RNF8- and RNF168-dependent degradation of KDM4A/JMJD2A triggers 53BP1 recruitment to DNA damage sites

In response to DNA damage, cells initiate complex signalling cascades leading to growth arrest and DNA repair. The recruitment of 53BP1 to damaged sites requires the activation of the ubiquitination cascade controlled by the E3 ubiquitin ligases RNF8 and RNF168, and methylation of histone H4 on lysine 20. However, molecular events that regulate the accessibility of methylated histones, to allow the recruitment of 53BP1 to DNA breaks, are unclear. Here, we show that like 53BP1, the JMJD2A (also known as KDM4A) tandem tudor domain binds dimethylated histone H4K20; however, JMJD2A is degraded by the proteasome following the DNA damage in an RNF8-dependent manner. We demonstrate that JMJD2A is ubiquitinated by RNF8 and RNF168. Moreover, ectopic expression of JMJD2A abrogates 53BP1 recruitment to DNA damage sites, indicating a role in antagonizing 53BP1 for methylated histone marks. The combined knockdown of JMJD2A and JMJD2B significantly rescued the ability of RNF8- and RNF168-deficient cells to form 53BP1 foci. We propose that the RNF8-dependent degradation of JMJD2A regulates DNA repair by controlling the recruitment of 53BP1 at DNA damage sites.     

Two novel genes expressed in Xenopus germ line: characteristic features of putative protein structures,their gene expression profiles and their possible roles in gametogenesis and embryogenesis

We compared the secondary spermatogonia and the primary spermatocytes of Xenopus for the proteins in their microsomal fractions and identified a newly synthesized protein (94 kDa) and three other proteins (99, 85, and 72 kDa) which increased their amount after entering the meiotic phase. These four proteins were used as antigens to produce polyclonal antibody which was found to react with the four proteins as well as two other proteins (208 and 60 kDa). Immunoscreening of Xenopus testis cDNA library with this polyclonal antibody yielded two cDNA clones (Xmegs and Xtr) encoding novel proteins. Xmegs mRNA was specifically expressed in the spermatogenic cells from the mid-pachytene stage to completion of two meiotic divisions. The putative Xmegs protein contained 19 tandem repeats of 26 amino acid residues rich in proline as well as potential phosphorylation sites (i.e., serine and threonine residues). Around this repetitive area, we found five PEST sequences known as a proteolytic signal to target protein for degradation. The presence of PEST sequences was believed to allow protein levels to closely parallel mRNA abundance. These results suggested the possible role of this novel protein in the regulation of two meiotic divisions specific to the spermatogenesis in a phosphorylation- and/or dephosphorylation-dependent manner. On the other hand, Xtr mRNA was expressed in both spermatogenic and oogenic cells except for round spermatids and the later stage cells. This mRNA was also expressed in the early stage embryos and its amount was kept constant from the St. I oocyte to the gastrula stage and decreased thereafter. The putative Xtr protein contained four complete and one partial tudor-like domains that were discovered in Drosophila tudor protein which plays an important role in PGC differentiation and abdominal segmentation. The characteristic expression profile of Xtr and the protein structure similar to the Drosophila tudor protein suggested its possible role in the progression of meiosis and PGC differentiation.     

Xtr, a plural tudor domain-containing protein, is involved in the translational regulation of maternal mRNA during oocyte maturation in Xenopus laevis

Xtr in the fertilized eggs of Xenopus has been demonstrated to be a member of a messenger ribonucleoprotein (mRNP) complex that plays a crucial role in karyokinesis during cleavage. Since the Xtr is also present both in oocytes and spermatocytes and its amount increases immediately after spematogenic cells enter into the meiotic phase, this protein was also predicted to act during meiotic progression. Taking advantage of Xenopus oocytes' large size to microinject anti-Xtr antibody into them for inhibition of Xtr function, we examined the role of Xtr in meiotic progression of oocytes. Microinjection of anti-Xtr antibody into immature oocytes followed by reinitiation of oocyte maturation did not affect germinal vesicle break down and the oscillation of Cdc2/cyclin B activity during meiotic progression but caused abnormal spindle formation and chromosomal alignment at meiotic metaphase I and II. Immunoprecipitation of Xtr showed the association of Xtr with FRGY2 and mRNAs such as RCC1 and XL-INCENP mRNAs, which are involved in the progression of karyokinesis. When anti-Xtr antibody was injected into oocytes, translation of XL-INCENP mRNA, which is known to be repressed in immature oocytes and induced after reinitiation of oocyte maturation, was inhibited even if the oocytes were treated with progesterone. A similar translational regulation was observed in oocytes injected with a reporter mRNA, which was composed of an enhanced green fluorescent protein open reading frame followed by the 3' untranslational region (3'UTR) of XL-INCENP mRNA. These results indicate that Xtr regulates the translation of XL-INCENP mRNA through its 3'UTR during meiotic progression of oocyte.     

Epigenetic virtues of chromodomains

The chromatin organization modifier domain (chromodomain) was first identified as a motif associated with chromatin silencing in Drosophila. There is growing evidence that chromodomains are evolutionary conserved across different eukaryotic species to control diverse aspects of epigenetic regulation. Although originally reported as histone H3 methyllysine readers, the chromodomain functions have now expanded to recognition of other histone and non-histone partners as well as interaction with nucleic acids. Chromodomain binding to a diverse group of targets is mediated by a conserved substructure called the chromobox homology region. This motif can be used to predict methyllysine binding and distinguish chromodomains from related Tudor “Royal” family members. In this review, we discuss and classify various chromodomains according to their context, structure and the mechanism of target recognition.     

DMA-tudor interaction modules control the specificity of in vivo condensates

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Drosophila tudor is essential for polar granule assembly and pole cell specification, but not for posterior patterning

Pole cells and posterior segmentation in Drosophila are specified by maternally encoded genes whose products accumulate at the posterior pole of the oocyte. Among these genes is tudor (tud). Progeny of hypomorphic tud mothers lack pole cells and have variable posterior patterning defects. We have isolated a null allele to further investigate tud function. While no pole cells are ever observed in embryos from tud-null mothers, 15% of these embryos have normal posterior patterning. OSKAR (OSK) and VASA (VAS) proteins, and nanos (nos) RNA, all initially localize to the pole plasm of tud-null oocytes and embryos from tud-null mothers, while localization of germ cell-less (gcl) and polar granule component (pgc), is undetectable or severely reduced. In embryos from tud-null mothers, polar granules are greatly reduced in number, size, and electron density. Thus, tud is dispensable for somatic patterning, but essential for pole cell specification and polar granule formation.     

Proteomic Discovery of Plasma Protein Biomarkers and Development of Models Predicting Prognosis of High-Grade Serous Ovarian Carcinoma

Ovarian cancer is one of the most lethal female cancers. For accurate prognosis prediction, this study aimed to investigate novel, blood-based prognostic biomarkers for high-grade serous ovarian carcinoma (HGSOC) using mass spectrometry–based proteomics methods. We conducted label-free liquid chromatography–tandem mass spectrometry using frozen plasma samples obtained from patients with newly diagnosed HGSOC (n = 20). Based on progression-free survival (PFS), the samples were divided into two groups: good (PFS ≥18 months) and poor prognosis groups (PFS <18 months). Proteomic profiles were compared between the two groups. Referring to proteomics data that we previously obtained using frozen cancer tissues from chemotherapy-naïve patients with HGSOC, overlapping protein biomarkers were selected as candidate biomarkers. Biomarkers were validated using an independent set of HGSOC plasma samples (n = 202) via enzyme-linked immunosorbent assay (ELISA). To construct models predicting the 18-month PFS rate, we performed stepwise selection based on the area under the receiver operating characteristic curve (AUC) with 5-fold cross-validation. Analysis of differentially expressed proteins in plasma samples revealed that 35 and 61 proteins were upregulated in the good and poor prognosis groups, respectively. Through hierarchical clustering and bioinformatic analyses, GSN, VCAN, SND1, SIGLEC14, CD163, and PRMT1 were selected as candidate biomarkers and were subjected to ELISA. In multivariate analysis, plasma GSN was identified as an independent poor prognostic biomarker for PFS (adjusted hazard ratio, 1.556; 95% confidence interval, 1.073–2.256; p = 0.020). By combining clinical factors and ELISA results, we constructed several models to predict the 18-month PFS rate. A model consisting of four predictors (FIGO stage, residual tumor after surgery, and plasma levels of GSN and VCAN) showed the best predictive performance (mean validated AUC, 0.779). The newly developed model was converted to a nomogram for clinical use. Our study results provided insights into protein biomarkers, which might offer clues for developing therapeutic targets.     

Xtr, a plural tudor domain-containing protein, coexists with FRGY2 both in cytoplasmic mRNP particle and germ plasm in Xenopus embryo: its possible role in translational regulation of maternal mRNAs

Xtr is present exclusively in early embryonic and germline cells. We have previously shown that loss-of-function of the Xtr in embryos causes arrest of karyokinesis progression. Since Xtr contains plural tudor domains, which are known to associate with target proteins directly, we examined Xtr-interacting proteins by immunoprecipitation with an anti-Xtr monoclonal antibody and detected a few RNA-binding proteins such as FRGY2, a component of messenger ribonucleoprotein (mRNP) particle. The coexistence of Xtr with FRGY2 by constituting an mRNP particle was further confirmed by gel filtration assay. Search of mRNAs in the immunoprecipitate with Xtr suggested that the Xtr-associated molecules included several mRNAs, of which translational products were known to play crucial roles in karyokinesis progression (RCC1, XRHAMM, and so on) and in germ cell development (XDead end). Immunohistochemical observation clearly showed the co-localization of Xtr with FRGY2 also in germ plasm, in which XDead end mRNA has been shown to be localized specifically. Taken together, we proposed the possible role of Xtr in translational activation of the maternal mRNAs repressed in mRNP particle.