLRT1 is expressed at brain compartmental boundaries and FLRT2 is expressed in a subset of the sclerotome, adjacent to the region that forms the syndetome, suggesting that interaction with FGF signalling may be a general property of FLRT proteins. We confirmed this by showing that all FLRTs can interact with FGFR1 and FLRTs can be induced by the activation of FGF signalling by FGF-2. We conclude that FLRT proteins act as regulators of FGF signalling, being induced by the signal and then able to interact with the signalling receptor, in many tissues during mouse embryogenesis. This process may, in part, be dependent on homophilic intercellular interactions between FLRT molecules.

Regulated expression of FLRT genes implies a functional role in the regulation of FGF signalling during mouse development.

Fibroblast growth factor receptors (FGFRs) are a family of four transmembrane (TM) receptor tyrosine kinases (RTKs) which bind to a large family of fibroblast growth factor (FGF) ligands with varying affinity and specificity. FGFR signaling regulates many physiological and pathological processes in development and tissue homeostasis. Understanding FGFR signaling processes requires the identification of partner proteins which regulate receptor function and biological outputs. In this study, we employ an epitope-tagged, covalently dimerized, and constitutively activated form of FGFR1 to identify potential protein partners by MS. By this approach, we sample candidate FGFR effectors throughout the life history of the receptor. Functional classification of the partners identified revealed specific subclasses involved in protein biosynthesis and folding; structural and regulatory components of the cytoskeleton; known signaling effectors and small GTPases implicated in endocytosis and vesicular trafficking. The kinase dependency of the interaction was determined for a subset of previously unrecognized partners by coimmunoprecipitation, Western blotting, and immunocytochemistry. From this group, the small GTPase Rab5 was selected for functional interrogation. We show that short hairpin (sh) RNA-mediated depletion of Rab5 attenuates the activation of the extracellular-regulated kinase (ERK) 1/2 pathway by FGFR signaling. The strategic approach adopted in this study has revealed bona fide novel effectors of the FGFR signaling pathway.

Protein partners in the life history of activated fibroblast growth factor receptors.

The transmembrane receptor 'ROR2' resembles members of the receptor tyrosine kinase family of signalling receptors in sequence but its' signal transduction mechanisms remain enigmatic. This problem has particular importance because mutations in ROR2 are associated with two human skeletal dysmorphology syndromes, recessive Robinow Syndrome (RS) and dominant acting Brachydactyly type B (BDB). Here we show, using a constitutive dimerisation approach, that ROR2 exhibits dimerisation-induced tyrosine kinase activity and the ROR2 C-terminal domain, which is deleted in BDB, is required for recruitment and activation of the non-receptor tyrosine kinase Src. Native ROR2 phosphorylation is induced by the ligand Wnt5a and is blocked by pharmacological inhibition of Src kinase activity. Eight sites of Src-mediated ROR2 phosphorylation have been identified by mass spectrometry. Activation via tyrosine phosphorylation of ROR2 receptor leads to its internalisation into Rab5 positive endosomes. These findings show that BDB mutant receptors are defective in kinase activation as a result of failure to recruit Src.

The deleted in brachydactyly B domain of ROR2 is required for receptor activation by recruitment of Src.

Fibronectin leucine rich transmembrane (FLRT) proteins have dual properties as regulators of cell adhesion and potentiators of fibroblast growth factor (FGF) mediated signalling. The mechanism by which the latter is achieved is still unknown and is the subject of this investigation.

Critical role of FLRT1 phosphorylation in the interdependent regulation of FLRT1 function and FGF receptor signalling.

AS (Apert syndrome) is a congenital disease composed of skeletal, visceral and neural abnormalities, caused by dominant-acting mutations in FGFR2 [FGF (fibroblast growth factor) receptor 2]. Multiple FGFR2 splice variants are generated through alternative splicing, including PTC (premature termination codon)-containing transcripts that are normally eliminated via the NMD (nonsense-mediated decay) pathway. We have discovered that a soluble truncated FGFR2 molecule encoded by a PTC-containing transcript is up-regulated and persists in tissues of an AS mouse model. We have termed this IIIa-TM as it arises from aberrant splicing of FGFR2 exon 7 (IIIa) into exon 10 [TM (transmembrane domain)]. IIIa-TM is glycosylated and can modulate the binding of FGF1 to FGFR2 molecules in BIAcore-binding assays. We also show that IIIa-TM can negatively regulate FGF signalling in vitro and in vivo. AS phenotypes are thought to result from gain-of-FGFR2 signalling, but our findings suggest that IIIa-TM can contribute to these through a loss-of-FGFR2 function mechanism. Moreover, our findings raise the interesting possibility that FGFR2 signalling may be a regulator of the NMD pathway.

Identification and characterization of an inhibitory fibroblast growth factor receptor 2 (FGFR2) molecule, up-regulated in an Apert Syndrome mouse model.

Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae are major bacterial agents of meningitis. They each bind the 37/67-kDa laminin receptor (LamR) via the surface protein adhesins: meningococcal PilQ and PorA, H. influenzae OmpP2 and pneumococcal CbpA. We have previously reported that a surface-exposed loop of the R2 domain of CbpA mediates LamR-binding. Here we have identified the LamR-binding regions of PorA and OmpP2. Using truncated recombinant proteins we show that binding is dependent on amino acids 171-240 and 91-99 of PorA and OmpP2, respectively, which are predicted to localize to the fourth and second surface-exposed loops, respectively, of these proteins. Synthetic peptides corresponding to the loops bound LamR and could block LamR-binding to bacterial ligands in a dose dependant manner. Meningococci expressing PorA lacking the apex of loop 4 and H. influenzae expressing OmpP2 lacking the apex of loop 2 showed significantly reduced LamR binding. Since both loops are hyper-variable, our data may suggest a molecular basis for the range of LamR-binding capabilities previously reported among different meningococcal and H. influenzae strains.

Mapping the laminin receptor binding domains of Neisseria meningitidis PorA and Haemophilus influenzae OmpP2.

5-&#x7532;&#x57FA;&#x80DE;&#x5627;&#x5576; (5-mC) &#x662F;&#x8868;&#x89C2;&#x9057;&#x4F20;&#x4FEE;&#x9970;&#x4E0E;&#x57FA;&#x56E0;&#x6291;&#x5236;&#x6709;&#x5173;&#x3002;&#x6700;&#x8FD1;&#x7684;&#x7814;&#x7A76;&#x8868;&#x660E;&#xFF0C;5 mC &#x53EF;&#x4EE5;&#x662F;&#x9176;&#x6C27;&#x5316;&#x6210; 5 hydroxymethylcytosine &#x548C;&#x8FDB;&#x4E00;&#x6B65;&#x7EB3;&#x5165; 5-formylcytosine &#xFF08;5-fC) &#x548C; 5-carboxylcytsine &#xFF08;5-caC)&#x3002;5 caC &#x5DF2;&#x88AB;&#x53D1;&#x73B0;&#x5728;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;&#x5728;&#x5C0F;&#x9F20;&#x65E9;&#x671F;&#x80DA;&#x80CE;&#x4E2D;&#xFF0C;&#x4F46;&#x6B64;&#x4FEE;&#x6539;&#x6CA1;&#x6709;&#x63A2;&#x6D4B;&#x6C34;&#x5E73;&#x8FC4;&#x4ECA;&#x62A5;&#x544A;&#x4F9B;&#x4F53;&#x7EC6;&#x80DE;&#x7EC4;&#x7EC7;&#x3002;&#x800C;&#x8BA4;&#x4E3A;&#x8BE5; 5 caC &#x53EF;&#x4EE5;&#x4F5C;&#x4E3A;&#x4E2D;&#x95F4;&#x79EF;&#x6781;&#x53BB;&#x7532;&#x57FA;&#x7684;&#x8FC7;&#x7A0B;&#x4E2D;&#xFF0C;&#x8FD9;&#x79CD;&#x5F62;&#x5F0F;&#x7684;&#x6539;&#x6027;&#x80DE;&#x5627;&#x5576;&#x7684;&#x4F5C;&#x7528;&#x4ECD;&#x7136;&#x4E0D;&#x660E;&#x6717;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x663E;&#x793A;&#x8BE5; 5 caC &#x662F; immunochemically &#x63A2;&#x6D4B;&#x8788;&#x5375;&#x5DE2;&#x4F53;&#x7EC6;&#x80DE;&#x4E2D;&#x3002;&#x6211;&#x4EEC;&#x8BC1;&#x660E; 5 hmC &#x548C; 5 caC &#x5DF2;&#x672C;&#x5730;&#x5316;&#x5230;&#x7C7B;&#x4F3C;&#x7684;&#x7A7A;&#x95F4;&#x5206;&#x5E03;&#x6A21;&#x5F0F;&#x4E0E;&#x8788;&#x6EE4;&#x6CE1;&#x7EC6;&#x80DE;&#x7684;&#x7EC6;&#x80DE;&#x6838;&#x67D3;&#x8272;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x7814;&#x7A76;&#x7ED3;&#x679C;&#x8868;&#x660E;&#x8FD9; 5 carboxylcytosine &#x53EF;&#x80FD;&#x5728;&#x67D0;&#x4E9B;&#x751F;&#x7269;&#x7684;&#x4E0A;&#x4E0B;&#x6587;&#x4E2D;&#x53D1;&#x6325;&#x72EC;&#x7279;&#x7684;&#x529F;&#x80FD;&#x4F5C;&#x7528;&#x3002;

5 Carboxylcytosine &#x4EC5;&#x5C40;&#x9650;&#x4E8E; euchromatic &#x5730;&#x533A;&#x8788;&#x5375;&#x5DE2;&#x4E2D;&#x5375;&#x6CE1;&#x7EC6;&#x80DE;&#x7684;&#x7EC6;&#x80DE;&#x6838;&#x4E2D;&#x3002;

5-Methylcytosine (5-mC) is an epigenetic modification associated with gene repression. Recent studies demonstrated that 5-mC can be enzymatically oxidised into 5-hydroxymethylcytosine and further into 5-formylcytosine (5-fC) and 5-carboxylcytsine (5-caC). 5-caC has been found in embryonic stem cells and in mouse pre-implantation embryos but no detectable levels of this modification have been reported for somatic tissues to date. Whereas it has been suggested that 5-caC can serve as an intermediate in the process of active demethylation, the function of this form of modified cytosine remains obscure. Here we show that 5-caC is immunochemically detectable in somatic cells of axolotl ovary. We demonstrate that both 5-hmC and 5-caC are localized to the euchromatin in the nuclei of axolotl follicular cells with similar patterns of spatial distribution. Our results suggest that 5-carboxylcytosine may play a distinct functional role in certain biological contexts.

5-Carboxylcytosine is localized to euchromatic regions in the nuclei of follicular cells in axolotl ovary.

5-M&eacute;thylcytosine (5-mC) est une modification &eacute;pig&eacute;n&eacute;tique associ&eacute;e de r&eacute;pression g&eacute;nique. &Eacute;tudes r&eacute;centes ont d&eacute;montr&eacute; que mC-5 peut &ecirc;tre enzymatiquement oxyd&eacute; dans 5-hydroxym&eacute;thylcytosine et plus loin dans le 5-formylcytosine (5-fC) et 5-carboxylcytsine (5-caC). 5-ACC a &eacute;t&eacute; trouv&eacute; dans les cellules souches embryonnaires et chez les embryons pr&eacute;implantatoires, mais aucun des niveaux d&eacute;tectables de cette modification n&#39;ont &eacute;t&eacute; rapport&eacute;s pour les tissus somatiques &agrave; ce jour. Consid&eacute;rant qu&#39;il a &eacute;t&eacute; sugg&eacute;r&eacute; que 5-caC peut servir d&#39;interm&eacute;diaire dans le processus de d&eacute;m&eacute;thylation active, la fonction de cette forme de cytosine mis &agrave; jour le reste obscure. Nous montrons ici que 5-caC est immunochimiquement d&eacute;tectable dans les cellules somatiques de l&#39;ovaire de l&#39;axolotl. Nous d&eacute;montrons que les hmC-5 et 5-caC sont localis&eacute;es &agrave; l&#39;euchromatine dans les noyaux des cellules folliculaires de l&#39;axolotl avec des profils similaires de r&eacute;partition spatiale. Nos r&eacute;sultats sugg&egrave;rent que 5-carboxylcytosine peut jouer un r&ocirc;le fonctionnel distinct dans certains contextes biologiques.

5-Carboxylcytosine est localis&eacute;e dans des r&eacute;gions euchromatiques dans les noyaux des cellules folliculaires de l&#39;ovaire de l&#39;axolotl.

5-Methylcytosine (5-mC) ist eine epigenetische Modifikation gen Unterdr&uuml;ckung zugeordnet. J&uuml;ngste Studien gezeigt, dass 5-mC enzymatisch oxidiert in 5-Hydroxymethylcytosine und weiter in 5-Formylcytosine (5-fC) und 5-Carboxylcytsine (5-CaC) sein kann. 5-CaC in embryonale Stammzellen und Maus Pr&auml;implantations Embryonen gefunden wurde, aber keine nachweisbaren Niveaus dieser &Auml;nderung wurden bisher f&uuml;r somatische Gewebe beschrieben. W&auml;hrend es vorgeschlagen hat, dass diese 5-CaC als Zwischenprodukt in den Prozess der aktiven Demethylierung dienen kann, bleibt die Funktion dieser Form der modifizierten Cytosin obskuren. Hier zeigen wir, dass diese 5-CaC an somatischen Zellen der Eierst&ouml;cke Axolotl immunochemically nachweisbar ist. Wir zeigen, dass 5-HmC und 5-CaC an das Euchromatin in den Kernen der Axolotl follikul&auml;ren Zellen mit &auml;hnlichen Mustern der r&auml;umlichen Verteilung lokalisiert werden. Unsere Ergebnisse legen nahe, dass dieser 5-Carboxylcytosine in einem bestimmten biologischen Kontext eine unterschiedliche funktionale Rolle spielen kann.

5-Carboxylcytosine ist Histons Regionen in den Kernen der follikul&auml;ren Zellen in Axolotl Eierstock lokalisiert.

5-Metilcitosina (5-mC) &egrave; una modificazione epigenetica associata repressione genica. Recenti studi hanno dimostrato che 5-mC pu&ograve; essere enzimaticamente ossidato in 5-idrossimetilcitosina e ulteriormente in 5-formylcytosine (5-fC) e 5-carboxylcytsine (5-caC). 5-caC &egrave; stato trovato nelle cellule staminali embrionali e in preimpianto embrioni del mouse ma livelli rilevabili di questa modifica non sono stati segnalati per tessuti somatici per Data. Considerando che &egrave; stato suggerito che 5-caC pu&ograve; servire come un intermedio nel processo di demetilazione attiva, la funzione di questa forma di citosina modificato rimane oscura. Qui vi mostriamo che quello 5-caC &egrave; immunochemically rilevabile nelle cellule somatiche dell&#39;ovaia axolotl. Dimostriamo che sia hmC-5 e 5-caC sono localizzate a euchromatin nei nuclei delle cellule follicolari axolotl con simili modelli di distribuzione spaziale. I nostri risultati suggeriscono che 5-carboxylcytosine possa avere un ruolo funzionale distinto in determinati contesti biologici.

5-Carboxylcytosine &egrave; localizzata alle regioni eucromatina nei nuclei delle cellule follicolari nell&#39;ovaio axolotl.

5-&#x30E1;&#x30C1;&#x30EB;&#x30B7;&#x30C8;&#x30B7;&#x30F3; (5-mC) &#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x6291;&#x5236;&#x3068;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x3001;&#x30A8;&#x30D4;&#x30B8;&#x30A7;&#x30CD;&#x30C6;&#x30A3;&#x30C3;&#x30AF;&#x306A;&#x4FEE;&#x98FE;&#x3067;&#x3059;&#x3002;&#x6700;&#x8FD1;&#x306E;&#x8ABF;&#x67FB;&#x306F;&#x305D;&#x306E; 5 mC &#x9175;&#x7D20;&#x306B;&#x3088;&#x3063;&#x3066; 5-&#x30D2;&#x30C9;&#x30ED;&#x30AD;&#x30B7;&#x30E1;&#x30C1;&#x30EB;&#x30B7;&#x30C8;&#x30B7;&#x30F3;&#x306B;&#x9178;&#x5316;&#x304A;&#x3088;&#x3073; 5-formylcytosine &#xFF08;5-fC&#xFF09; &#x3068; 5-carboxylcytsine &#xFF08;5-caC) &#x306B;&#x3055;&#x3089;&#x306B;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3092;&#x793A;&#x3057;&#x305F;&#x3002;5 caC &#x306F;&#x3001;&#x80DA;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x3068;&#x30DE;&#x30A6;&#x30B9;&#x7740;&#x5E8A;&#x524D;&#x80DA;&#x767A;&#x898B;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x304C;&#x3001;&#x3053;&#x306E;&#x5909;&#x66F4;&#x306E;&#x691C;&#x51FA;&#x30EC;&#x30D9;&#x30EB;&#x4F53;&#x7D30;&#x80DE;&#x7D44;&#x7E54;&#x306E;&#x65E5;&#x4ED8;&#x306B;&#x5831;&#x544A;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x305B;&#x3093;&#x3002;&#x305D;&#x306E; 5 caC &#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x8131;&#x30E1;&#x30C1;&#x30EB;&#x5316;&#x306E;&#x904E;&#x7A0B;&#x3067;&#x4E2D;&#x9593;&#x4F53;&#x3068;&#x3057;&#x3066;&#x4F7F;&#x7528;&#x3067;&#x304D;&#x308B;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x306B;&#x5BFE;&#x3057;&#x3001;&#x5909;&#x66F4;&#x30B7;&#x30C8;&#x30B7;&#x30F3;&#x306E;&#x3053;&#x306E;&#x5F62;&#x5F0F;&#x306E;&#x95A2;&#x6570;&#x306F;&#x3042;&#x3044;&#x307E;&#x3044;&#x306A;&#x307E;&#x307E;&#x306B;&#x306A;&#x308A;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x305D;&#x306E; 5 caC &#x304C;&#x4E21;&#x30A2;&#x30DB;&#x30ED;&#x30FC;&#x30C8;&#x30EB;&#x5375;&#x5DE3;&#x306E;&#x4F53;&#x7D30;&#x80DE;&#x306E;&#x691C;&#x51FA;&#x3067;&#x3042;&#x308B;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; 5 hmC &#x3068; 5 caC &#x30E6;&#x30FC;&#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3; &#x30A2;&#x30DB;&#x30ED;&#x30FC;&#x30C8;&#x30EB;&#x6FFE;&#x80DE;&#x7D30;&#x80DE;&#x306E;&#x6838;&#x306E;&#x7A7A;&#x9593;&#x5206;&#x5E03;&#x306E;&#x985E;&#x4F3C;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x306B;&#x30ED;&#x30FC;&#x30AB;&#x30E9;&#x30A4;&#x30BA;&#x3092;&#x30C7;&#x30E2;&#x30F3;&#x30B9;&#x30C8;&#x30EC;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x3057;&#x307E;&#x3059;&#x3002;&#x305D;&#x306E; 5 carboxylcytosine &#x7279;&#x5B9A;&#x751F;&#x7269;&#x306E;&#x30B3;&#x30F3;&#x30C6;&#x30AD;&#x30B9;&#x30C8;&#x3067;&#x660E;&#x78BA;&#x306A;&#x6A5F;&#x80FD;&#x7684;&#x5F79;&#x5272;&#x3092;&#x518D;&#x751F;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;

5 Carboxylcytosine &#x30E6;&#x30FC;&#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3;&#x9818;&#x57DF;&#x30A2;&#x30DB;&#x30ED;&#x30FC;&#x30C8;&#x30EB;&#x5375;&#x5DE3;&#x306B;&#x304A;&#x3051;&#x308B;&#x5375;&#x80DE;&#x7D30;&#x80DE;&#x306E;&#x6838;&#x306B;&#x30ED;&#x30FC;&#x30AB;&#x30E9;&#x30A4;&#x30BA;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

5-Methylcytosine (5-&#xC5E0;&#xC528;) &#xAD00;&#xB828; &#xC720;&#xC804;&#xC790; &#xC5B5;&#xC81C; &#xB41C; epigenetic &#xC218;&#xC815;&#xC785;&#xB2C8;&#xB2E4;. &#xCD5C;&#xADFC; &#xD559;&#xBB38;&#xC740; &#xADF8; 5 mC enzymatically 5 hydroxymethylcytosine&#xC5D0; &#xC2DC;&#xACC4;&#xC640; 5-formylcytosine (5-fC)&#xC640; 5-carboxylcytsine (5-caC)&#xC5D0; &#xCD94;&#xAC00; &#xB420; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xC124;&#xBA85; &#xD588;&#xB2E4;. 5-caC &#xB9C8;&#xC6B0;&#xC2A4; pre-implantation &#xBC30;&#xC544; &#xBC0F; &#xBC30;&#xC544; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xBC1C;&#xACAC; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4; &#xC788;&#xC9C0;&#xB9CC;&#xC774; &#xC218;&#xC815; &#xC5C6;&#xC74C; &#xAC10;&#xC9C0; &#xB808;&#xBCA8;&#xAE4C;&#xC9C0; &#xCCB4;&#xC138;&#xD3EC; &#xC870;&#xC9C1;&#xC5D0; &#xB300; &#xD55C; &#xBCF4;&#xACE0; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xADF8; 5-caC &#xD65C;&#xC131; demethylation &#xACFC;&#xC815; &#xC911;&#xAC04;&#xC774; &#xB420; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xC81C;&#xC548; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;, &#xBC18;&#xBA74;&#xC774; &#xD615;&#xD0DC;&#xC758; &#xC218;&#xC815;&#xB41C; &#xC2DC; &#xD568;&#xC218; &#xC560;&#xB9E4; &#xD55C; &#xB0A8;&#xC544; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC;&#xAC00; &#xADF8; 5 Cac&#xB294; immunochemically axolotl &#xB09C;&#xC18C;&#xC758; &#xCCB4;&#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xAC10;&#xC9C0; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. 5 HmC &#xBC0F; 5-caC &#xACF5;&#xAC04; &#xBD84;&#xD3EC;&#xC758; &#xBE44;&#xC2B7;&#xD55C; &#xD328;&#xD134;&#xC744; &#xAC00;&#xC9C4; axolotl follicular &#xC138;&#xD3EC;&#xC758; &#xD575;&#xC5D0; &#xC788;&#xB294; euchromatin&#xC5D0; &#xC9C0;&#xC5ED;&#xD654; &#xB429;&#xB2C8;&#xB2E4; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xC758; &#xACB0;&#xACFC; 5-carboxylcytosine &#xC0DD;&#xBB3C; &#xD559;&#xC801; &#xD2B9;&#xC815; &#xCEE8;&#xD14D;&#xC2A4;&#xD2B8;&#xC5D0;&#xC11C; &#xBCC4;&#xAC1C; &#xAE30;&#xB2A5; &#xC5ED;&#xD560;&#xC744; &#xD560; &#xC218; &#xAC83;&#xC774; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4;.

5 Carboxylcytosine axolotl &#xB09C;&#xC18C;&#xC5D0; &#xC18C; &#xB0AD; &#xBAA8;&#xC591; &#xC138;&#xD3EC;&#xC758; &#xD575;&#xC5D0; &#xC788;&#xB294; euchromatic &#xC9C0;&#xC5ED;&#xC73C;&#xB85C; &#xC9C0;&#xC5ED;&#xD654; &#xB429;&#xB2C8;&#xB2E4;.

5-METILCITOSINA (5-mC) es una modificaci&oacute;n epigen&eacute;tica asociado con represi&oacute;n g&eacute;nica. Estudios recientes demostraron que 5-mC puede ser enzim&aacute;ticamente oxidado en 5-hidroximetilcitosina y m&aacute;s en 5-formylcytosine (5-fC) y 5-carboxylcytsine (5-caC). 5-caC se ha encontrado en las c&eacute;lulas madre embrionarias y en embriones de preimplantaci&oacute;n de rat&oacute;n pero no niveles detectables de esta modificaci&oacute;n se han divulgado para tejidos som&aacute;ticos hasta la fecha. Considerando que se ha sugerido que 5-caC puede servir como intermediario en el proceso de desmetilaci&oacute;n activa, la funci&oacute;n de esta forma de citosina modificado sigue siendo obscura. A continuaci&oacute;n os mostramos que eso 5-caC es inmunoqu&iacute;mica detectable en las c&eacute;lulas som&aacute;ticas del ovario de ajolote. Demostramos que tanto hmC-5 y 5-caC se localizan a la eucromatina en los n&uacute;cleos de las c&eacute;lulas foliculares de ajolote con similares patrones de distribuci&oacute;n espacial. Nuestros resultados sugieren que 5-carboxylcytosine puede jugar un papel funcional distinto en ciertos contextos biol&oacute;gicos.

5-Carboxylcytosine se localiza a regiones euchromatic en los n&uacute;cleos de las c&eacute;lulas foliculares en el ovario de ajolote.

Interactions between commensal pathogens and hosts are critical for disease development but the underlying mechanisms for switching between the commensal and virulent states are unknown. We show that the human pathogen Neisseria meningitidis, the leading cause of pyogenic meningitis, can modulate gene expression via uptake of host pro-inflammatory cytokines leading to increased virulence. This uptake is mediated by type IV pili (Tfp) and reliant on the PilT ATPase activity. Two Tfp subunits, PilE and PilQ, are identified as the ligands for TNF-α and IL-8 in a glycan-dependent manner, and their deletion results in decreased virulence and increased survival in a mouse model. We propose a novel mechanism by which pathogens use the twitching motility mode of the Tfp machinery for sensing and importing host elicitors, aligning with the inflamed environment and switching to the virulent state.

Pro-inflammatory cytokines can act as intracellular modulators of commensal bacterial virulence.

5-Methylcytosine (5mC) is an epigenetic modification involved in regulation of gene activity during differentiation. Tet dioxygenases oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be excised from DNA by thymine-DNA glycosylase (TDG) followed by regeneration of unmodified cytosine via the base excision repair pathway. Despite evidence that this mechanism is operative in embryonic stem cells, the role of TDG-dependent demethylation in differentiation and development is currently unclear. Here, we demonstrate that widespread oxidation of 5hmC to 5caC occurs in postimplantation mouse embryos. We show that 5fC and 5caC are transiently accumulated during lineage specification of neural stem cells (NSCs) in culture and in vivo. Moreover, 5caC is enriched at the cell-type-specific promoters during differentiation of NSCs, and TDG knockdown leads to increased 5fC/5caC levels in differentiating NSCs. Our data suggest that active demethylation contributes to epigenetic reprogramming determining lineage specification in embryonic brain.

Transient accumulation of 5-carboxylcytosine indicates involvement of active demethylation in lineage specification of neural stem cells.

The non-integrin laminin receptor (LAMR1/RPSA) and galectin-3 (Gal-3) are multi-functional host molecules with roles in diverse pathological processes, particularly of infectious or oncogenic origins. Using bimolecular fluorescence complementation and confocal imaging, we demonstrate that the two proteins homo- and heterodimerize, and that each isotype forms a distinct cell surface population. We present evidence that the 37 kDa form of LAMR1 (37LRP) is the precursor of the previously described 67 kDa laminin receptor (67LR), whereas the heterodimer represents an entity that is distinct from this molecule. Site-directed mutagenesis confirmed that the single cysteine (C(173)) of Gal-3 or lysine (K(166)) of LAMR1 are critical for heterodimerization. Recombinant Gal-3, expressed in normally Gal-3-deficient N2a cells, dimerized with endogenous LAMR1 and led to a significantly increased number of internalized bacteria (Neisseria meningitidis), confirming the role of Gal-3 in bacterial invasion. Contact-dependent cross-linking determined that, in common with LAMR1, Gal-3 binds the meningococcal secretin PilQ, in addition to the major pilin PilE. This study adds significant new mechanistic insights into the bacterial-host cell interaction by clarifying the nature, role and bacterial ligands of LAMR1 and Gal-3 isotypes during colonization.

Deciphering the complex three-way interaction between the non-integrin laminin receptor, galectin-3 and Neisseria meningitidis.

DNA methylation (5-methylcytosine (5mC)) patterns are often altered in cancers. Ten-eleven translocation (Tet) proteins oxidise 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). In addition to their presumptive specific biological roles, these oxidised forms of 5mC may serve as intermediates in demethylation process. According to several reports, 5hmC levels are strongly decreased in cancers; however, the distribution of 5fC and 5caC in malignant tissue has not been studied.

Lee Wheldon

Medical Molecular Sciences,
Centre for Biomolecular Sciences,
Medical Molecular Sciences, Centre for Biomolecular Sciences

University of Nottingham

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Lee Wheldon

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Medical Molecular Sciences,Centre for Biomolecular Sciences,Medical Molecular Sciences, Centre for Biomolecular Sciences

University of Nottingham

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Medical Molecular Sciences,
Centre for Biomolecular Sciences,
Medical Molecular Sciences, Centre for Biomolecular Sciences