Structure and function of Spc42 coiled-coils in yeast centrosome assembly and duplication.

Centrosomes and spindle pole bodies (SPBs) are membraneless organelles whose duplication and assembly is necessary for bipolar mitotic spindle formation. The structural organization and functional roles of major proteins in these organelles can provide critical insights into cell division control. Spc42, a phosphoregulated protein with an N-terminal dimeric coiled-coil (DCC), assembles into a hexameric array at the budding yeast SPB core, where it functions as a scaffold for SPB assembly. Here, we present in vitro and in vivo data to elucidate the structural arrangement and biological roles of Spc42 elements. Crystal structures reveal details of two additional coiled-coils in Spc42: a central trimeric coiled-coil and a C-terminal antiparallel DCC. Contributions of the three Spc42 coiled-coils and adjacent undetermined regions to the formation of an ∼145 Šhexameric lattice in an in vitro lipid monolayer assay and to SPB duplication and assembly in vivo reveal structural and functional redundancy in Spc42 assembly. We propose an updated model that incorporates the inherent symmetry of these Spc42 elements into a lattice, and thereby establishes the observed sixfold symmetry. The implications of this model for the organization of the central SPB core layer are discussed.

Gene regulation and suppression of type I interferon signaling by STAT3 in diffuse large B cell lymphoma.

STAT3 is constitutively activated in many cancers and regulates gene expression to promote cancer cell survival, proliferation, invasion, and migration. In diffuse large B cell lymphoma (DLBCL), activation of STAT3 and its kinase JAK1 is caused by autocrine production of IL-6 and IL-10 in the activated B cell-like subtype (ABC). However, the gene regulatory mechanisms underlying the pathogenesis of this aggressive lymphoma by STAT3 are not well characterized. Here we performed genome-wide analysis and identified 2,251 STAT3 direct target genes, which involve B cell activation, survival, proliferation, differentiation, and migration. Whole-transcriptome profiling revealed that STAT3 acts as both a transcriptional activator and a suppressor, with a comparable number of up- and down-regulated genes. STAT3 regulates multiple oncogenic signaling pathways, including NF-κB, a cell-cycle checkpoint, PI3K/AKT/mTORC1, and STAT3 itself. In addition, STAT3 negatively regulates the lethal type I IFN signaling pathway by inhibiting expression of IRF7, IRF9, STAT1, and STAT2 Inhibition of STAT3 activity by ruxolitinib synergizes with the type I IFN inducer lenalidomide in growth inhibition of ABC DLBCL cells in vitro and in a xenograft mouse model. Therefore, this study provides a mechanistic rationale for clinical trials to evaluate ruxolitinib or a specific JAK1 inhibitor combined with lenalidomide in ABC DLBCL.

HiJAKing the epigenome in leukemia and lymphoma.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway is central to signaling by receptors of diverse cytokines, growth factors, and other related molecules. Many of these receptors transmit anti-apoptosis, proliferation, and differentiation signals that are critical for normal hematopoiesis and immune response. However, the JAK/STAT signaling pathway is deregulated in many hematologic malignancies, and as such is co-opted by malignant cells to promote their survival and proliferation. It has recently come to light that an alternative mechanism, wherein nuclear JAKs epigenetically modify the chromatin to increase gene expression independent of STATs, also plays an important role in the pathogenesis of many hematologic malignancies. In this review, we will focus on common genetic alterations of the JAK family members in leukemia and lymphoma, and provide examples in which JAKs regulate gene expression by targeting the cancer epigenome.

Epigenetic gene regulation by Janus kinase 1 in diffuse large B-cell lymphoma.

Janus kinases (JAKs) classically signal by activating STAT transcription factors but can also regulate gene expression by epigenetically phosphorylating histone H3 on tyrosine 41 (H3Y41-P). In diffuse large B-cell lymphomas (DLBCLs), JAK signaling is a feature of the activated B-cell (ABC) subtype and is triggered by autocrine production of IL-6 and IL-10. Whether this signaling involves STAT activation, epigenetic modification of chromatin, or both mechanisms is unknown. Here we use genetic and pharmacological inhibition to show that JAK1 signaling sustains the survival of ABC DLBCL cells. Whereas STAT3 contributed to the survival of ABC DLBCL cell lines, forced STAT3 activity could not protect these cells from death following JAK1 inhibition, suggesting epigenetic JAK1 action. JAK1 regulated the expression of nearly 3,000 genes in ABC DLBCL cells, and the chromatin surrounding many of these genes was modified by H3Y41-P marks that were diminished by JAK1 inhibition. These JAK1 epigenetic target genes encode important regulators of ABC DLBCL proliferation and survival, including IRF4, MYD88, and MYC. A small molecule JAK1 inhibitor cooperated with the BTK inhibitor ibrutinib in reducing IRF4 levels and acted synergistically to kill ABC DLBCL cells, suggesting that this combination should be evaluated in clinical trials.

E. coli RNA Polymerase Determinants of Open Complex Lifetime and Structure.

In transcription initiation by Escherichia coli RNA polymerase (RNAP), initial binding to promoter DNA triggers large conformational changes, bending downstream duplex DNA into the RNAP cleft and opening 13bp to form a short-lived open intermediate (I2). Subsequent conformational changes increase lifetimes of λPR and T7A1 open complexes (OCs) by >10(5)-fold and >10(2)-fold, respectively. OC lifetime is a target for regulation. To characterize late conformational changes, we determine effects on OC dissociation kinetics of deletions in RNAP mobile elements σ(70) region 1.1 (σ1.1), ß' jaw and ß' sequence insertion 3 (SI3). In very stable OC formed by the wild type WT RNAP with λPR (RPO) and by Δσ1.1 RNAP with λPR or T7A1, we conclude that downstream duplex DNA is bound to the jaw in an assembly with SI3, and bases -4 to +2 of the nontemplate strand discriminator region are stably bound in a positively charged track in the cleft. We deduce that polyanionic σ1.1 destabilizes OC by competing for binding sites in the cleft and on the jaw with the polyanionic discriminator strand and downstream duplex, respectively. Examples of σ1.1-destabilized OC are the final T7A1 OC and the λPR I3 intermediate OC. Deleting σ1.1 and either ß' jaw or SI3 equalizes OC lifetimes for λPR and T7A1. DNA closing rates are similar for both promoters and all RNAP variants. We conclude that late conformational changes that stabilize OC, like early ones that bend the duplex into the cleft, are primary targets of regulation, while the intrinsic DNA opening/closing step is not.