WDHD1 modulates the post-transcriptional step of the centromeric silencing pathway

The centromere is a highly specialized chromosomal element that is essential for chromosome segregation during mitosis. Centromere integrity must therefore be properly preserved and is strictly dependent upon the establishment and maintenance of surrounding chromatin structure. Here we identify WDHD1, a WD40-domain and HMG-domain containing protein, as a key regulator of centromere function. We show that WDHD1 associates with centromeres in a cell cycle-dependent manner, coinciding with mid-to-late S phase. WDHD1 down-regulation compromises HP1α localization to pericentric heterochromatin and leads to altered expression of epigenetic markers associated with this chromatin region. As a consequence, such reduced epigenetic silencing is manifested in disrupted heterochromatic state of the centromere and a defective mitosis. Moreover, we demonstrate that a possible underlying mechanism of WDHD1’s involvement lies in the proper generation of the small non-coding RNAs encoded by the centromeric satellite repeats. This role is mediated at the post-transcriptional level and likely through stabilizing Dicer association with centromeric RNA. Collectively, these findings suggest that WDHD1 may be a critical component of the RNA-dependent epigenetic control mechanism that sustains centromere integrity and genomic stability

Differential regulation of CHOP translation by phosphorylated eIF4E under stress conditions

Cells respond to environmental stress by inducing translation of a subset of mRNAs important for survival or apoptosis. CHOP, a downstream transcriptional target of stress-induced ATF4, is also regulated translationally in a uORF-dependent manner under stress. Low concentration of anisomycin induces CHOP expression at both transcriptional and translational levels. To study specifically the translational aspect of CHOP expression, and further clarify the regulatory mechanisms underlying stress-induced translation initiation, we developed a CMV promoter-regulated, uORFchop-driven reporter platform. Here we show that anisomycin-induced CHOP expression depends on phosphorylated eIF4E/S209 and eIF2α/S51. Contrary to phospho-eIF2α/S51, phospho-eIF4E/S209 is not involved in thapsigargin-induced CHOP expression. Studies using various kinase inhibitors and mutants uncovered that both the p38MAPK-Mnk and mTOR signaling pathways contribute to stress-responsive reporter and CHOP expression. We also demonstrated that anisomycin-induced translation is tightly regulated by partner binding preference of eIF4E. Furthermore, mutating the uORF sequence abolished the anisomycin-induced association of chop mRNA with phospho-eIF4E and polysomes, thus demonstrating the significance of this cis-regulatory element in conferring on the transcript a stress-responsive translational inducibility. Strikingly, although insulin treatment activated ERK-Mnk and mTOR pathways, and consequently eIF4E/S209 phosphorylation, it failed to induce phospho-eIF2α/S51 and reporter translation, thus pinpointing a crucial determinant in stress-responsive translation

Dephosphorylation of Nucleophosmin by PP1β Facilitates pRB Binding and Consequent E2F1-dependent DNA Repair

We report a new pathway through which PP1β signals to nucleophosmin (NPM) in response to DNA damage. UV induces dephosphorylation of NPM at multiple sites, leading to enhancement of complex formation between NPM and retinoblastoma tumor suppressor protein and the subsequent upregulation of E2F1. Consequently, such signaling pathway potentiates the cellular DNA repair capacity

Nek9, a Novel Fact-Associated Protein, Modulates Interphase Progression

The heterodimeric Spt16-Pob3/DUF/FACT complex is a class of chromatin structure modulators with important roles in replication and transcription . Although regarded as a transcription elongator for chromatin template, little is known about the mode of action and involvement in other molecular processes of the mammalian FACT. Here we report the identification of a novel interacting and functional partner of FACT, Nek9 . Nek9 forms a stable, similar to600- kDa complex with FACT in the interphase nuclei. Its active form is characterized by phosphorylation- dependent electrophoretic mobility shift and phosphorylation at a conserved residue within the activation loop (Thr(210)). When complexed with FACT, Nek9 exhibits markedly elevated phosphorylation on Thr(210). Cell cycle analysis on the Nek9 (dsRNAi) cells directly implicated Nek9 in maintaining proper G(1) and S progression, a role temporally correlated to the formation of a phospho-Nek9-FACT complex. Collectively, these observations provide evidence that Nek9, potentially as an active enzymatic partner of FACT, mediates certain FACT-associated cellular processes, which are ultimately essential for interphase progression

Size scaling of nucleolus in Caenorhabditis elegans embryos

Nucleolus is viewed as a plurifunctional center in the cell, tightly linked to ribosome biosynthesis. As a non-membranous structure, how the size of nucleolus is determined is a long outstanding question, and the possibility of “direct size scaling to the nucleus” was raised by genetic studies in fission yeast. Here, we used the model organism Caenorhabditis elegans to test this hypothesis in multi-cellular organisms. We depleted ani-2, ima-3, or C27D9.1 by RNAi feeding, which altered embryo sizes to different extents in ncl-1 mutant worms. DIC imaging provided evidence that in size-altering embryo nucleolar size decreases in small cells and increases in large cells. Furthermore, analyses of nucleolar size in four blastomeres (ABa, ABp, EMS, and P2) within the same embryo of ncl-1 mutants consistently demonstrated the correspondence between cell and nucleolar sizes – the small cells (EMS and P2) have smaller nucleoli in comparison to the large cells (ABa). Keywords: Nucleolar size, Embryonic size, ani-2, ima-3, C27D9.

Nucleophosmin acts as a novel AP2α-binding transcriptional corepressor during cell differentiation

Nucleophosmin (NPM) is an important nucleolar phosphoprotein with pleiotropic functions in various cellular processes. In this study, we have further examined the largely uncharacterized role of NPM in transcriptional regulation by uncovering novel NPM-binding transcriptional factors. Among potential interactors, we found that activating protein transcription factor 2 (AP2)α forms a complex with NPM during retinoic-acid-induced cell differentiation. We show that this complex is recruited to the promoters of certain retinoic-acid-responsive genes, including NPM itself. Such binding of AP2α, and consequent recruitment of NPM, is selective and dependent on a consensus AP2α-binding sequence. Remarkably, suppression of NPM by RNA interference alleviates the repression of gene expression mediated by retinoic acid and AP2α. Our findings further show that, on promoter binding, NPM probably exerts its repressive effect by inducing a change in local chromatin structure that also engages histone deacetylases. This study unveils a hitherto unrecognized transcriptional corepressor function of the NPM protein, and highlights a novel mechanism by which NPM regulates cell growth and differentiation

Evolutionarily significant A-to-I RNA editing events originated through G-to-A mutations in primates

Abstract Background Recent studies have revealed thousands of A-to-I RNA editing events in primates, but the origination and general functions of these events are not well addressed. Results Here, we perform a comparative editome study in human and rhesus macaque and uncover a substantial proportion of macaque A-to-I editing sites that are genomically polymorphic in some animals or encoded as non-editable nucleotides in human. The occurrence of these recent gain and loss of RNA editing through DNA point mutation is significantly more prevalent than that expected for the nearby regions. Ancestral state analyses further demonstrate that an increase in recent gain of editing events contribute to the over-representation, with G-to-A mutation site as a favorable location for the origination of robust A-to-I editing events. Population genetics analyses of the focal editing sites further reveal that a portion of these young editing events are evolutionarily significant, indicating general functional relevance for at least a fraction of these sites. Conclusions Overall, we report a list of A-to-I editing events that recently originated through G-to-A mutations in primates, representing a valuable resource to investigate the features and evolutionary significance of A-to-I editing events at the population and species levels. The unique subset of primate editome also illuminates the general functions of RNA editing by connecting it to particular gene regulatory processes, based on the characterized outcome of a gene regulatory level in different individuals or primate species with or without these editing events