CELF1 represses Doublesex1 expression via its 5' UTR in the crustacean Daphnia magna.

In sex determination of the crustacean Daphnia magna, male-specific expression of DM-domain transcription factor Doublesex1 (Dsx1) orchestrates the male developmental program triggered by environmental stimuli. We previously identified the CELF1 ortholog as a candidate of proteins associated with the 5' UTR of the Dsx1α isoform. Here we report the CELF1-dependent suppression of Dsx1 expression in D. magna. During embryogenesis, CELF1 expression was not sexually dimorphic. Silencing of CELF1 led to the activation of Dsx1 expression both in female and male embryos. Overexpression of CELF1 in male embryos resulted in a reduction of Dsx1 expression. By these manipulations of CELF1 expression, the Dsx1 transcript level was not significantly changed. To investigate whether the CELF1 controls Dsx1 expression via its 5' UTR, we injected the GFP reporter mRNA having intact Dsx1α 5' UTR or mutated one lacking the GU-rich element (GRE) that is known as a binding site of the CELF1 ortholog. We found that deletion of the GRE significantly increased the reporter gene expression. These results indicate that CELF1 suppresses Dsx1 expression both in females and males, possibly at the post-transcriptional level. We speculate that CELF1 may avoid unintended Dsx1 expression and generation of sexual ambiguity by setting a threshold of Dsx1 expression.

Identification of CUL4A-DDB1-WDFY1 as an E3 ubiquitin ligase complex involved in initiation of lysophagy.

Macroautophagy is a bulk degradation system in which double membrane-bound structures called autophagosomes to deliver cytosolic materials to lysosomes. Autophagy promotes cellular homeostasis by selectively recognizing and sequestering specific targets, such as damaged organelles, protein aggregates, and invading bacteria, termed selective autophagy. We previously reported a type of selective autophagy, lysophagy, which helps clear damaged lysosomes. Damaged lysosomes become ubiquitinated and recruit autophagic machinery. Proteomic studies using transfection reagent-coated beads and further evaluations reveal that a CUL4A-DDB1-WDFY1 E3 ubiquitin ligase complex is essential to initiate lysophagy and clear damaged lysosomes. Moreover, we show that LAMP2 is ubiquitinated by the CUL4A E3 ligase complex as a substrate on damaged lysosomes. These results reveal how cells selectively tag damaged lysosomes to initiate autophagy for the clearance of lysosomes.

Sense-overlapping lncRNA as a decoy of translational repressor protein for dimorphic gene expression.

Long noncoding RNAs (lncRNAs) are vastly transcribed and extensively studied but lncRNAs overlapping with the sense orientation of mRNA have been poorly studied. We analyzed the lncRNA DAPALR overlapping with the 5´ UTR of the Doublesex1 (Dsx1), the male determining gene in Daphnia magna. By affinity purification, we identified an RNA binding protein, Shep as a DAPALR binding protein. Shep also binds to Dsx1 5´ UTR by recognizing the overlapping sequence and suppresses translation of the mRNA. In vitro and in vivo analyses indicated that DAPALR increased Dsx1 translation efficiency by sequestration of Shep. This regulation was impaired when the Shep binding site in DAPALR was deleted. These results suggest that Shep suppresses the unintentional translation of Dsx1 by setting a threshold; and when the sense lncRNA DAPALR is expressed, DAPALR cancels the suppression caused by Shep. This mechanism may be important to show dimorphic gene expressions such as sex determination and it may account for the binary expression in various developmental processes.

Genome-Wide Chromatin Analysis of FFPE Tissues Using a Dual-Arm Robot with Clinical Potential.

Although chromatin immunoprecipitation and next-generation sequencing (ChIP-seq) using formalin-fixed paraffin-embedded tissue (FFPE) has been reported, it remained elusive whether they retained accurate transcription factor binding. Here, we developed a method to identify the binding sites of the insulator transcription factor CTCF and the genome-wide distribution of histone modifications involved in transcriptional activation. Importantly, we provide evidence that the ChIP-seq datasets obtained from FFPE samples are similar to or even better than the data for corresponding fresh-frozen samples, indicating that FFPE samples are compatible with ChIP-seq analysis. H3K27ac ChIP-seq analyses of 69 FFPE samples using a dual-arm robot revealed that driver mutations in EGFR were distinguishable from pan-negative cases and were relatively homogeneous as a group in lung adenocarcinomas. Thus, our results demonstrate that FFPE samples are an important source for epigenomic research, enabling the study of histone modifications, nuclear chromatin structure, and clinical data.

ARE-binding protein ZFP36L1 interacts with CNOT1 to directly repress translation via a deadenylation-independent mechanism.

Eukaryotic gene expression can be spatiotemporally tuned at the post-transcriptional level by cis-regulatory elements in mRNA sequences. An important example is the AU-rich element (ARE), which induces mRNA destabilization in a variety of biological contexts in mammals and can also mediate translational control. Regulation is mediated by trans-acting factors that recognize the ARE, such as Tristetraprolin (TTP) and BRF1/ZFP36L1. Although both proteins can destabilize their target mRNAs through the recruitment of the CCR4-NOT deadenylation complex, TTP also directly regulates translation. Whether ZFP36L1 can directly repress translation remains unknown. Here, we used an in vitro translation system derived from mammalian cell lines to address this key mechanistic issue in ARE regulation by ZFP36L1. Functional assays with mutant proteins reveal that ZFP36L1 can repress translation via AU-Rich elements independent of deadenylation. ZFP36L1-mediated translation repression requires interaction between ZFP36L1 and CNOT1, suggesting that it might use a repression mechanism similar to either TPP or miRISC. However, several lines of evidence suggest that the similarity ends there. Unlike, TTP, it does not efficiently interact with either 4E-HP or GIGYF2, suggesting it does not repress translation by recruiting these proteins to the mRNA cap. Moreover, ZFP36L1 could not repress ECMV-IRES driven translation and was resistant to pharmacological eIF4A inhibitor silvestrol, suggesting fundamental differences with miRISC repression via eIF4A. Collectively, our results reveal that ZFP36L1 represses translation directly and suggest that it does so via a novel mechanism distinct from other translational regulators that interact with the CCR4-NOT deadenylase complex.

Ubiquitylation of Ku80 by RNF126 Promotes Completion of Nonhomologous End Joining-Mediated DNA Repair.

Repair of damaged DNA is critical for maintenance of genetic information. In eukaryotes, DNA double-strand breaks (DSBs) are recognized by the Ku70-Ku80 heterodimer, which then recruits proteins that mediate repair by nonhomologous end joining (NHEJ). Prolonged retention of Ku70/80 at DSBs prevents completion of repair, however, with ubiquitylation of Ku80 having been implicated in Ku70/80 dissociation from DNA. Here, we identify RNF126 as a ubiquitin ligase that is recruited to DSBs and ubiquitylates Ku80, with UBE2D3 serving as an E2 enzyme. Knockdown of RNF126 prevented Ku70/80 dissociation from DSBs and inhibited break repair. Attenuation of Ku80 ubiquitylation by replacement of ubiquitylation site lysines with arginine residues delayed Ku70/80 release from chromatin after DSB induction by genotoxic insults. Together, our data indicate that RNF126 is a novel regulator of NHEJ that promotes completion of DNA repair by ubiquitylating Ku80 and releasing Ku70/80 from damaged DNA.

The subcellular localization and activity of cortactin is regulated by acetylation and interaction with Keap1.

Cortactin is an F-actin-binding protein that localizes to the cell cortex, where the actin remodeling that is required for cell migration occurs. We found that cortactin shuttled between the cytoplasm and the nucleus under basal conditions. We identified Kelch-like ECH-associated protein 1 (Keap1), a cytosolic protein that is involved in oxidant stress responses, as a binding partner of cortactin that promoted the cortical localization of cortactin and cell migration. The ability of cortactin to promote cell migration is regulated by various posttranslational modifications, including acetylation. We showed that the acetylated form of cortactin was mainly localized to the nucleus and that acetylation of cortactin decreased cell migration by inhibiting the binding of cortactin to Keap1. Our findings reveal that Keap1 regulates cell migration by affecting the subcellular localization and activity of cortactin independently of its role in oxidant stress responses.

CACUL1/CAC1 Regulates the Antioxidant Response by Stabilizing Nrf2.

Nrf2 is the pre-dominant transcription activator responsible for coordinated up-regulation of ARE-driven antioxidant and detoxification genes. The activity of Nrf2 is tightly regulated at basal levels through its ubiquitination by Cul3-Keap1 and consequential degradation. Upon exposure to stress, the Cul3-Keap1 ligase is inhibited, leading to Nrf2 stabilization and activation. Here we describe CACUL1/CAC1 as a positive regulator of the Nrf2 pathway. We found that CACUL1 is up-regulated by Nrf2-activating oxidative stresses in cells and in mice. The association of CACUL1 with the Cul3-Keap1 complex led to a decrease in Nrf2 ubiquitination levels at non-stressed as well as stressed conditions, and sensitized cells for higher Nrf2 activation. Furthermore, CACUL1 knock-down led to a decrease in Nrf2 activity and cell viability under stress. Our results show that CACUL1 is a regulator of Nrf2 ubiquitination, adding another regulatory layer to the Nrf2 antioxidant stress response.

TBL2 is a novel PERK-binding protein that modulates stress-signaling and cell survival during endoplasmic reticulum stress.

Under ER stress, PKR-like ER-resident kinase (PERK) phosphorylates translation initiation factor eIF2α, resulting in repression of global protein synthesis and concomitant upregulation of the translation of specific mRNAs such as activating transcription factor 4 (ATF4). This PERK function is important for cell survival under ER stress and poor nutrient conditions. However, mechanisms of the PERK signaling pathway are not thoroughly understood. Here we identify transducin (beta)-like 2 (TBL2) as a novel PERK-binding protein. We found that TBL2 is an ER-localized type-I transmembrane protein and preferentially binds to the phosphorylated form of PERK, but not another eIF2α kinase GCN2 or ER-resident kinase IRE1, under ER stress. Immunoprecipitation analysis using various deletion mutants revealed that TBL2 interacts with PERK via the N-terminus proximal region and also associates with eIF2α via the WD40 domain. In addition, TBL2 knockdown can lead to impaired ATF4 induction under ER stress or poor nutrient conditions such as glucose and oxygen deprivation. Consistently, TBL2 knockdown rendered cells vulnerable to stresses similarly to PERK knockdown. Thus, TBL2 serves as a potential regulator of the PERK pathway.

The role of acetylation in the subcellular localization of an oncogenic isoform of translation factor eIF5A.

Mammalian cells express two isoforms of eIF5A, eIF5A1 and eIF5A2, but little is known about the function of eIF5A2. Here we report that eIF5A2 is reversibly acetylated at lysine-47. HDAC6 and SIRT2 were identified as the enzymes responsible for deacetylating eIF5A2. Analysis using acetylation-deficient mutants indicated that acetylation regulates the subcellular localization of eIF5A2.

Acetylation regulates subcellular localization of eukaryotic translation initiation factor 5A (eIF5A).

Eukaryotic translation initiation factor 5A (eIF5A) is a protein subject to hypusination, which is essential for its function. eIF5A is also acetylated, but the role of that modification is unknown. Here, we report that acetylation regulates the subcellular localization of eIF5A. We identified PCAF as the major cellular acetyltransferase of eIF5A, and HDAC6 and SIRT2 as its major deacetylases. Inhibition of the deacetylases or impaired hypusination increased acetylation of eIF5A, leading to nuclear accumulation. As eIF5A is constitutively hypusinated under physiological conditions, we suggest that reversible acetylation plays a major role in controlling the subcellular localization of eIF5A.

Identification and functional analysis of Zranb2 as a novel Smad-binding protein that suppresses BMP signaling.

Smads 1/5/8 transduce the major intracellular signaling of bone morphogenetic proteins (BMPs). In the present study, we analyzed Smad1-binding proteins in HEK293T cells using a proteomic technique and identified the protein, zinc-finger, RAN-binding domain-containing protein 2 (ZRANB2). Zranb2 interacted strongly with Smad1, Smad5, and Smad8 and weakly with Smad4. The overexpression of Zranb2 inhibited BMP activities in C2C12 myoblasts in vitro, and the injection of Zranb2 mRNA into zebrafish embryos induced weak dorsalization. Deletion analyses of Zranb2 indicated that the serine/arginine-rich (SR) domain and the glutamine-rich domain were required for the inhibition of BMP activity and the interaction with Smad1, respectively. Zranb2 was found to be localized in the nucleus; however, the SR domain-deleted mutant localized to the cytoplasm. The knockdown of endogenous Zranb2 in C2C12 cells enhanced BMP activity. Zranb2 suppressed Smad transcriptional activity without affecting Smad phosphorylation, nuclear localization, or DNA binding. Taken together, these findings suggested that Zranb2 is a novel BMP suppressor that forms a complex with Smads in the nucleus.

The PX-RICS-14-3-3zeta/theta complex couples N-cadherin-beta-catenin with dynein-dynactin to mediate its export from the endoplasmic reticulum.

We have recently shown that beta-catenin-facilitated export of cadherins from the endoplasmic reticulum requires PX-RICS, a beta-catenin-interacting GTPase-activating protein for Cdc42. Here we show that PX-RICS interacts with isoforms of 14-3-3 and couples the N-cadherin-beta-catenin complex to the microtubule-based molecular motor dynein-dynactin. Similar to knockdown of PX-RICS, knockdown of either 14-3-3zeta or - resulted in the disappearance of N-cadherin and beta-catenin from the cell-cell boundaries. Furthermore, we found that PX-RICS and 14-3-3zeta/ are present in a large multiprotein complex that contains dynein-dynactin components as well as N-cadherin and beta-catenin. Both RNAi- and dynamitin-mediated inhibition of dynein-dynactin function also led to the absence of N-cadherin and beta-catenin at the cell-cell contact sites. Our results suggest that the PX-RICS-14-3-3zeta/ complex links the N-cadherin-beta-catenin cargo with the dynein-dynactin motor and thereby mediates its endoplasmic reticulum export.