Molecular and Cellular Biology最新文献

CCN2/connective tissue growth factor (CTGF) potentially serves as a therapeutic target for chronic kidney disease. Here we investigated CCN2 module-4, encoded by Ccn2 exon 5, through the generation of Ccn2 exon 5 knockout mice (Ex5^-/- mice). To investigate renal fibrosis pathogenesis, Ex5^-/- mice were employed to model unilateral ureteral obstruction (UUO), unilateral ischemic-reperfusion injury (UIRI), and 5/6 nephrectomy. Interstitial fibrosis was significantly attenuated in the Ex5^-/- mice in the three models. Furthermore, phosphorylated focal adhesion kinase (FAK) levels in tubular epithelial cells were significantly lower in the kidneys of the UUO- and UIRI-Ex5^-/- mice than those of the Ex5^+/+ mice. Moreover, CCN2 module 4-mediated renal tubule FAK and promoted fibrosis. These findings indicate that CCN2 module-4-FAK pathway components will serve as therapeutic targets for effectively attenuating renal fibrosis.

Claspin plays multiple important roles in regulation of DNA replication as a mediator for the cellular response to replication stress, an integral replication fork factor that facilitates replication fork progression and a factor that promotes initiation by recruiting Cdc7 kinase. Here, we report a novel role of Claspin in growth recovery from serum starvation, which requires the activation of PI3 kinase (PI3K)-PDK1-Akt-mTOR pathways. In the absence of Claspin, cells do not proceed into S phase and eventually die partially in a ROS- and p53-dependent manner. Claspin directly interacts with PI3K and mTOR, and is required for activation of PI3K-PDK1-mTOR and for that of mTOR downstream factors, p70S6K and 4EBP1, but not for p38 MAPK cascade during the recovery from serum starvation. PDK1 physically interacts with Claspin, notably with CKBD, in a manner dependent on phosphorylation of the latter protein, and is required for interaction of mTOR with Claspin. Thus, Claspin plays a novel role as a key regulator for nutrition-induced proliferation/survival signaling by activating the mTOR pathway. The results also suggest a possibility that Claspin may serve as a common mediator that receives signals from different PI3K-related kinases and transmit them to specific downstream kinases.

The physiological roles of aryl hydrocarbon receptor (AhR) in the small intestine have been revealed as immunomodulatory and barrier functions. However, its contributions to cell fate regulation are incompletely understood. The Notch-activated signaling cascade is a central component of intestinal cell fate determinations. The lateral inhibitory mechanism governed by Notch directs cell fates toward distinct cell lineages (i.e., absorptive and secretory cell lineages) through its downstream effector, mouse atonal homolog 1 (MATH1). An investigation employing cell lines and intestinal crypt cells revealed that AhR regulates Math1 expression in a xenobiotic response element (XRE)-dependent manner. The AhR-Math1 axis was further addressed using intestinal organoids, where AhR-Math1 and HES1-Math1 axes appeared to coexist within the underlying Math1 transcriptional machinery. When the HES1-Math1 axis was pharmacologically suppressed, β-naphthoflavone-mediated AhR activation increased the number of goblet and Math1⁺ progenitor cells in the organoids. The same pharmacological dissection of the AhR-Math1 axis was applied in vivo, demonstrating an enhanced number of Math1⁺ progenitor cells in the small intestine following AhR activation. We report here that AhR-Math1 is a direct transcriptional axis with effects on Math1⁺ progenitor cells in the small intestine, highlighting a novel molecular basis for fine-tuning Notch-mediated cell fate regulation.

Rif1 mediates telomere length, DNA replication, and DNA damage responses in budding yeast. Previous work identified several posttranslational modifications of Rif1, however none of these was shown to mediate the molecular or cellular responses to DNA damage, including telomere damage. We searched for such modifications using immunoblotting methods and the cdc13-1 and tlc1Δ models of telomere damage. We found that Rif1 is phosphorylated during telomere damage, and that serines 57 and 110 within a novel phospho-gate domain (PGD) of Rif1 are important for this modification, in cdc13-1 cells. The phosphorylation of Rif1 appeared to inhibit its accumulation on damaged chromosomes and the proliferation of cells with telomere damage. Moreover, we found that checkpoint kinases were upstream of this Rif1 phosphorylation and that the Cdk1 activity was essential for maintaining it. Apart from telomere damage, S57 and S110 were essential for Rif1 phosphorylation during the treatment of cells with genotoxic agents or during mitotic stress. We propose a speculative "Pliers" model to explain the role of the PGD phosphorylation during telomere and other types of damage.

IRES mediated translation initiation requires a different repertoire of factors than canonical cap-dependent translation. Treatments that inhibit the canonical translation factor EIF4G1 have little or no effect on the ability of the Insr and Igf1r cellular IRESes to promote translation. Transcripts for two cellular receptors contain RNA elements that facilitate translation initiation without intact EIF4G1. Cellular IRES mechanisms may resemble viral type III IRESes allowing them to promote translate with a limited number of initiation factors allowing them to work under stress conditions when canonical translation is repressed.

PURPL is a p53-induced lncRNA that suppresses basal p53 levels. Here, we investigated PURPL upon p53 activation in liver cancer cells, where it is expressed at significantly higher levels than other cell types. Using isoform sequencing, we discovered novel PURPL transcripts that have a retained intron and/or previously unannotated exons. To determine PURPL function upon p53 activation, we performed transcriptome sequencing (RNA-Seq) after depleting PURPL using CRISPR interference (CRISPRi), followed by Nutlin treatment to induce p53. Strikingly, although loss of PURPL in untreated cells altered the expression of only 7 genes, loss of PURPL resulted in altered expression of ~800 genes upon p53 activation, revealing a context-dependent function of PURPL. Pathway analysis suggested that PURPL is important for fine-tuning the expression of specific genes required for mitosis. Consistent with these results, we observed a significant decrease in the percentage of mitotic cells upon PURPL depletion. Collectively, these data identify novel transcripts from the PURPL locus and suggest that PURPL delicately moderates the expression of mitotic genes in the context of p53 activation to control cell cycle arrest.

Transcriptional gene silencing by small interfering RNAs (siRNAs) has been widely described in various species, including plants and yeast. In mammals, its extent remains somewhat debated. Previous studies showed that siRNAs targeting gene promoters could induce the silencing of the targeted promoter, although the involvement of off-target mechanisms was also suggested. Here, by using nascent RNA capture and RNA polymerase II chromatin immunoprecipitation, we show that siRNAs targeting a chromatin-associated noncoding RNA induced its transcriptional silencing. Deletion of the sequence targeted by one of these siRNAs on the two alleles by genome editing further showed that this silencing was due to base-pairing of the siRNA to the target. Moreover, by using cells with heterozygous deletion of the target sequence, we showed that only the wild-type allele, but not the deleted allele, was silenced by the siRNA, indicating that transcriptional silencing occurred only in cis. Finally, we demonstrated that both Ago1 and Ago2 are involved in this transcriptional silencing. Altogether, our data demonstrate that siRNAs targeting a chromatin-associated RNA at a distance from its promoter induce its transcriptional silencing. Our results thus extend the possible repertoire of endogenous or exogenous interfering RNAs.

Mitochondria play essential and specific roles during erythroid differentiation. Recently, FAM210B, encoding a mitochondrial inner membrane protein, has been identified as a novel target of GATA-1, as well as an erythropoietin-inducible gene. While FAM210B protein is involved in regulate mitochondrial metabolism and heme biosynthesis, its detailed function remains unknown. Here, we generated both knockout and knockdown of endogenous FAM210B in human induced pluripotent stem-derived erythroid progenitor (HiDEP) cells using CRISPR/Cas9 methodology. Intriguingly, erythroid differentiation was more pronounced in the FAM210B-depleted cells, and this resulted in increased frequency of orthochromatic erythroblasts and decreased frequencies of basophilic/polychromatic erythroblasts. Comprehensive metabolite analysis and functional analysis indicated that oxygen consumption rates and the NAD (NAD⁺)/NADH ratio were significantly decreased, while lactate production was significantly increased in FAM210B deletion HiDEP cells, indicating involvement of FAM210B in mitochondrial energy metabolism in erythroblasts. Finally, we purified FAM210B-interacting protein from K562 cells that stably expressed His/biotin-tagged FAM210B. Mass spectrometry analysis of the His/biotin-purified material indicated interactions with multiple subunits of mitochondrial ATP synthases, such as subunit alpha (ATP5A) and beta (ATP5B). Our results suggested that FAM210B contributes prominently to erythroid differentiation by regulating mitochondrial energy metabolism. Our results provide insights into the pathophysiology of dysregulated hematopoiesis.