APOBEC3B expression is promoted by lincNMR collaborating with TGF-ß-Smad pathway.

Long non-coding RNAs (lncRNAs) participate in carcinogenesis and cancer malignancies. Transforming growth factor-ß (TGF-ß) is involved in various cellular processes including cancer progression. We performed comprehensive RNA sequencing analyses to identify lncRNAs regulated by TGF-ß and found that lincNMR (long intergenic noncoding RNA-nucleotide metabolism regulator, also identified as MAP3K9-DT) was induced by TGF-ß in various cell lines. There are several variants of lincNMR (hereafter lincNMRs) in the lincNMR/MAP3K9-DT locus, and their expression was increased by TGF-ß. TGF-ß-mediated induction of lincNMRs was decreased by depletion of Smad2/3 in Huh7, suggesting that the TGF-ß-Smad pathway is involved in lincNMRs expression. We also found that APOBEC3B but not other APOBEC family members were a target gene of lincNMRs. APOBEC3B, a cytidine deaminase, promotes C to U mutation and highly expressed in various human cancers. Although it is associated with cancer progression, regulatory mechanisms of APOBEC3B expression have not been fully elucidated. We performed RNA immunoprecipitation assays and proved that lincNMRs bound to endogenous Smad2 in Huh7 cells. The increased activity of the promoter of APOBEC3B induced by overexpression of Smad2/3 was inhibited by depletion of lincNMRs. These data suggest that lincNMRs participate in APOBEC3B expression by collaborating with TGF-ß-Smad pathway. High expression of lincNMRs was positively correlated with high expression of APOBEC3B in various cancer cell lines. Overexpression of APOBEC3B as well as lincNMR was found in human cancers such as hepatic and lung cancers and was associated with their poor prognosis, suggesting that lincNMR may contribute to tumor malignancy via enhanced expression of APOBEC3B.

Homologous recombination is reduced in female embryonic stem cells by two active X chromosomes.

The reactivation of X-linked genes is observed in some primary breast tumors. Two active X chromosomes are also observed in female embryonic stem cells (ESCs), but whether double doses of X-linked genes affect DNA repair efficiency remains unclear. Here, we establish isogenic female/male ESCs and show that the female ESCs are more sensitive to camptothecin and have lower gene targeting efficiency than male ESCs, suggesting that homologous recombination (HR) efficiency is reduced in female ESCs. We also generate Xist-inducible female ESCs and show that the lower HR efficiency is restored when X chromosome inactivation is induced. Finally, we assess the X-linked genes with a role in DNA repair and find that Brcc3 is one of the genes involved in a network promoting proper HR. Our findings link the double doses of X-linked genes with lower DNA repair activity, and this may have relevance for common diseases in female patients, such as breast cancer.

Expression of Tumor Suppressor FHIT Is Regulated by the LINC00173-SNAIL Axis in Human Lung Adenocarcinoma.

Long non-coding RNAs (lncRNAs) play a critical role in a variety of human diseases such as cancer. Here, to elucidate a novel function of a lncRNA called LINC00173, we investigated its binding partner, target gene, and its regulatory mechanism in lung adenocarcinoma, including the A549 cell line and patients. In the A549 cell line, RNA immunoprecipitation (RIP) assays revealed that LINC00173 efficiently binds to SNAIL. RNA-seq and RT-qPCR analyses revealed that the expression of FHIT was decreased upon LINC00173 depletion, indicating that FHIT is a target gene of LINC00173. Overexpression of SNAIL suppressed and depletion of SNAIL increased the expression of FHIT, indicating that SNAIL negatively regulates FHIT. The downregulation of FHIT expression upon LINC00173 depletion was restored by additional SNAIL depletion, revealing a LINC00173-SNAIL-FHIT axis for FHIT regulation. Data from 501 patients with lung adenocarcinoma also support the existence of a LINC00173-SNAIL-FHIT axis, as FHIT expression correlated positively with LINC00173 (p = 1.75 × 10-6) and negatively with SNAIL (p = 7.00 × 10-5). Taken together, we propose that LINC00173 positively regulates FHIT gene expression by binding to SNAIL and inhibiting its function in human lung adenocarcinoma. Thus, this study sheds light on the LINC00173-SNAIL-FHIT axis, which may be a key mechanism for carcinogenesis and progression in human lung adenocarcinoma.

Molecular Pathogenesis of Pulmonary Fibrosis, with Focus on Pathways Related to TGF-ß and the Ubiquitin-Proteasome Pathway.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease. During the past decade, novel pathogenic mechanisms of IPF have been elucidated that have shifted the concept of IPF from an inflammatory-driven to an epithelial-driven disease. Dysregulated repair responses induced by recurrent epithelial cell damage and excessive extracellular matrix accumulation result in pulmonary fibrosis. Although there is currently no curative therapy for IPF, two medications, pirfenidone and nintedanib, have been introduced based on understanding the pathogenesis of the disease. In this review, we discuss advances in understanding IPF pathogenesis, highlighting epithelial-mesenchymal transition (EMT), the ubiquitin-proteasome system, and endothelial cells. TGF-ß is a central regulator involved in EMT and pulmonary fibrosis. HECT-, RING finger-, and U-box-type E3 ubiquitin ligases regulate TGF-ß-Smad pathway-mediated EMT via the ubiquitin-proteasome pathway. p27 degradation mediated by the SCF-type E3 ligase, Skp2, contributes to the progression of pulmonary fibrosis by promotion of either mesenchymal fibroblast proliferation, EMT, or both. In addition to fibroblasts as key effector cells in myofibroblast differentiation and extracellular matrix deposition, endothelial cells also play a role in the processes of IPF. Endothelial cells can transform into myofibroblasts; therefore, endothelial-mesenchymal transition can be another source of myofibroblasts.

Inhibiting Skp2 E3 Ligase Suppresses Bleomycin-Induced Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with poor prognosis and no curative therapies. SCF-Skp2 E3 ligase is a target for cancer therapy, but there have been no reports about Skp2 as a target for IPF. Here we demonstrate that Skp2 is a promising therapeutic target for IPF. We examined whether disrupting Skp2 suppressed pulmonary fibrosis in a bleomycin (BLM)-induced mouse model and found that pulmonary fibrosis was significantly suppressed in Skp2-deficient mice compared with controls. The pulmonary accumulation of fibrotic markers such as collagen type 1 and fibronectin in BLM-infused mice was decreased in Skp2-deficient mice. Moreover, the number of bronchoalveolar lavage fluid cells accompanied with pulmonary fibrosis was significantly diminished. Levels of the Skp2 target p27 were significantly decreased by BLM-administration in wild-type mice, but recovered in Skp2-/- mice. In vimentin-positive mesenchymal fibroblasts, the decrease of p27-positive cells and increase of Ki67-positive cells by BLM-administration was suppressed by Skp2-deficency. As these results suggested that inhibiting Skp2 might be effective for BLM-induced pulmonary fibrosis, we next performed a treatment experiment using the Skp2 inhibitor SZL-P1-41. As expected, BLM-induced pulmonary fibrosis was significantly inhibited by SZL-P1-41. Moreover, p27 levels were increased by the SZL-P1-41 treatment, suggesting p27 may be an important Skp2 target for BLM-induced pulmonary fibrosis. Our study suggests that Skp2 is a potential molecular target for human pulmonary fibrosis including IPF.

Tenascin C in colorectal cancer stroma is a predictive marker for liver metastasis and is a potent target of miR-198 as identified by microRNA analysis.

BACKGROUND: Tumour stroma has important roles in the development of colorectal cancer (CRC) metastasis. We aimed to clarify the roles of microRNAs (miRNAs) and their target genes in CRC stroma in the development of liver metastasis. METHODS: Tumour stroma was isolated from formalin-fixed, paraffin-embedded tissues of primary CRCs with or without liver metastasis by laser capture microdissection, and miRNA expression was analysed using TaqMan miRNA arrays. RESULTS: Hierarchical clustering classified 16 CRCs into two groups according to the existence of synchronous liver metastasis. Combinatory target prediction identified tenascin C as a predicted target of miR-198, one of the top 10 miRNAs downregulated in tumour stroma of CRCs with synchronous liver metastasis. Immunohistochemical analysis of tenascin C in 139 primary CRCs revealed that a high staining intensity was correlated with synchronous liver metastasis (P<0.001). Univariate and multivariate analyses revealed that the tenascin C staining intensity was an independent prognostic factor to predict postoperative overall survival (P=0.005; n=139) and liver metastasis-free survival (P=0.001; n=128). CONCLUSIONS: Alterations of miRNAs in CRC stroma appear to form a metastasis-permissive environment that can elevate tenascin C to promote liver metastasis. Tenascin C in primary CRC stroma has the potential to be a novel biomarker to predict postoperative prognosis.

Regulation of GATA-binding protein 2 levels via ubiquitin-dependent degradation by Fbw7: involvement of cyclin B-cyclin-dependent kinase 1-mediated phosphorylation of THR176 in GATA-binding protein 2.

A GATA family transcription factor, GATA-binding protein 2 (GATA2), participates in cell growth and differentiation of various cells, such as hematopoietic stem cells. Although its expression level is controlled by transcriptional induction and proteolytic degradation, the responsible E3 ligase has not been identified. Here, we demonstrate that F-box/WD repeat-containing protein 7 (Fbw7/Fbxw7), a component of Skp1, Cullin 1, F-box-containing complex (SCF)-type E3 ligase, is an E3 ligase for GATA2. GATA2 contains a cell division control protein 4 (Cdc4) phosphodegron (CPD), a consensus motif for ubiquitylation by Fbw7, which includes Thr(176). Ectopic expression of Fbw7 destabilized GATA2 and promoted its proteasomal degradation. Substitution of threonine 176 to alanine in GATA2 inhibited binding with Fbw7, and the ubiquitylation and degradation of GATA2 by Fbw7 was suppressed. The CPD kinase, which mediates the phosphorylation of Thr(176), was cyclin B-cyclin-dependent kinase 1 (CDK1). Moreover, depletion of endogenous Fbw7 stabilized endogenous GATA2 in K562 cells. Conditional Fbw7 depletion in mice increased GATA2 levels in hematopoietic stem cells and myeloid progenitors at the early stage. Increased GATA2 levels in Fbw7-conditional knock-out mice were correlated with a decrease in a c-Kit high expressing population of myeloid progenitor cells. Our results suggest that Fbw7 is a bona fide E3 ubiquitin ligase for GATA2 in vivo.

Chk1 phosphorylates the tumour suppressor Mig-6, regulating the activation of EGF signalling.

The tumour suppressor gene product Mig-6 acts as an inhibitor of epidermal growth factor (EGF) signalling. However, its posttranslational modifications and regulatory mechanisms have not been elucidated. Here, we investigated the phosphorylation of human Mig-6 and found that Chk1 phosphorylated Mig-6 in vivo as well as in vitro. Moreover, EGF stimulation promoted phosphorylation of Mig-6 without DNA damage and the phosphorylation was inhibited by depletion of Chk1. EGF also increased Ser280-phosphorylated Chk1, a cytoplasmic-tethering form, via PI3K pathway. Mass spectrometric analyses suggested that Ser 251 of Mig-6 was a major phosphorylation site by Chk1 in vitro and in vivo. Substitution of Ser 251 to alanine increased inhibitory activity of Mig-6 against EGF receptor (EGFR) activation. Moreover, EGF-dependent activation of EGFR and cell growth were inhibited by Chk1 depletion, and were rescued by co-depletion of Mig-6. Our results suggest that Chk1 phosphorylates Mig-6 on Ser 251, resulting in the inhibition of Mig-6, and that Chk1 acts as a positive regulator of EGF signalling. This is a novel function of Chk1.

Interaction between RB protein and NuMA is required for proper alignment of spindle microtubules.

Retinoblastoma protein (pRB) controls cell cycle progression and cell cycle exit through interactions with cellular proteins. Many pRB-binding proteins, which function in gene transcription or modulation of chromatin structure, harbor LXCXE motifs in their binding domain for pRB. In this study, we found that nuclear mitotic apparatus protein (NuMA), a mitotic spindle organizer, interacts with pRB through LSCEE sequences located in its C-terminal region. siRNA-mediated down-regulation of pRB caused aberrant distribution of NuMA and alignment of spindle microtubules in mitotic cells. Abnormal organization of spindle microtubules was also accompanied by misalignment of an over-expressed NuMA mutant (mut-NuMA) with a defect in pRB binding caused by an LSGEK mutation. The mut-NuMA-over-expressing cells showed lower potency for survival than wild-type NuMA (wt-NuMA)-over-expressing cells during 2 weeks of culture. Interestingly, knockdown of pRB reduced the population of wt-NuMA-over-expressing cells to the same level as mut-NuMA cells after 2 weeks. Taken together, pRB may have a novel function in regulating the mitotic function of NuMA and spindle organization, which are required for proper cell cycle progression.

YB-1 promotes transcription of cyclin D1 in human non-small-cell lung cancers.

Cyclin D1, an oncogenic G1 cyclin, and YB-1, a transcription factor involved in cell growth, are both over-expressed in several human cancers. In human lung cancer, the functional association between YB-1 and cyclin D1 has never been elucidated. In this study, we show YB-1 is involved in the transcription of cyclin D1 in human lung cancer. Depletion of endogenous YB-1 by siRNA inhibited progression of G1 phase and down-regulated both the protein and mRNA levels of cyclin D1 in human lung cancer cells. Forced over-expression of YB-1 with a cyclin D1 reporter plasmid increased luciferase activity, and ChIP assay results showed YB-1 bound to the cyclin D1 promoter. Moreover, the amount of YB-1 mRNA positively correlated with cyclin D1 mRNA levels in clinical non-small-cell lung cancer (NSCLC) specimens. Immunohistochemical analysis also indicated YB-1 expression correlated with cyclin D1 expression in NSCLC specimens. In addition, most of the cases expressing both cyclin D1 and CDC6, another molecule controlled by YB-1, had co-existing YB-1 over-expression. Together, our results suggest that aberrant expression of both cyclin D1 and CDC6 by YB-1 over-expression may collaboratively participate in lung carcinogenesis.

Anti-VEGF antibody therapy induces tumor hypoxia and stanniocalcin 2 expression and potentiates growth of human colon cancer xenografts.

Tumor angiogenesis plays a critical role in colorectal cancer progression. Recent randomized clinical trials have revealed the additive effect of bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF)-A, to conventional chemotherapy in the improved survival of patients with metastatic colorectal cancer. However, a number of preclinical reports indicate the development of resistance to anti-angiogenic therapy. In this study, we addressed the effects of anti-VEGF antibodies on the growth and malignant behavior of colorectal cancer cells. TK-4, a solid tumor strain derived from a colon cancer patient, was subcutaneously or orthotopically implanted into nude mice. Short-term administration of anti-VEGF antibodies inhibited the growth of cecal tumors at day 14 by suppressing mitosis, but prolonged treatment resulted in the recovery of cellular proliferation and suppression of apoptosis at day 35. Intratumoral hypoxia induced by anti-VEGF antibody treatment resulted in activation of hypoxia inducible factor-1α protein and an increased number of aldehyde dehydrogenase 1-positive tumor cells. In microarray analysis, stanniocalcin 2 (STC2) was the most highly upregulated gene in anti-VEGF antibody-treated tumors. In vitro analyses showed that the growth and migration of SW480 colon cancer cells under hypoxic conditions were significantly inhibited by knockdown of STC2. In vivo serial transplantation of TK-4 revealed that long-term administration of anti-VEGF antibodies increased the tumorigenicity of colon cancers and accelerated tumor growth when transplanted into secondary recipient mice. Our data provide a potential molecular explanation for the limited clinical effectiveness of anti-VEGF antibodies.

Coordinated regulation of differentiation and proliferation of embryonic cardiomyocytes by a jumonji (Jarid2)-cyclin D1 pathway.

In general, cell proliferation and differentiation show an inverse relationship, and are regulated in a coordinated manner during development. Embryonic cardiomyocytes must support embryonic life by functional differentiation such as beating, and proliferate actively to increase the size of the heart. Therefore, progression of both proliferation and differentiation is indispensable. It remains unknown whether proliferation and differentiation are related in these embryonic cardiomyocytes. We focused on abnormal phenotypes, such as hyperproliferation, inhibition of differentiation and enhanced expression of cyclin D1 in cardiomyocytes of mice with mutant jumonji (Jmj, Jarid2), which encodes the repressor of cyclin D1. Analysis of Jmj/cyclin D1 double mutant mice showed that Jmj was required for normal differentiation and normal expression of GATA4 protein through cyclin D1. Analysis of transgenic mice revealed that enhanced expression of cyclin D1 decreased GATA4 protein expression and inhibited the differentiation of cardiomyocytes in a CDK4/6-dependent manner, and that exogenous expression of GATA4 rescued the abnormal differentiation. Finally, CDK4 phosphorylated GATA4 directly, which promoted the degradation of GATA4 in cultured cells. These results suggest that CDK4 activated by cyclin D1 inhibits differentiation of cardiomyocytes by degradation of GATA4, and that initiation of Jmj expression unleashes the inhibition by repression of cyclin D1 expression and allows progression of differentiation, as well as repression of proliferation. Thus, a Jmj-cyclin D1 pathway coordinately regulates proliferation and differentiation of cardiomyocytes.

YB1 binds to and represses the p16 tumor suppressor gene.

Y box binding protein 1 (YB1) has multiple functions associated with drug resistance, cell proliferation and metastasis through transcriptional and translational regulation. Increased expression of YB1 is closely related to tumor growth and aggressiveness. We showed that YB1 protein levels were decreased through replicative and premature senescence and were correlated with increased expression levels of p16(INK) (4A) tumor suppressor gene. Depletion of YB1 was associated with increased levels of p16 in human and murine primary cells. Forced expression of YB1 in mouse embryonic fibroblasts resulted in decreased expression of p16 and increased cell proliferation. Senescence-associated expression of ß-galactosidase was repressed in YB1-over-expressing cells. Chromatin immunoprecipitation assays showed that YB1 directly associates with the p16 promoter. Taken together, all our findings indicate that YB1 directly binds to and represses p16 transcription, subsequently resulting in the promotion of cell growth and prevention of cellular senescence.

Roles of the Skp2/p27 axis in the progression of chronic nephropathy.

S-phase kinase-associated protein 2 (Skp2) is an F-box protein component of the Skp/Cullin/F-box-type E3 ubiquitin ligase that targets several cell cycle regulatory proteins for degradation through the ubiquitin-dependent pathway. Skp2-mediated degradation of p27, a cyclin-dependent kinase inhibitor, is involved in cell cycle regulation. Tubular epithelial cell proliferation is a characteristic feature of renal damage that is apparent in the early stages of nephropathy. The p27 level is associated with the progression of renal injury, and increased Skp2 expression in progressive nephropathy is implicated in decreases of p27 expression. In Skp2(-/-) mice, renal damage caused by unilateral ureteral obstruction (UUO) was ameliorated by p27 accumulation, mainly in tubular epithelial cells. However, the amelioration of UUO-induced renal injury in Skp2(-/-) mice was prevented by p27 deficiency in Skp2(-/-)/p27(-/-) mice. These results suggest that the Skp2-mediated reduction in p27 is a pathogenic activity that occurs during the progression of nephropathy. Here, we discuss the roles of the Skp2/p27 axis and/or related signaling pathways/components in the progression of chronic nephropathy.

Cell cycle regulation by long non-coding RNAs.

The mammalian cell cycle is precisely controlled by cyclin-dependent kinases (CDKs) and related pathways such as the RB and p53 pathways. Recent research on long non-coding RNAs (lncRNAs) indicates that many lncRNAs are involved in the regulation of critical cell cycle regulators such as the cyclins, CDKs, CDK inhibitors, pRB, and p53. These lncRNAs act as epigenetic regulators, transcription factor regulators, post-transcription regulators, and protein scaffolds. These cell cycle-regulated lncRNAs mainly control cellular levels of cell cycle regulators via various mechanisms, and may provide diversity and reliability to the general cell cycle. Interestingly, several lncRNAs are induced by DNA damage and participate in cell cycle arrest or induction of apoptosis as DNA damage responses. Therefore, deregulations of these cell cycle regulatory lncRNAs may be involved in tumorigenesis, and they are novel candidate molecular targets for cancer therapy and diagnosis.

p130RB2 positively contributes to ATR activation in response to replication stress via the RPA32-ETAA1 axis.

Ataxia-telangiectasia mutated and Rad3-related (ATR) kinase is a crucial regulator of the cell cycle checkpoint and activated in response to DNA replication stress by two independent pathways via RPA32-ETAA1 and TopBP1. However, the precise activation mechanism of ATR by the RPA32-ETAA1 pathway remains unclear. Here, we show that p130RB2, a member of the retinoblastoma protein family, participates in the pathway under hydroxyurea-induced DNA replication stress. p130RB2 binds to ETAA1, but not TopBP1, and depletion of p130RB2 inhibits the RPA32-ETAA1 interaction under replication stress. Moreover, p130RB2 depletion reduces ATR activation accompanied by phosphorylation of its targets RPA32, Chk1, and ATR itself. It also causes improper re-progression of S phase with retaining single-stranded DNA after cancelation of the stress, which leads to an increase in the anaphase bridge phenotype and a decrease in cell survival. Importantly, restoration of p130RB2 rescued the disrupted phenotypes of p130RB2 knockdown cells. These results suggest positive involvement of p130RB2 in the RPA32-ETAA1-ATR axis and proper re-progression of the cell cycle to maintain genome integrity.

CCIVR2 facilitates comprehensive identification of both overlapping and non-overlapping antisense transcripts within specified regions.

Pairs of sense and antisense transcriptions that are adjacent at their 5' and 3' regions are called divergent and convergent transcription, respectively. However, the structural properties of divergent/convergent transcription in different species or RNA biotypes are poorly characterized. Here, we developed CCIVR2, a program that facilitates identification of both overlapping and non-overlapping antisense transcripts produced from divergent/convergent transcription whose transcription start sites (TSS) or transcript end sites (TES) are located within a specified region. We used CCIVR2 to analyze antisense transcripts starting around the sense TSS (from divergent transcription) or ending around the sense TES (from convergent transcription) in 11 different species and found species- and RNA biotype-specific features of divergent/convergent transcription. Furthermore, we confirmed that CCIVR2 enables the identification of multiple sense/antisense transcript pairs from divergent transcription, including those with known functions in processes such as embryonic stem cell differentiation and TGFß stimulation. CCIVR2 is therefore a valuable bioinformatics tool that facilitates the characterization of divergent/convergent transcription in different species and aids the identification of functional sense/antisense transcript pairs from divergent transcription in specified biological processes.

Up-regulation of Cks1 and Skp2 with TNFα/NF-κB signaling in chronic progressive nephropathy.

The cyclin-dependent kinase (CDK) inhibitor p27 level is associated with progression of renal damage. We previously reported that mRNA of Skp2, a component of Skp/Cullin/F-box (SCF)-ubiquitin ligase which targets to p27, was increased in unilateral ureteral obstructive kidneys in mice and that the nephritis was attenuated in Skp2-deficient mice. However, the details have not been fully clarified. Here, we found that not only Skp2 but also cdc kinase subunit 1 (Cks1), an essential cofactor for the SCF-Skp2 ubiquitin ligase in targeting p27, was increased in another chronic progressive model, anti-thymocyte serum (ATS) rat nephropathy. After induction of ATS nephropathy, Skp2(+) /Cks1(+) /Ki67(+) tubular epithelial cell numbers increased, and p27(+) tubular epithelial cells decreased transiently. Moreover, we found that TNFα was involved in expression of both Skp2 and Cks1 in NRK cell line as well as the in ATS nephropathy. Nuclear accumulations of NF-κB subunits RelB and p52 were increased in the tubular epithelial cells of the nephritic kidney. Both Skp2 and Cks1 were colocalized with RelB in these cells. These data suggest that both Skp2 and Cks1 are up-regulated by the TNFα-RelB/p52 pathway in the early stages of renal damage and are collaboratively involved in down-regulation of p27 in proliferative tubular dilation and the progression of chronic nephropathy.

CCIVR facilitates comprehensive identification of cis-natural antisense transcripts with their structural characteristics and expression profiles.

Cis-natural antisense transcripts (cis-NATs) are transcribed from the same genomic locus as their partner gene but from the opposite DNA strand and overlap with the partner gene transcript. Here, we developed a simple and convenient program termed CCIVR (comprehensive cis-NATs identifier via RNA-seq data) that comprehensively identifies all kinds of cis-NATs based on genome annotation with expression data obtained from RNA-seq. Using CCIVR with genome databases, we demonstrated total cis-NAT pairs from 11 model organisms. CCIVR analysis with RNA-seq data from parthenogenetic and androgenetic embryonic stem cells identified well-known imprinted cis-NAT pair, KCNQ1/KCNQ1OT1, ensuring the availability of CCIVR. Finally, CCIVR identified cis-NAT pairs that demonstrate inversely correlated expression upon TGFß stimulation including cis-NATs that functionally repress their partner genes by introducing epigenetic alteration in the promoters of partner genes. Thus, CCIVR facilitates the investigation of structural characteristics and functions of cis-NATs in numerous processes in various species.

Stabilization of CDK6 by ribosomal protein uS7, a target protein of the natural product fucoxanthinol.

Cyclins and cyclin-dependent kinases (CDKs) regulate the cell cycle, which is important for cell proliferation and development. Cyclins bind to and activate CDKs, which then drive the cell cycle. The expression of cyclins periodically changes throughout the cell cycle, while that of CDKs remains constant. To elucidate the mechanisms underlying the constant expression of CDKs, we search for compounds that alter their expression and discover that the natural product fucoxanthinol downregulates CDK2, 4, and 6 expression. We then develop a method to immobilize a compound with a hydroxyl group onto FG beads® and identify human ribosomal protein uS7 (also known as ribosomal protein S5) as the major fucoxanthinol-binding protein using the beads and mass spectrometry. The knockdown of uS7 induces G1 cell cycle arrest with the downregulation of CDK6 in colon cancer cells. CDK6, but not CDK2 or CDK4, is degraded by the depletion of uS7, and we furthermore find that uS7 directly binds to CDK6. Fucoxanthinol decreases uS7 at the protein level in colon cancer cells. By identifying the binding proteins of a natural product, the present study reveals that ribosomal protein uS7 may contribute to the constant expression of CDK6 via a direct interaction.