Global Analysis of CPEBs Reveals Sequential and Non-Redundant Functions in Mitotic Cell Cycle

<div><p>CPEB (Cytoplasmic Polyadenylation Element Binding) proteins are a family of four RNA-binding proteins that regulate the translation of maternal mRNAs controlling meiotic cell cycle progression. But CPEBs are not limited to the transcriptionally silent germline; they are also expressed, in various combinations, in somatic cells, yet their role in regulation of mitosis-related gene expression is largely unknown. Deregulation of CPEB1 and CPEB4 have been linked to tumor development. However, a systematic analysis addressing their requirements for the temporal regulation of mitotic gene expression has yet to be performed. This study addresses the requirements of each of the four CPEBs for mitotic phase transitions, with a particular focus on cytoplasmic polyadenylation and translational regulation. We demonstrate that CPEB3 is the only member dispensable for mitotic cell division, whereas the other three members, CPEB1, 2, and 4, are essential to successful mitotic cell division. Thus, CPEB1 is required for prophase entry, CPEB2 for metaphase and CPEB4 for cytokinesis. These three CPEBs have sequential non-redundant functions that promote the phase-specific polyadenylation and translational activation of CPE-regulated transcripts in the mitotic cell cycle.</p></div

CPEB1 is required for prophase entry; CPEB2 for metaphase-to-anaphase transition and CPEB4 for cytokinesis.

<p>(A) HEK-293 cells stably expressing an IPTG-inducible system for each CPEB knock-down were induced or not with IPTG. Two days after induction, cells were transfected with a plasmid encoding fluorescent histon H2B. After one additional day cells were recorded by live imaging experiments. Images were acquired every 10 minutes and analyzed for mitotic progression. Representative images are shown for individual cells in interphase or during specific phases of mitosis that are readily identified by chromosome condensation state and organisation. (B) Mitotic-stage analysis of 50 cells from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138794#pone.0138794.s002" target="_blank">S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138794#pone.0138794.s006" target="_blank">S5</a> Videos as in Fig 2A. On Y axis, each lane represents one cell. On X axis, time is represented as minutes. Colors represent the indicated mitotic phases. Mitotic entry was determined by analyzing the first signs of DNA condensation cross-reinforced with cell-rounding. Mitotic exit was scored based on chromosome segregation at anaphase and DNA decondensation. Sh, short-hairpin; CTRL, control.</p

CPEB1, CPEB2 and CPEB4 are required for proper GFP translation at G2 and M phases.

<p>(A) HEK-293 cells stably expressing an IPTG-inducible system for each CPEB knock-down and carrying a GFP-3'UTR +/-CPE together with a RFP-3'UTR were induced or not with IPTG. Two days after induction, cells were recorded by live imaging experiments and analyzed for GFP expression (see Fig I in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138794#pone.0138794.s001" target="_blank">S1 File</a>, for RFP expression). Representative images from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138794#pone.0138794.s007" target="_blank">S6</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138794#pone.0138794.s012" target="_blank">S11</a> Videos are shown. (B) HEK-293 cells stably expressing an IPTG-inducible system for each CPEB knock-down and cells carrying a GFP-3'UTR with mutated CPE (CPE-) were induced or not with IPTG. Two days after induction cells were synchronized by double thymidine blockade (DTB). Samples were collected at the indicated phases of the cell cycle and GFP expression was measured by FACS. Results are shown as the mean value from six experiments, error bars indicate s.d; au, arbitrary units; Sh, short-hairpin; CTRL, control.</p

CPEB1, CPEB2 and CPEB4 are required for cell cycle progression.

<p>(A) HEK-293 cells stably expressing an IPTG-inducible system for CPEB1,2 and 4 knock-down were induced or not with IPTG. Two days after induction cells were synchronized through double thymidine blockade (DTB). Samples were harvested at the indicated time points and protein lysates were analyzed on SDS–PAGE followed by immunoblotting for the indicated proteins. α-tubulin was used as a loading control. (B) HEK-293 cells stably expressing an IPTG-inducible system for each CPEB knock-down were induced or not with IPTG. Three days after induction, cells were marked with EdU for two hours and then released. Samples were harvested at the indicated time points, stained for DNA content with PI and analyzed by FACS. Mean EdU⁺ cell-population: 23100 for control, 10800 for CPEB1-KD cells, 16400 for CPEB2-KD cells, 20900 for CPEB3-KD cells, 18900 for CPEB4-KD cells). Sh, short-hairpin; CTRL, control; IPTG, Isopropyl β-D-1-thiogalactopyranoside; DTB, double-thymidine block; Edu, 5-ethynyl-2'-deoxyuridine; PI, propidium iodide; ES, early S-phase; MS, middle S-phase; LS, late S-phase; FACS, fluorescence-activated cell sorter.</p

Sequential functions of CPEB1 and CPEB4 regulate pathologic expression of vascular endothelial growth factor and angiogenesis in chronic liver disease.

BACKGROUND & AIMS: Vascular endothelial growth factor (VEGF) regulates angiogenesis, yet therapeutic strategies to disrupt VEGF signaling can interfere with physiologic angiogenesis. In a search for ways to inhibit pathologic production or activities of VEGF without affecting its normal production or functions, we investigated the post-transcriptional regulation of VEGF by the cytoplasmic polyadenylation element-binding proteins CPEB1 and CPEB4 during development of portal hypertension and liver disease. METHODS: We obtained transjugular liver biopsies from patients with hepatitis C virus-associated cirrhosis or liver tissues removed during transplantation; healthy human liver tissue was obtained from a commercial source (control). We also performed experiments with male Sprague-Dawley rats and CPEB-deficient mice (C57BL6 or mixed C57BL6/129 background) and their wild-type littermates. Secondary biliary cirrhosis was induced in rats by bile duct ligation, and portal hypertension was induced by partial portal vein ligation. Liver and mesenteric tissues were collected and analyzed in angiogenesis, reverse transcription polymerase chain reaction, polyA tail, 3' rapid amplification of complementary DNA ends, Southern blot, immunoblot, histologic, immunohistochemical, immunofluorescence, and confocal microscopy assays. CPEB was knocked down with small interfering RNAs in H5V endothelial cells, and translation of luciferase reporters constructs was assessed. RESULTS: Activation of CPEB1 promoted alternative nuclear processing within noncoding 3'-untranslated regions of VEGF and CPEB4 messenger RNAs in H5V cells, resulting in deletion of translation repressor elements. The subsequent overexpression of CPEB4 promoted cytoplasmic polyadenylation of VEGF messenger RNA, increasing its translation; the high levels of VEGF produced by these cells led to their formation of tubular structures in Matrigel assays. We observed increased levels of CPEB1 and CPEB4 in cirrhotic liver tissues from patients, compared with control tissue, as well as in livers and mesenteries of rats and mice with cirrhosis or/and portal hypertension. Mice with knockdown of CPEB1 or CPEB4 did not overexpress VEGF or have signs of mesenteric neovascularization, and developed less-severe forms of portal hypertension after portal vein ligation. CONCLUSIONS: We identified a mechanism of VEGF overexpression in liver and mesentery that promotes pathologic, but not physiologic, angiogenesis, via sequential and nonredundant functions of CPEB1 and CPEB4. Regulation of CPEB4 by CPEB1 and the CPEB4 autoamplification loop induces pathologic angiogenesis. Strategies to block the activities of CPEBs might be developed to treat chronic liver and other angiogenesis-dependent diseases.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO; SAF2011-29491 and SAF2014-55473-R to MF; BFU2011-30121, BFU2014-54122-P and Consolider RNAREG CSD2009-00080 to RM; PI13/00341 to JB; PI11/01562 and PI14/00125 to PN), Generalitat de Catalunya (SGR1436 to RM; SGR1108 to JB), Fundación Botín by Banco Santander through its Santander Universities Global Division (to RM), AECC Scientific Foundation (to RM and MF), Worldwide Cancer Research (to RM and MF) and RETIC Cancer RD12/0036/0051/FEDER to PN. GFM is funded by a Juan de la Cierva contract from MINECO. IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (Government of Spain). CIBERehd is an initiative from the Instituto de Salud Carlos III. We also thank Dr Francisco X Real for his contribution to knockout mice generation