No abstract

The mammalian SWI/SNF chromatin remodeling complex, whose function is of critical importance in transcriptional regulation, contains approximately 10 protein components. The expression levels of the core SWI/SNF subunits, including BRG1/Brm, BAF155, BAF170, BAF60, hSNF/Ini1, and BAF57, are stoichiometric, with few to no unbound molecules in the cell. Here we report that exogenous expression of the wild type or certain deletion mutants of BAF57, a key subunit that mediates the interaction between the remodeling complex and transcription factors, results in diminished expression of endogenous BAF57. This down-regulation process is mediated by an increase in proteasome-dependent degradation of the BAF57 protein. Furthermore, the protein levels of BAF155/170 dictate the maximum cellular amount of BAF57. We mapped the domains responsible for the interaction between BAF57 and BAF155 and demonstrated that protein-protein interactions between them play an important role in this regulatory process. These findings provide insights into the physiological mechanisms responsible for maintaining the proper stoichiometric levels of the protein components comprising multimeric enzyme complexes.The SWI/SNF chromatin remodeling complexes are evolutionarily conserved multimeric enzymatic machines that alter the nucleosomal structure using energy derived from ATP hydrolysis (34). Ample experimental evidence suggests that the SWI/SNF complexes play important roles in fundamental cellular processes such as transcription, replication, and the repair of chromatin (24, 28). As a result, mammalian SWI/SNF complexes have been implicated in diverse physiological and pathological processes, including cell proliferation and differentiation, retrovirus infection, and carcinogenesis (17,21,25).The human SWI/SNF complexes contain either BRG1 or Brm as the catalytic ATPase subunit and approximately 10

IntroductionPhiladelphia chromosome-negative myeloproliferative neoplasms (MPNs) are a group of clonal hematopoietic disorders that includes polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). 1,2 Recent studies have confirmed the pathogenetic involvement of an acquired, somatic, gain-offunction, activating, point mutation JAK2V617F in MPNs. [3][4][5][6] This represents a guanine to thymidine mutation in exon 14 resulting in a valine to phenylalanine substitution at codon 617 in the JH2 or pseudokinase domain of the JAK2 gene (a member of the Janus kinase [JAK] family of nonreceptor tyrosine kinases, JAK1, JAK2, JAK3, and TYK2). 2,6 Highly sensitive assays for JAK2 have determined that the JAK2V617F mutation is present in 90% of patients with PV and approximately 50% to 60% of patients with ET or PMF. 7 In addition, a subset of patients, most commonly with PV, are homozygous for the JAK2V617F allele, the result of copy-neutral loss of heterozygosity at the JAK2 locus, especially in patients with PV. 2,7,8 Mutations in exon 12 of JAK2 are present in almost all patients with PV who are JAK2V617F negative. 9,10 The JAK proteins function in the cytoplasm to relay signals initiated by membrane-bound cytokine receptors. Engagement of the receptor results in the phosphorylation of the receptor and JAK2, which recruits its substrate proteins such as signal transducers and activators of transcription (STATs). 11,12 STATs, especially STAT3 and STAT5, translocate to the nucleus and transactivate many genes involved in cell proliferation and survival (eg, Bcl-xL, cyclin D1, and PIM1). 8,11,12 The V617F mutation in JAK2 also activates the downstream signaling pathways through the phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK). This contributes to diminished apoptosis of the hematopoietic progenitor cells (HPCs). 2,8 Overexpression of JAK2V617F in murine Ba/F3 cells with coexpression of the erythropoietin receptor (EpoR) confers in vitro cytokine-independent growth. 3,13 Recently, it was shown that enforced expression of JAK2V617F in human hematopoietic stem cells and myeloid progenitors directed differentiation toward the erythroid lineage, along with increased expression and phosphorylation of GATA-1 and decreased expression of PU.1. 14-16 JAK2V617F expression in retroviral models and in transgenic mice is sufficient to cause myeloproliferative disorders in the mice that recapitulate many clinicopathologic features observed in human PV, ET, and PMF. [17][18][19][20][21] 22,23 In vivo studies in mouse models have also shown that mutant JAK2V617F represents a novel target for therapeutic intervention with JAK2-selective tyrosine kinase inhibitors in MPNs. 21,24 For example, TG101348 inhibits myeloproliferation and myelofibrosis in a murine model of JAK2V617F-induced polycythemia. 21,22 Early clinical trials of several JAK2-selective kinase inhibitors (eg, XL019, TG101348, and INCB18424) are under way in JAK2-driven MPNs with poor prognosis (eg, PMF). ...

The PRC2 complex protein EZH2 is a histone methyltransferase that is known to bind and recruit DNMT1 to the DNA to modulate DNA methylation. Here, we determined that the pan-HDAC inhibitor panobinostat (LBH589) treatment depletes DNMT1 and EZH2 protein levels, disrupts the interaction of DNMT1 with EZH2, as well as de-represses JunB in human acute leukemia cells. Similar to treatment with the hsp90 inhibitor 17-DMAG, treatment with panobinostat also inhibited the chaperone association of heat shock protein 90 with DNMT1 and EZH2, which promoted the proteasomal degradation of DNMT1 and EZH2. Unlike treatment with the DNA methyltransferase inhibitor decitabine, which demethylates JunB promoter DNA, panobinostat treatment mediated chromatin alterations in the JunB promoter. Combined treatment with panobinostat and decitabine caused greater attenuation of DNMT1 and EZH2 levels than either agent alone, which was accompanied by more JunB de-repression and loss of clonogenic survival of K562 cells. Co-treatment with panobinostat and decitabine also caused more loss of viability of primary AML but not normal CD34+ bone marrow progenitor cells. Collectively, these findings indicate that co-treatment with panobinostat and decitabine targets multiple epigenetic mechanisms to de-repress JunB and exerts antileukemia activity against human acute myeloid leukemia cells.

This report describes the cloning and characterization of a pseudouridine (psi) synthase from mouse that we have named mouse pseudouridine synthase 1 (mpus1p). The cDNA is approximately 1.5 kb and when used as a probe on a Northern blot of mouse RNA from tissues and cultured cells, several bands were detected. The open reading frame is 393 amino acids and has 35% identity over its length with yeast psi synthase 1 (pus1p). The recombinant protein was expressed in Escherichia coli and the purified protein converted specific uridines to psi in a number of tRNA substrates. The positions modified in stoichiometric amounts in vitro were 27/28 in the anticodon stem and also positions 34 and 36 in the anticodon of an intron containing tRNA. A human cDNA was also cloned and the smaller open reading frame (348 amino acids) was 92% identical over its length with mpus1p but is shorter by 45 amino acids at the amino terminus. The expressed recombinant human protein has no activity on any of the tRNA substrates, most probably the result of the truncated open reading frame.