Polycomb RING1A- and RING1B-dependent histone H2A monoubiquitylation at pericentromeric regions promotes S-phase progression

The functions of polycomb products extend beyond their well-known activity as transcriptional regulators to include genome duplication processes. Polycomb activities during DNA replication and DNA damage repair are unclear, particularly without induced replicative stress.We have used a cellularmodel of conditionally inactive polycomb E3 ligases (RING1A and RING1B), which monoubiquitylate lysine 119 of histone H2A (H2AK119Ub), to examine DNA replication in unperturbed cells. We identify slow elongation and fork stalling during DNA replication that is associated with the accumulation of mid and late S-phase cells. Signs of replicative stress and colocalisation of double-strand breaks with chromocenters, the sites of coalesced pericentromeric heterocromatic (PCH) domains, were enriched in cells at mid S-phase, the stage at which PCH is replicated. Altered replication was rescued by targeted monoubiquitylation of PCH through methyl- CpG binding domain protein 1. The acute senescence associated with the depletion of RING1 proteins, which is mediated by p21 (also known as CDKN1A) upregulation, could be uncoupled from a response to DNA damage. These findings link cell proliferation and the polycomb proteins RING1A and RING1B to S-phase progression through a specific function in PCH replication.Ministerio de Economı́a y Competitividad BFU2010-18146, SAF2013-47997, BFU2013-42918-

Regulatory effects of miR-19a on MAD2 expression and tumorigenesis in gastric cancer

Gastric cancer (GC) is worldwide the sixth most diagnosed and third leading cause of cancer deaths, with poor and late prognosis, probably due to post-surgery adjuvant treatment resistance and lack of a thorough panel of prognostic markers. We have previously shown that mitotic arrest deficient 2 (MAD2, encoded by MAD2L1), a key protein of the spindle assembly checkpoint, is relevant in GC cells; its interference impairs migration and growth, while its overexpression correlates with tumorigenesi

Isolation and characterization of casein kinase I from Dictyostelium discoideum.

In the present study, the molecular cloning and characterization of a 49-kDa form of casein kinase (CK)I from Dictyostelium discoideum is reported. The predicted amino acid sequence shares 70% identity with the catalytic domain of the mammalian delta and epsilon isoforms, Drosophila CKIepsilon and Schizosaccharomyces pombe Hhp1, and 63% identity with Hrr25, a 57-kDa form of yeast CK involved in DNA repair. D. discoideum CKI (DdCKI) was expressed in vegetative asynchronous cells as well as in differentiated cells, as detected by Northern-blot analysis. The level of DdCKI expression did not change during the cell cycle. Antibodies raised against a truncated version of the protein recognized a 49-kDa protein from D. discoideum extracts. Protein expression paralleled the pattern found for the RNA. The expression of DdCKI in Escherichia coli resulted in an active enzyme that autophosphorylated and phosphorylated casein. Immunofluorescence assays showed that DdCKI was localized in the cytoplasm and nuclei of Dictyostelium cells. The lack of disruptants of the CKI gene suggests that this protein is essential for the vegetative growth of D. discoideum. Overexpression of DdCKI resulted in cells with increased resistance to hydroxyurea, suggesting a potential role for this kinase in DNA repair

Expression of normal and truncated forms of human endoglin.

Endoglin is a transmembrane glycoprotein 633 residues in length expressed at the surface of endothelial cells as a disulphide-linked homodimer; the specific cysteine residues involved in endoglin dimerization are unknown. Mutations in the coding region of the endoglin gene are responsible for hereditary haemorrhagic telangiectasia type 1 (HHT1), a dominantly inherited vascular disorder. Many of these mutations, if translated, would lead to truncated forms of the protein. It is therefore of interest to assess the protein expression of different truncated forms of endoglin. Infections in vitro or in vivo with recombinant vaccinia virus, as well as transient transfections with expression vectors, were used to express normal and truncated forms of endoglin. Truncated mutants could be classified into three different groups: (1) those that did not produce stable transcripts; (2) those that produced stable transcripts but did not secrete the protein; and (3) those that secreted a soluble dimeric protein. This is the first time that a recombinant truncated form of endoglin has been found to be expressed in a soluble form. Because a chimaeric construct encoding the N-terminal sequence of platelet/endothelial cell adhesion molecule (PECAM-1) antigen fused to residues Ile281-Ala658 of endoglin also yielded a dimeric surface protein, these results suggest that cysteine residues contained within the fragment Cys330-Cys412 are involved in disulphide bond formation. Infection with vaccinia recombinants encoding an HHT1 mutation did not affect the expression of the normal endoglin, and did not reveal an association of the recombinant soluble form with the transmembrane endoglin, supporting a haploinsufficiency model for HHT1