Ramesh Subrahmanyam

p16INK4a translation suppressed by miR-24

Background Expression of the tumor suppressor p16INK4a increases during aging and replicative senescence. Methodology/Principal Findings Here, 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/Significance Together, our results suggest that miR-24 represses the initiation and elongation phases of p16 translation.

The NH 2 -Terminal Coiled-Coil Domain and Tyrosine 177 Play Important Roles in Induction of a Myeloproliferative Disease in Mice by Bcr-Abl

ABSTRACT 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 NH2-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.

β-catenin expression results in p53-independent DNA damage and oncogene-induced senescence in prelymphomagenic thymocytes in vivo

ABSTRACT The expression of β-catenin, a potent oncogene, is causally linked to tumorigenesis. Therefore, it was surprising that the transgenic expression of oncogenic β-catenin in thymocytes resulted in thymic involution instead of lymphomagenesis. In this report, we demonstrate that this is because the expression of oncogenic β-catenin induces DNA damage, growth arrest, oncogene-induced senescence (OIS), and apoptosis of immature thymocytes. In p53-deficient mice, the expression of oncogenic β-catenin still results in DNA damage and OIS, but the thymocytes survive and eventually progress to thymic lymphoma. β-Catenin-induced thymic lymphomas are distinct from lymphomas that arise in p53−/− mice. They are CD4− CD8−, while p53-dependent lymphomas are largely CD4+ CD8+, and they develop at an earlier age and in the absence of c-Myc expression or Notch1 signaling. Thus, we report that oncogenic β-catenin-induced, p53-independent growth arrest and OIS and p53-dependent apoptosis protect developing thymocytes from transformation by oncogenic β-catenin.

A 220-nucleotide deletion of the intronic enhancer reveals an epigenetic hierarchy in immunoglobulin heavy chain locus activation.

A tissue-specific transcriptional enhancer, Eμ, has been implicated in developmentally regulated recombination and transcription of the immunoglobulin heavy chain (IgH) gene locus. We demonstrate that deleting 220 nucleotides that constitute the core Eμ results in partially active locus, characterized by reduced histone acetylation, chromatin remodeling, transcription, and recombination, whereas other hallmarks of tissue-specific locus activation, such as loss of H3K9 dimethylation or gain of H3K4 dimethylation, are less affected. These observations define Eμ-independent and Eμ-dependent phases of locus activation that reveal an unappreciated epigenetic hierarchy in tissue-specific gene expression.

Cutting Edge: SWI/SNF Mediates Antisense Igh Transcription and Locus-Wide Accessibility in B Cell Precursors

The stepwise process of Ag receptor gene assembly, termed V(D)J recombination, is coordinated during lymphocyte development by sweeping changes in chromatin that permit or deny access to a single recombinase enzyme. We now show that switching/sucrose nonfermenting (SWI/SNF) chromatin remodeling complexes are recruited to the Igh locus by an enhancer-dependent process and that these complexes are essential for generating recombinase accessibility throughout the locus. Depletion of SWI/SNF in pro-B cells also inhibits antisense transcription through all clusters of Igh gene segments, a pioneering process that has been implicated in the initial opening of chromatin. We conclude that SWI/SNF complexes play multiple roles in Igh gene assembly, ranging from initial locus activation to the spreading and maintenance of chromatin accessibility over large VH, DH, and JH domains.

Localized epigenetic changes induced by DH recombination restricts recombinase to DJH junctions

Genes encoding immunoglobulin heavy chains (Igh) are assembled by rearrangement of variable (VH), diversity (DH) and joining (JH) gene segments. Three critical constraints govern VH recombination. These include timing (VH recombination follows DH recombination), precision (VH gene segments recombine only to DJH junctions) and allele specificity (VH recombination is restricted to DJH-recombined alleles). Here we provide a model for these universal features of VH recombination. Analyses of DJH-recombined alleles showed that DJH junctions were selectively epigenetically marked, became nuclease sensitive and bound RAG recombinase proteins, which thereby permitted DH-associated recombination signal sequences to initiate the second step of Igh gene assembly. We propose that VH recombination is precise, because these changes did not extend to germline DH segments located 5′ of the DJH junction.

Localized DNA Demethylation at Recombination Intermediates during Immunoglobulin Heavy Chain Gene Assembly

The dynamics of DNA methylation during the complex genomic rearrangement of antigen receptor genes in developing B lymphocytes reveal localized demethylation of the first recombination product that may serve as a mark necessary for the second step of rearrangement.

Epigenetic Features that Regulate IgH Locus Recombination and Expression

Precisely regulated rearrangements that yield imprecise recombination junctions are hallmarks of antigen receptor gene assembly. At the immunoglobulin heavy chain (IgH) gene locus this is initiated by rearrangement of a D (H) gene segment to a J (H) gene segment to generate DJ(H) junctions, followed by rearrangement of a V (H) gene segment to the DJ(H) junction to generate fully recombined VDJ alleles. In this review we discuss the regulatory features of each step of IgH gene assembly and the role of epigenetic mechanisms in achieving regulatory precision.

RAGs’ eye view of the immunoglobulin heavy chain gene locus

The immunoglobulin heavy chain (IgH) gene locus is activated at a precise stage of B lymphocyte development to undergo gene rearrangements that assemble the functional gene. In this review we summarize our current understanding of the chromatin state of the IgH as it appears just prior to the initiation of V(D)J recombination, and the implications of this structure for regulation of recombination. We also examine the role of the intron enhancer, Eμ, in establishing the pre-rearrangement chromatin structure. The emerging picture shows that the IgH locus consists of independently regulated domains, each of which requires multiple levels of epigenetic changes to reach the fully activated state.