Orderly termination of sister-chromatid cohesion during mitosis is critical for accurate chromosome segregation. During prophase, mitotic kinases phosphorylate cohesin and its protector sororin, triggering Wapl-dependent cohesin release from chromosome arms. The shugoshin (Sgo1)–PP2A complex protects centromeric cohesin until its cleavage by separase at anaphase onset. Here, we report the crystal structure of a human cohesin subcomplex comprising SA2 and Scc1. Multiple HEAT repeats of SA2 form a dragon-shaped structure. Scc1 makes extensive contacts with SA2, with one binding hotspot. Sgo1 and Wapl compete for binding to a conserved site on SA2–Scc1. Mutations of SA2 residues at this site that disrupt Wapl binding bypass Sgo1 requirement in cohesion protection. Thus, besides recruiting PP2A to dephosphorylate cohesin and sororin, Sgo1 physically shields cohesin from Wapl. This unexpected, direct antagonism between Sgo1 and Wapl augments centromeric cohesion protection.

SUMMARY The ring-shaped cohesin complex regulates transcription, DNA repair, and chromosome segregation by dynamically entrapping chromosomes to promote chromosome compaction and sister-chromatid cohesion. The cohesin ring needs to open and close to allow its loading to and release from chromosomes. Cohesin dynamics are controlled by the releasing factors Pds5 and Wapl and the cohesin stabilizer Sororin. Here, we report the crystal structure of human Pds5B bound to a conserved peptide motif found in both Wapl and Sororin. Our structure establishes the basis for how Wapl and Sororin antagonistically influence cohesin dynamics. The structure further reveals that Pds5 can bind inositol hexakisphosphate (IP6). The IP6-binding segment of Pds5B is shaped like the jaw of a plier lever and inhibits the binding of Scc1 to Smc3. We propose that Pds5 stabilizes a transient, open state of cohesin to promote its release from chromosomes.

• Ikaros controls cellular proliferation by repressing genes that regulate cell cycle progression and the PI3K pathway in leukemia.• CK2 inhibitor restores Ikaros tumor suppressor function in high-risk B-ALL with IKZF1 deletion and has a strong therapeutic effect in vivo.Ikaros (IKZF1) is a tumor suppressor that binds DNA and regulates expression of its target genes. The mechanism of Ikaros activity as a tumor suppressor and the regulation of Ikaros function in leukemia are unknown. Here, we demonstrate that Ikaros controls cellular proliferation by repressing expression of genes that promote cell cycle progression and the phosphatidylinositol-3 kinase (PI3K) pathway. We show that Ikaros function is impaired by the pro-oncogenic casein kinase II (CK2), and that CK2 is overexpressed in leukemia. CK2 inhibition restores Ikaros function as transcriptional repressor of cell cycle and PI3K pathway genes, resulting in an antileukemia effect. In high-risk leukemia where one IKZF1 allele has been deleted, CK2 inhibition restores the transcriptional repressor function of the remaining wild-type IKZF1 allele. CK2 inhibition demonstrated a potent therapeutic effect in a panel of patient-derived primary high-risk B-cell acute lymphoblastic leukemia xenografts as indicated by prolonged survival and a reduction of leukemia burden. We demonstrate the efficacy of a novel therapeutic approach for high-risk leukemia: restoration of Ikaros tumor suppressor activity via inhibition of CK2. These results provide a rationale for the use of CK2 inhibitors in clinical trials for high-risk leukemia, including cases with deletion of one IKZF1 allele. (Blood. 2015;126(15):1813-1822 Introduction Ikaros (IKZF1) activity is essential for normal hematopoiesis and immune development. [1][2][3][4] Ikaros knockout mice have severely impaired hematopoiesis, 5-7 whereas mice with the heterozygous loss of Ikaros develop T-cell leukemia. 8 In humans, impaired Ikaros activity due to the deletion or inactivating mutation of a single IKZF1 allele results in high-risk B-cell leukemia that is resistant to treatment.9-14 Ikaros regulates transcription of target genes via chromatin remodeling. [15][16][17] Ikaros activity is controlled through multiple mechanisms. Mouse studies suggest that the transcription of IKZF1 during normal hematopoiesis is regulated by a complex network. 18 However, Ikaros protein is expressed at high levels in most hematopoietic cells, and posttranslational modifications are hypothesized to play a critical role in regulating Ikaros activity. 19 Several groups have shown that phosphorylation, [19][20][21][22][23][24] sumoylation, 25 and ubiquitination 22 can regulate Ikaros function as a transcriptional repressor. However, the role of posttranslational modification in the regulation of Ikaros tumor suppressor activity in leukemia is unknown.Despite extensive global analyses of Ikaros DNA binding in normal murine hematopoietic cells, 26-28 the molecular mechanisms by which Ikaros exerts its tumor suppressor effects in human leukemia ...

No abstract

Purpose: Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncogenic factor stabilizing c-MYC protein and driving cellular transformation.We determine whether CIP2A expression can serve as marker for gastric cancer and investigate the mechanism underlying CIP2A-mediated transformation and cell proliferation. Experimental Design: Normal and malignant gastric tissues derived from 37 patients with gastric cancer were analyzed for CIP2A expression using reverse transcription-PCR and immunohistochemical staining. Gastric and other cell lines with different p53 and pRB backgrounds were used to inhibit CIP2A expression using small interfering RNA and then examined for clonogenic potentials, senescence, or differentiation. Results: CIP2A mRNA was present in 34 of 37 (90%) of tumor specimens but absent in 27 of 37 (73%) of matched normal gastric mucosa. In 10 adjacent normal tissues with detectable CIP2A mRNA, 6 of them exhibited much weaker levels of CIP2A compared with their corresponding tumors. Thus, a total of 32 (87%) gastric cancer samples overexpressed CIP2A. CIP2A protein expression was readily detectable in the tumor tissues but absent in normal gastric mucosa. Depleting CIP2A expression substantially inhibited growth and clonogenic capabilities of tumor cell lines independently of p53 and pRB pathways. Gastric cancer^derived AGS cells underwent senescence following the inhibition of CIP2A expression. Moreover, CIP2A depletion triggered partial differentiation of leukemic HL60 cells. Conclusion: CIP2A in tumor cells is required for sustained proliferation by preventing cell growth arrest, senescence, or differentiation and its expression is significantly (P < 0.001) discriminatory between normal and cancerous gastric tissue.

The 5' end of avian sarcoma and leukosis virus RNA near the primer binding site forms two RNA secondary structures, U5-inverted repeat (U5-IR) and U5-leader stems, which are required for efficient initiation of reverse transcription. Lying between these two secondary structures is a 7-base sequence that can anneal to the T*C loop of the tRNATrP primer. Base substitutions in U5 RNA which disrupt this potential interaction result in a defect in the initiation of reverse transcription both in vivo and in vitro. The defect can be complemented in vitro by base substitutions in the primer. The U5 RNA-T4C interaction is also dependent upon the presence of both the U5-IR and the U5-leader structures. These RNA secondary structures and primer interactions are conserved in other type C and D retroviruses, suggesting that there is a common mechanism for the initiation of reverse transcription in all of these retroviruses.

Recent evidence has accumulated that the dynamic histone methylation mediated by histone methyltransferases and demethylases plays key roles in regulation of chromatin structure and transcription. In the present study, we show that SET and MYND domain-containing protein 3 (SMYD3), a histone methyltransferase implicated in oncogenesis, directly trans-activates the telomerase reverse transcriptase (hTERT) gene that is essential for cellular immortalization and transformation. SMYD3 occupies its binding motifs on the hTERT promoter and is required for maintenance of histone H3-K4 trimethylation, thereby contributing to inducible and constitutive hTERT expression in normal and malignant human cells. Knocking down SMYD3 in tumor cells abolished trimethylation of H3-K4, attenuated the occupancy by the transactivators c-MYC and Sp1, and led to diminished histone H3 acetylation in the hTERT promoter region, which was coupled with down-regulation of hTERT mRNA and telomerase activity. These results suggest that SMYD3-mediated trimethylation of H3-K4 functions as a licensing element for subsequent transcription factor binding to the hTERT promoter. The present findings provide significant insights into regulatory mechanisms of hTERT/telomerase expression; moreover, identification of the hTERT gene as a direct target of SMYD3 contributes to a better understanding of SMYD3-mediated cellular transformation. [Cancer Res 2007;67(6):2626-31]

Cohesin, along with positive regulators, establishes sister-chromatid cohesion by forming a ring to circle chromatin. The wings apart-like protein (Wapl) is a key negative regulator of cohesin and forms a complex with precocious dissociation of sisters protein 5 (Pds5) to promote cohesin release from chromatin. Here we report the crystal structure and functional characterization of human Wapl. Wapl contains a flexible, variable N-terminal region (Wapl-N) and a conserved C-terminal domain (Wapl-C) consisting of eight HEAT (Huntingtin, Elongation factor 3, A subunit, and target of rapamycin) repeats. Wapl-C folds into an elongated structure with two lobes. Structure-based mutagenesis maps the functional surface of Wapl-C to two distinct patches (I and II) on the N lobe and a localized patch (III) on the C lobe. Mutating critical patch I residues weaken Wapl binding to cohesin and diminish sister-chromatid resolution and cohesin release from mitotic chromosomes in human cells and Xenopus egg extracts. Surprisingly, patch III on the C lobe does not contribute to Wapl binding to cohesin or its known regulators. Although patch I mutations reduce Wapl binding to intact cohesin, they do not affect Wapl-Pds5 binding to the cohesin subcomplex of sister chromatid cohesion protein 1 (Scc1) and stromal antigen 2 (SA2) in vitro, which is instead mediated by Wapl-N. Thus, Wapl-N forms extensive interactions with Pds5 and Scc1-SA2. Wapl-C interacts with other cohesin subunits and possibly unknown effectors to trigger cohesin release from chromatin.chromosome segregation | crystallography | genomic stability | mitosis | protein-protein interaction P roper chromosome segregation during mitosis maintains genomic stability. Errors in this process cause aneuploidy, which contributes to tumorigenesis under certain contexts (1). Timely establishment and dissolution of sister-chromatid cohesion are critical for accurate chromosome segregation and require the cell-cycle-regulated interactions between cohesin and its regulators (2-4).In human cells, cohesin consists of four core subunits: Structural maintenance of chromosomes 1 (Smc1), Smc3, sister chromatid cohesion protein 1 (Scc1), and stromal antigen 1 or 2 (SA1/2). Smc1 and Smc3 are related ATPases, and each contains an ATPase head domain, a long coiled-coil domain, and a hinge domain that mediates Smc1-Smc3 heterodimerization. The Smc1-Smc3 heterodimer associates with the Scc1-SA1/2 heterodimer to produce the intact cohesin. Specifically, the N-and C-terminal winged helix domains (WHDs) of Scc1 connect the ATPase domains of Smc3 and Smc1, respectively, forming a ring (4).Cohesin is loaded onto chromatin in telophase/G1, but the chromatin-bound cohesin at this stage is highly dynamic and is actively removed from chromatin by the cohesin inhibitor Wings apart-like protein (Wapl) (5-7). During DNA replication in S phase, the ATPase head domain of Smc3 is acetylated by the acetyltransferase establishment of cohesion protein 1 (Eco1) (8-13). In vertebrates, replication-coupled Smc3 acet...