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BBA - Gene Regulatory Mechanisms (v.1809, #7)

Editorial Board (pp. i).

Identification of Evi-1 as a novel effector of PKCδ in the apoptotic response to DNA damage by Hoi Chin Hew; Hanshao Liu; Zheng-Guang Lu; Junko Kimura; Yoshio Miki; Kiyotsugu Yoshida (pp. 285-294).

Protein kinase C delta (PKCδ), a PKC family isoform, regulates diverse signal transduction pathways during DNA damage to induce apoptosis. To explore the apoptosis mechanism that PKCδ modulates, we sought to uncover transcription factor targets of PKCδ by devising a screening strategy that utilizes ChIP-cloning and microarray analysis. Transcription factor candidates were generated with the application of public access data-mining tools and this resulted in the identification of Evi-1 as a novel PKCδ-mediated DNA damage responsive molecule. The results demonstrated that PKCδ is constitutively associated with Evi-1. PKCδ regulated Evi-1 to activate PLZF transcription upon genotoxic stress. Furthermore, both Evi-1 and PLZF were associated with DNA damage-stimulated apoptosis. Taken together, we have discovered a novel regulation of Evi-1, which transactivates PLZF, by PKCδ to induce cell death in response to genotoxic stress.Display Omitted► PKCδ induces apoptosis in response to DNA damage. ► PKCδ regulates Evi-1 to activate PLZF transcription. ► Evi-1 is associated with DNA damage-stimulated cell death.

Keywords: Abbreviations; ChIP; chromatin immunoprecipitation; HSC; haematopoietic stem cells; PKCδ; protein kinase C δ; PLZF; promyotic leukemic zinc fingerPKCδ; Evi-1; PLZF; DNA damage; Apoptosis

Identification and characterization of the major alternative promoter regulating Bcrp1/Abcg2 expression in the mouse intestine by Karthika Natarajan; Yi Xie; Takeo Nakanishi; William T. Beck; Kenneth S. Bauer; Douglas D. Ross (pp. 295-305).

Mouse models are often used to predict drug absorption in humans. Mouse Bcrp1 protein exhibits sequence and functional homology with human BCRP protein. Additionally, BCRP/Bcrp1 expression is regulated by alternative promoter usage in humans and mice; however, the precise intestine-specific alternative promoter utilized in either species is yet to be determined. Therefore we sought to identify and characterize the mouse intestinal Bcrp1 promoter. Using real-time quantitative RT-PCR and 5′ RACE PCR we first established the predominance of a single Bcrp1 first exon (E1b) in the Bcrp1 mRNA isolated throughout the mouse intestine. Simultaneously using 5′ RACE PCR we identified E1C as the predominant BCRP 5′ UTR expressed in the human intestine. Next we established functional activity for the murine promoter upstream of E1b using reporter assays. Subsequently using deletion-construct analysis we found the core promoter region to span −231 to −42bps from the transcriptional start site of E1b. We then predicted a cAMP response element (CRE) as a transcription factor binding site unique only to the E1b promoter region, using in silico methods. We finally established functional interaction of phospho-CREB (p-CREB) protein with the CRE on the E1b promoter using both functional assays and chromatin immunoprecipitation assays. In conclusion, mouse intestinal Bcrp1 expression is regulated by a single alternative promoter upstream of E1b, the predominant Bcrp1 mRNA isoform expressed in the mouse intestine. Furthermore, Bcrp1 E1b mRNA expression is regulated by binding of p-CREB to its cis site on the mouse E1b promoter region.► Human intestinal BCRP expression is regulated by the alternative promoter E1C. ► Mouse intestinal Bcrp1 expression is regulated by the alternative promoter E1b. ► A p-CREB response element (CRE) was found in mouse E1b promoter but not in the other mouse alternative Bcrp1 promoters. ► p-CREB directly binds and activates transcription from Bcrp1 E1b promoter. ► cAMP plays a role in regulation of mouse small intestinal Bcrp1 transcription.

Keywords: Bcrp1/Abcg2 alternative promoter; Mouse small intestine; BCRP/ABCG2 alternative promoter; Human intestine; p-CREB; CRE

Dickkopf homolog 1, a Wnt signaling antagonist, is transcriptionally up-regulated via an ATF4-independent and MAPK/ERK-dependent pathway following amino acid deprivation by Dan Zhou; Yukun Zhang; Yuan-Xiang Pan; Hong Chen (pp. 306-315).

Amino acid response (AAR) pathway is activated when cells are deprived of amino acids. In the present study, using the human colon cancer cell line SW480, we observed that DKK1, an antagonist of the Wnt pathway, was significantly induced at the mRNA level after the removal of amino acids from the medium. Addition of the amino alcohol histidinol, which prevents the formation of histidinyl-tRNA^His, also increased DKK1 mRNA to a level similar to that observed when cells were deprived of all amino acids. Transcriptional activity and stability of DKK1 mRNA were both increased in the amino acid-deprived condition. The induction of DKK1 gene expression was confirmed by the increased immunofluorescent staining of the DKK1 protein in the amino acid deprived condition. Although chromatin immunoprecipitation assays showed increased RNA Polymerase II binding at the DKK1 promoter in amino acid-limited conditions, ATF4 binding to the promoter is absent. Luciferase reporter assays did not detect any functional AARE within the DKK1 gene structure. Knockdown of ATF4 by siRNA did not affect the increase of DKK1 mRNA during amino acid limitation. Inhibition of ERK phosphorylation abolished the induction of DKK1. Our study revealed that DKK1 is a novel target gene in the response to amino acid deficiency and that the expression of DKK1 is up-regulated through an ATF4-independent and an ERK-dependent pathway.► The Wnt signaling pathway was affected by amino acid deprivation through the induction of DKK1. ► Transcriptional regulation of DKK1 by amino acid deficiency was by the phosphorylation of ERK. ► Knockdown of ATF4 does not inhibit the induction of DKK1 mRNA. ► The study demonstrated the interactions among Wnt, AAR, and MAPK/ERK signaling pathways in a colon cancer cell line. ► DKK1 is regulated by a novel amino acid response pathway that is independent of the master regulator ATF4.

Keywords: Abbreviations; AAR; amino acid response; AARE; amino acids response elements; ATF; activating transcription factor; ASNS; asparagines synthetase; DKK1; dickkopf homolog 1; eIF2α; eukaryotic initiation factor 2 alpha; ERK; extracellular signal-regulated kinase; GCN2; general control non-derepressible 2; HisOH; histidinol; MAPK; mitogen-activated protein kinase; p-eIF2α; phosphorylated eukaryotic initiation factor 2 alpha; p-ERK; phosphorylated extracellular signal-regulated kinase; Pol II; RNA Polymerase II; Raf; rapidly accelerated fibrosarcoma proteinAmino acid deficiency; ATF4; DKK1; Phosphorylated ERK; Transcription; Wnt

The nuclear exporter, Crm1, is regulated by NFY and Sp1 in cancer cells and repressed by p53 in response to DNA damage by Pauline J. van der Watt; Virna D. Leaner (pp. 316-326).

The nuclear exporter protein, Crm1, plays a key role in normal cell functioning, mediating the nucleo-cytoplasmic transport of cargo proteins. Elevated Crm1 expression has recently been identified in various tumours; however, the mechanisms driving its expression have not been investigated to date. In this study we identified the Crm1 promoter and factors associated with its elevated expression and with its repression under conditions of DNA damage. The −1405 to +99 Crm1 promoter region was found to be significantly more active in cancer and transformed cells compared to normal, and the −175 to +99 region identified as responsible for the differential activity. Mutation of two CCAAT boxes and a GC box within this region significantly diminished Crm1 promoter activity and ChIP analysis revealed binding of NFY and Sp1 to these sites, with increased binding in transformed and cancer cells. In addition, p53 was found to repress Crm1 promoter activity, after induction with doxorubicin, with p53 siRNA blocking the effect. Crm1 promoter constructs with mutated CCAAT boxes were significantly less responsive to p53 repression, and in vivo binding of NFY to the CCAAT boxes was diminished upon p53 binding, suggesting that p53 mediates repression of the Crm1 promoter via interfering with NFY. This was confirmed using NFY knock-down cells, in which Crm1 promoter activity was significantly less responsive to p53. In vitro EMSAs revealed that NFY and p53 bind the CCAAT boxes as a single complex under conditions of DNA damage. In summary, this study is a first to analyse Crm1 promoter regulation and reveals NFY and Sp1 as contributors to Crm1 overexpression in cancer. In addition, this study reveals that Crm1 transcription is inhibited by DNA damage and that the mechanism of inhibition involves p53 interfering with NFY function.► Crm1 expression is elevated in cancer cells and inhibited in response to DNA damage. ► Sp1 positively regulates Crm1 expression through a GC box at +24 to +36. ► NFY binding at two CCAAT boxes is necessary for Crm1 expression. ► p53 induction negatively regulates Crm1 expression via interfering with NFY activity.

Keywords: Crm1; NFY; Sp1; p53; Cancer

Changes in gene expression induced by Sp1 knockdown differ from those caused by challenging Sp1 binding to gene promoters by Sylvia Mansilla; Waldemar Priebe; José Portugal (pp. 327-336).

C/G-rich DNA regions, which include those recognized by the Sp1 transcription factor in several gene promoters, also encompass potential binding sites for the DNA-intercalating anthracyclines doxorubicin and WP631. We explored the differences between changes in gene expression caused by the ability of these drugs to compete with Sp1 for binding to DNA and those produced by Sp1 knockdown. By quantitative RT-PCR of around 100 genes, most of them involved in control of cell cycle progression, we found that the treatment of human MDA-MB231 breast carcinoma cells with bis-anthracycline WP631 for 24h produced a profile of gene down-regulation markedly different from the profile caused by doxorubicin treatment or by stable Sp1 knockdown. These observations are rationalized by considering a near-specific effect of WP631 on Sp1 interaction with several gene promoters, thus representing potential therapeutic targets for WP631, in contrast to a less specific effect of reducing the availability of Sp1 through RNA interference. Genes down-regulated upon each treatment were mapped to their molecular and biological functions, which documented the down-regulation, among other things, of genes involved in mRNA transcription regulation, granting us insights into the effects of challenging the transactivation of gene expression by Sp1.► In MDA-MB231 cells, bis-anthracycline WP631 produced gene down-regulation different from that caused by Sp1 knockdown. ► A near-specific effect of WP631 on Sp1 interaction with promoters was observed. ► Genes down-regulated upon treatment with WP631 or doxorubicin, or after Sp1 knockdown, were mapped to their functions. ► Down-regulation was observed in genes involved in mRNA transcription regulation.

Keywords: Gene expression; MDA-MB231 cells; shRNA; Sp1 knockdown; WP631

Choreographing pluripotency and cell fate with transcription factors by Kevin Andrew Uy Gonzales; Huck-Hui Ng (pp. 337-349).

The cellular identity of both pluripotent and differentiated cells is defined by the concerted interplay of transcriptional factors as well as other modulators such as epigenetic and signaling mediators. Therefore, the manipulation of a cell's transcriptional network directly facilitates inter-conversion between cellular identities. Understanding the molecular regulation of cell fate changes, including those involved in pluripotency, is crucial in realizing the practical potential of pluripotent and induced cell types. Here we review the advancements in the role of transcription factors in pluripotency, as well as in the conversion between and within pluripotent and somatic cell types.► Regulation of cellular identity by transcription factors. ► Transition between cellular identities by manipulating transcriptional networks. ► Developments in unraveling the transcriptional circuitry of pluripotent states. ► Recent advancements in reprogramming and transdifferentiation.

Keywords: Abbreviations; ChIP; chromatin immunoprecipitation; ESC; embryonic stem cell; EpiSC; epiblast stem cell; FGF; fibroblast growth factor; hESC; human ESC; hiPSC; human iPSC; ICM; inner cell mass; iPSC; induced pluripotent stem cell; LIF; leukemia inhibitory factor; lincRNA; large intergenic non-coding RNA; mESC; mouse ESC; miPSC; mouse iPSC; miRNA; microRNA; ncRNA; non-coding RNA; NOD; non-obese diabetic; OSKM; Oct4, Sox2, Klf4 and c-Myc; RNAi; RNA interference; SCNT; somatic cell nuclear transfer; siRNA; small interfering RNA; TGF-β; transforming growth factor betaTranscription factor; Stem cell; Pluripotency; Reprogramming; Transdifferentiation

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BBA - Gene Regulatory Mechanisms (v.1809, #7)