V(D)J recombination is initiated by the recombination activating gene (RAG) proteins RAG-1 and RAG-2. The ability of antigen-receptor-gene segments to undergo V(D)J recombination is correlated with spatially- and temporally-restricted chromatin modifications. We have found that RAG-2 bound specifically to histone H3 and that this binding was absolutely dependent on dimethylation or trimethylation at lysine 4 (H3K4me2 or H3K4me3). The interaction required a noncanonical plant homeodomain (PHD) that had previously been described within the noncore region of RAG-2. Binding of the RAG-2 PHD finger to chromatin across the IgH D-J(H)-C locus showed a strong correlation with the distribution of trimethylated histone H3 K4. Mutation of a conserved tryptophan residue in the RAG-2 PHD finger abolished binding to H3K4me3 and greatly impaired recombination of extrachromosomal and endogenous immunoglobulin gene segments. Together, these findings are consistent with the interpretation that recognition of hypermethylated histone H3 K4 promotes efficient V(D)J recombination in vivo.

BackgroundExpression of the tumor suppressor p16INK4a increases during aging and replicative senescence.Methodology/Principal FindingsHere, we report that the microRNA miR-24 suppresses p16 expression in human diploid fibroblasts and cervical carcinoma cells. Increased p16 expression with replicative senescence was associated with decreased levels of miR-24, a microRNA that was predicted to associate with the p16 mRNA coding and 3′-untranslated regions. Ectopic miR-24 overexpression reduced p16 protein but not p16 mRNA levels. Conversely, introduction of antisense (AS)-miR-24 blocked miR-24 expression and markedly enhanced p16 protein levels, p16 translation, and the production of EGFP-p16 reporter bearing the miR-24 target recognition sites.Conclusions/SignificanceTogether, our results suggest that miR-24 represses the initiation and elongation phases of p16 translation.

Bcr-Abl, a fusion protein generated by t(9;22)(q34;q11) translocation, plays a critical role in the pathogenesis of chronic myelogenous leukemia (CML). It has been shown that Bcr-Abl contains multiple functional domains and motifs and can disrupt regulation of many signaling pathways and cellular functions. However, the role of specific domains and motifs of Bcr-Abl or of specific signaling pathways in the complex in vivo pathogenesis of CML is not completely known. We have previously shown that expression of Bcr-Abl in bone marrow cells by retroviral transduction efficiently induces a myeloproliferative disorder (MPD) in mice resembling human CML. We have also shown that the Abl kinase activity within Bcr-Abl is essential for Bcr-Abl leukemogenesis, yet activation of the Abl kinase without Bcr sequences is not sufficient to induce MPD in mice. In this study we investigated the role of Bcr sequences within Bcr-Abl in inducing MPD using this murine model for CML. We found that the NH 2 -terminal coiled-coil (CC) domain was both essential and sufficient, even though not efficient, to activate Abl to induce an MPD in mice. Interestingly, deletion of the Src homology 3 domain complemented the deficiencies of the CC-deleted Bcr-Abl in inducing MPD in mice. We further demonstrated that the Grb2 binding site at Y177 played an important role in efficient induction of MPD. These studies directly demonstrated the important roles of Bcr sequences in induction of MPD by Bcr-Abl.The bcr-abl oncogene, produced from the t(9;22)(q34;q11) chromosomal translocation known as the Philadelphia chromosome (Ph), is associated with 95% of the cases of chronic myelogenous leukemia (CML) and also with 20% of the adult and 5% of the pediatric cases of acute lymphoblastic leukemia (ALL) (23,33). Depending on the nature of the translocation and exactly how the bcr and abl sequences become spliced into a final bcr-abl mRNA, various Bcr-Abl fusion proteins, including p185, p210, and p230, can be generated that show a preferential association with different types of leukemia (33). Clinical and laboratory studies indicate that Bcr-Abl plays an essential role in initiation of the chronic phase of CML and also plays a critical role in the maintenance and progression of the disease (10,20,50).Bcr-Abl contains multiple functional domains and motifs. Abl-derived sequences in Bcr-Abl contain Src homology 3 (SH3), SH2, and tyrosine kinase domains in their N-terminal half, as well as a DNA binding domain, an actin binding domain, nuclear localization signals, and SH3 binding sites in their C-terminal region (41). The Bcr region (in the major p210 form) contains a coiled-coil (CC) oligomerization domain, a serine/threonine kinase domain, a Pleckstrin homology (PH) domain, a Dbl guanine-nucleotide exchange factor homology domain, and binding sites for the Abl SH2 domain and Grb2, Grb10, and 14-3-3 proteins (2,25,41,49). The multiple domains of Bcr-Abl work cooperatively to activate intracellular signaling pathways commonly used in hematopoietic growth fa...

The first steps of murine immunoglobulin heavy-chain (IgH) gene recombination take place within a chromosomal domain that contains diversity (D(H)) and joining (J(H)) gene segments, but not variable (V(H)) gene segments. Here we show that the chromatin state of this domain is markedly heterogeneous. Specifically, only 5'- and 3'-most D(H) gene segments carry active chromatin modifications, whereas intervening D(H)s are associated with heterochromatic marks that are maintained by ongoing histone deacetylation. The intervening D(H)s form part of a tandemly repeated sequence that expresses tissue-specific, antisense oriented transcripts. We propose that the intervening D(H) genes are actively suppressed by repeat-induced epigenetic silencing, which is reflected in their infrequent representation in DJ(H) junctions compared to the flanking D(H) genes.