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
• Amino Acid Motifs MeSH
• Histone-Lysine N-Methyltransferase chemistry   metabolism   MeSH
• Protein Interaction Domains and Motifs MeSH
• Humans MeSH
• Tumor Suppressor Protein p53 chemistry   metabolism   MeSH
• NF-kappa B chemistry   metabolism   MeSH
• CREB-Binding Protein chemistry   metabolism   MeSH
• Myeloid-Lymphoid Leukemia Protein chemistry   metabolism   MeSH
• Basic Helix-Loop-Helix Transcription Factors chemistry   metabolism   MeSH
• Transcription Factors chemistry   metabolism   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Humoral immunity in mammals relies on the function of two developmentally and functionally distinct B-cell subsets-B1 and B2 cells. While B2 cells are responsible for the adaptive response to environmental antigens, B1 cells regulate the production of polyreactive and low-affinity antibodies for innate humoral immunity. The molecular mechanism of B-cell specification into different subsets is understudied. In this study, we identified lysine methyltransferase NSD2 (MMSET/WHSC1) as a critical regulator of B1 cell development. In contrast to its minor impact on B2 cells, deletion of the catalytic domain of NSD2 in primary B cells impairs the generation of B1 lineage. Thus, NSD2, a histone H3 K36 dimethylase, is the first-in-class epigenetic regulator of a B-cell lineage in mice.

• MeSH
• Survival Analysis MeSH
• B-Lymphocytes metabolism   MeSH
• Histone-Lysine N-Methyltransferase chemistry   metabolism   MeSH
• Histones metabolism   MeSH
• Immunity, Humoral MeSH
• Catalytic Domain * MeSH
• Lysine metabolism   MeSH
• Methylation MeSH
• Mice, Inbred C57BL MeSH
• Animals, Newborn MeSH
• Immunoglobulin Class Switching MeSH
• Structure-Activity Relationship MeSH
• Germinal Center metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural 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
• Chromatin Immunoprecipitation MeSH
• CpG Islands MeSH
• Transcription, Genetic MeSH
• Histone-Lysine N-Methyltransferase chemistry   MeSH
• Histones chemistry   MeSH
• Markov Chains MeSH
• DNA Methylation * MeSH
• Multivariate Analysis MeSH
• Mouse Embryonic Stem Cells cytology   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Oocytes cytology   MeSH
• Oogenesis MeSH
• Promoter Regions, Genetic MeSH
• Myeloid-Lymphoid Leukemia Protein chemistry   MeSH
• Sequence Analysis, RNA MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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
• Leukemia, Myeloid, Acute genetics   metabolism   MeSH
• Molecular Targeted Therapy MeSH
• Oncogene Proteins, Fusion genetics   metabolism   MeSH
• Histone-Lysine N-Methyltransferase chemistry   genetics   metabolism   MeSH
• HIV Integrase chemistry   metabolism   MeSH
• Humans MeSH
• Magnetic Resonance Spectroscopy MeSH
• Intercellular Signaling Peptides and Proteins chemistry   genetics   metabolism   MeSH
• MCF-7 Cells MeSH
• Models, Molecular MeSH
• Mice MeSH
• Myeloid-Lymphoid Leukemia Protein chemistry   genetics   metabolism   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Speciation genes restrict gene flow between the incipient species and related taxa. Three decades ago, we mapped a mammalian speciation gene, hybrid sterility 1 (Hst1), in the intersubspecific hybrids of house mouse. Here, we identify this gene as Prdm9, encoding a histone H3 lysine 4 trimethyltransferase. We rescued infertility in male hybrids with bacterial artificial chromosomes carrying Prdm9 from a strain with the "fertility" Hst1(f) allele. Sterile hybrids display down-regulated microrchidia 2B (Morc2b) and fail to compartmentalize gammaH2AX into the pachynema sex (XY) body. These defects, seen also in Prdm9-null mutants, are rescued by the Prdm9 transgene. Identification of a vertebrate hybrid sterility gene reveals a role for epigenetics in speciation and opens a window to a hybrid sterility gene network.

• MeSH
• Epigenesis, Genetic MeSH
• Financing, Organized MeSH
• Histone-Lysine N-Methyltransferase genetics   chemistry   metabolism   MeSH
• Histones metabolism   MeSH
• Hybridization, Genetic MeSH
• Crosses, Genetic MeSH
• Chromosome Mapping MeSH
• Meiosis MeSH
• Methylation MeSH
• Molecular Sequence Data MeSH
• Infertility, Male genetics   MeSH
• Mice, Inbred C3H MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Ovary enzymology   MeSH
• Gene Expression Regulation MeSH
• Amino Acid Sequence MeSH
• Testis enzymology   MeSH
• Chromosomes, Artificial, Bacterial MeSH
• Genetic Speciation MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH