The nine-amino-acid activation domain (9aaTAD) is defined by a short amino acid pattern including two hydrophobic regions (positions p3-4 and p6-7). The KIX domain of mediator transcription CBP interacts with the 9aaTAD domains of transcription factors MLL, E2A, NF-kB, and p53. In this study, we analyzed the 9aaTADs-KIX interactions by nuclear magnetic resonance. The positions of three KIX helixes α1-α2-α3 are influenced by sterically-associated hydrophobic I611, L628, and I660 residues that are exposed to solvent. The positions of two rigid KIX helixes α1 and α2 generate conditions for structural folding in the flexible KIX-L12-G2 regions localized between them. The three KIX I611, L628, and I660 residues interact with two 9aaTAD hydrophobic residues in positions p3 and p4 and together build a hydrophobic core of five residues (5R). Numerous residues in 9aaTAD position p3 and p4 could provide this interaction. Following binding of the 9aaTAD to KIX, the hydrophobic I611, L628, and I660 residues are no longer exposed to solvent and their position changes inside the hydrophobic core together with position of KIX α1-α2-α3 helixes. The new positions of the KIX helixes α1 and α2 allow the KIX-L12-G2 enhanced formation. The second hydrophobic region of the 9aaTAD (positions p6 and p7) provides strong binding with the KIX-L12-G2 region. Similarly, multiple residues in 9aaTAD position p6 and p7 could provide this interaction. In conclusion, both 9aaTAD regions p3, p4 and p6, p7 provide co-operative and highly universal binding to mediator KIX. The hydrophobic core 5R formation allows new positions of the rigid KIX α-helixes and enables the enhanced formation of the KIX-L12-G2 region. This contributes to free energy and is the key for the KIX-9aaTAD binding. Therefore, the 9aaTAD-KIX interactions do not operate under the rigid key-and-lock mechanism what explains the 9aaTAD natural variability.

• MeSH
• aminokyselinové motivy MeSH
• histonlysin-N-methyltransferasa chemie   metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• lidé MeSH
• nádorový supresorový protein p53 chemie   metabolismus   MeSH
• NF-kappa B chemie   metabolismus   MeSH
• protein vázající CREB chemie   metabolismus   MeSH
• protoonkogenní protein MLL chemie   metabolismus   MeSH
• transkripční faktory bHLH chemie   metabolismus   MeSH
• transkripční faktory chemie   metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Histone 3 K4 trimethylation (depositing H3K4me3 marks) is typically associated with active promoters yet paradoxically occurs at untranscribed domains. Research to delineate the mechanisms of targeting H3K4 methyltransferases is ongoing. The oocyte provides an attractive system to investigate these mechanisms, because extensive H3K4me3 acquisition occurs in nondividing cells. We developed low-input chromatin immunoprecipitation to interrogate H3K4me3, H3K27ac and H3K27me3 marks throughout oogenesis. In nongrowing oocytes, H3K4me3 was restricted to active promoters, but as oogenesis progressed, H3K4me3 accumulated in a transcription-independent manner and was targeted to intergenic regions, putative enhancers and silent H3K27me3-marked promoters. Ablation of the H3K4 methyltransferase gene Mll2 resulted in loss of transcription-independent H3K4 trimethylation but had limited effects on transcription-coupled H3K4 trimethylation or gene expression. Deletion of Dnmt3a and Dnmt3b showed that DNA methylation protects regions from acquiring H3K4me3. Our findings reveal two independent mechanisms of targeting H3K4me3 to genomic elements, with MLL2 recruited to unmethylated CpG-rich regions independently of transcription.

• MeSH
• chromatinová imunoprecipitace MeSH
• CpG ostrůvky MeSH
• genetická transkripce MeSH
• histonlysin-N-methyltransferasa chemie   MeSH
• histony chemie   MeSH
• Markovovy řetězce MeSH
• metylace DNA * MeSH
• multivariační analýza MeSH
• myší embryonální kmenové buňky cytologie   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• oocyty cytologie   MeSH
• oogeneze MeSH
• promotorové oblasti (genetika) MeSH
• protoonkogenní protein MLL chemie   MeSH
• sekvenční analýza RNA MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A number of transactivation domains for transcription factors including p53, E2A/HEB, MLL, cMyb, CREB, FOXO3, Gcn4, Oaf1 and Pdr1 have been reported to interact with the KIX domain of general transcriptional mediators CBP, p300 or MED15. Most of those factors belong to the already established Nine amino acid Transactivation Domain (9aaTAD) family. By using available structural data, we found binding analogy for the 9aaTAD in the MLL-KIX and also E2A/HEB-KIX complexes. We recognized two distinct TAD formations in the KIX complex. In the E2A/HEB-KIX complex, the leucine position is determined by the prolonged helical structure including the 9aaTAD and the leucine (long-helical TAD). However in the MLL-KIX complex, the equal position of 9aaTAD and proximal leucine is achieved differently by leucine-turn-helix structural architecture. Furthermore, the FOXO3-KIX complex shares structural analogy with the E2A-KIX complex in respect of both 9aaTAD and proximal leucine. Next, from (i) sequence alignment of the identified 9aaTADs in p53, E2A/HEB and MLL proteins and (ii) the resolved structure of the MLL-KIX and E2A/HEB-KIX complexes, we generated a plausible structural model for p53 that could be used also for other members of the 9aaTAD family. The position of 9aaTADs in Oaf1-, Pdr1- and Gcn4-MED15 KIX complexes and 9aaTAD composition are in good agreement with E2A, MLL, FOXO3 and p53. Analyses of structural data in this study define fundamental structural requirements and shed more light on the ambiguous 9aaTAD domain.

• MeSH
• aminokyselinové motivy MeSH
• databáze proteinů MeSH
• forkhead transkripční faktory chemie   metabolismus   MeSH
• interakční proteinové domény a motivy * MeSH
• molekulární konformace * MeSH
• molekulární modely * MeSH
• nádorový supresorový protein p53 chemie   metabolismus   MeSH
• protein vázající CREB chemie   metabolismus   MeSH
• protoonkogenní protein MLL chemie   MeSH
• sekvence aminokyselin MeSH
• transkripční faktory chemie   metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Mixed lineage leukemia (MLL) fusion-driven acute leukemias represent a genetically distinct subset of leukemias with poor prognosis. MLL forms a ternary complex with the lens epithelium-derived growth factor (LEDGF/p75) and MENIN. LEDGF/p75, a chromatin reader recognizing H3K36me3 marks, contributes to the association of the MLL multiprotein complex to chromatin. Formation of this complex is critical for the development of MLL leukemia. Available X-ray data represent only a partial structure of the LEDGF/p75-MLL-MENIN complex. Using nuclear magnetic resonance spectroscopy, we identified an additional LEDGF/p75-MLL interface, which overlaps with the binding site of known LEDGF/p75 interactors-HIV-1 integrase, PogZ, and JPO2. Binding of these proteins or MLL to LEDGF/p75 is mutually exclusive. The resolved structure, as well as mutational analysis, shows that the interaction is primarily sustained via two aromatic residues of MLL (F148 and F151). Colony-forming assays in MLL-AF9(+) leukemic cells expressing MLL interaction-defective LEDGF/p75 mutants revealed that this interaction is essential for transformation. Finally, we show that the clonogenic growth of primary murine MLL-AF9-expressing leukemic blasts is selectively impaired upon overexpression of a LEDGF/p75-binding cyclic peptide CP65, originally developed to inhibit the LEDGF/p75-HIV-1 integrase interaction. The newly defined protein-protein interface therefore represents a new target for the development of therapeutics against LEDGF/p75-dependent MLL fusion-driven leukemic disorders. Cancer Res; 74(18); 5139-51. ©2014 AACR.

• MeSH
• akutní myeloidní leukemie genetika   metabolismus   MeSH
• cílená molekulární terapie MeSH
• fúzní onkogenní proteiny genetika   metabolismus   MeSH
• histonlysin-N-methyltransferasa chemie   genetika   metabolismus   MeSH
• HIV-integrasa chemie   metabolismus   MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie MeSH
• mezibuněčné signální peptidy a proteiny chemie   genetika   metabolismus   MeSH
• MFC-7 buňky MeSH
• molekulární modely MeSH
• myši MeSH
• protoonkogenní protein MLL chemie   genetika   metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH