FBXL16 is a novel E2F1-regulated gene commonly upregulated in p16INK4A- and p14ARF-silenced HeLa cells.

Two crucial cell cycle regulators, p16INK4A and p14ARF, are produced from the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene locus by alternative reading frames; these regulators act as tumor suppressors during tumorigenesis. However, the molecular events incidental to the acute functional loss of CDKN2A remain a critical issue. Two pivotal regulatory pathways of cell fate determination involving p16INK4A/retinoblastoma protein (pRb)/E2F1 and p14ARF/p53 interact tightly with each other; however, novel factors with an integral or overlapping role in these two pathways remain incompletely defined. To this end, we specifically decreased the expression of p16INK4A or p14ARF proteins using RNA interference (RNAi) in HeLa cells. Using a DNA microarray approach, we showed that several genes are commonly regulated in both p16INK4A and p14ARF knockdown cells, compared with control RNA-treated cells. We focused on the FBXL16 (F-box and leucine-rich repeat protein 16) gene, the expression of which was reproducibly upregulated in p16INK4A and p14ARF knockdown cells, as evaluated using RT-PCR. Interestingly, the promoter region of FBXL16 was shown to be upregulated by activator E2Fs. Finally, RNAi-mediated knockdown of FBXL16 increased the cell proliferation rate of HeLa cells. Together, our results illustrate a unique aspect of the interdependence between the p16INK4A/pRb/E2F1 and p14ARF/p53 pathways at a molecular level.

Expression and tissue distribution of human X-linked armadillo repeat containing-6.

The armadillo family of proteins has been implicated in embryogenesis and tumorigenesis. Armadillo repeat containing X-linked (ARMCX)1-6 and its most closely related protein, ARMC10, share an uncharacterized domain in their carboxyl-terminal region and thereby constitute a unique subfamily. We previously detected an elevated ARMCX6 mRNA level in human cervical carcinoma (HeLa) cells in which the cyclin-dependent kinase inhibitor 2A products (p16INK4A and p14ARF) were silenced by RNA interference; however, the function of the ARMCX6 gene has yet to be determined. In the present study, we demonstrated that ARMCX6 tagged with green fluorescent protein was localized in the cytoplasm of HeLa cells. ARMCX6 mRNA was highly detected in the pancreas and spleen, indicating a tissue-specific expression pattern in human tissues. Taken together, these results suggest that ARMCX6 may be uniquely involved in a specific cellular function of certain tissues.

Transcriptional co-factor CDCA4 participates in the regulation of JUN oncogene expression.

CDCA4, a member of the TRIP-Br transcriptional co-factor family, has been shown to possess a unique role in regulating the transcriptional activities of p53 as well as E2F1 transcription factors. In this study, we aimed to identify a pivotal transcriptional target gene regulated by CDCA4, so we suppressed CDCA4 expression by CDCA4-specific short interference RNA (siRNA) in HeLa cells, and then performed a DNA microarray analysis. Among the identified genes, we focused on JUN, 14-3-3eta, and IL6ST (gp130) mRNAs which were up-regulated in CDCA4-specific siRNA-transfected cells compared to control siRNA-transfected cells. We confirmed that JUN, 14-3-3eta, and IL6ST proteins were up-regulated when cells were transfected with CDCA4-specific siRNA. 14-3-3eta and IL6ST protein levels were unchanged upon on transfection of cells with JUN-specific siRNA, indicating that 14-3-3eta and IL6ST genes are not a direct target of JUN. Serine 63 and 73 phosphorylation of JUN was unchanged when cells were transfected with CDCA4-specific siRNA. In addition, JUN-driven reporter activity was unaffected by CDCA4 co-transfection, suggesting that CDCA4 affects solely JUN mRNA expression. Finally, by preparing various JUN promoter reporter constructs, we minimized the JUN promoter sequence that was affected by CDCA4 co-expression. Together, these results add an important role of CDCA4 in the context of transcriptional regulation and cell fate determination through the JUN oncogene.

Transcriptional regulation of human polo-like kinases and early mitotic inhibitor.

Human polo-like kinases (PLK1-PLK4) have been implicated in mitotic regulation and carcinogenesis. PLK1 phosphorylates early mitotic inhibitor 1 (Emi1) to ensure mitosis entry, whereas Emi2 plays a key role during the meiotic cell cycle. Transcription factor E2F is primarily considered to regulate the G(1)/S transition of the cell cycle but its involvement in the regulation of mitosis has also been recently suggested. A gap still exists between the molecular basis of E2F and mitotic regulation. The present study was designed to characterize the transcriptional regulation of human PLK and Emi genes. Adenoviral overexpression of E2F1 increased PLK1 and PLK3 mRNA levels in A549 cells. A reporter gene assay revealed that the putative promoter regions of PLK1, PLK3, and PLK4 genes were responsive to activators E2F, E2F1-E2F3. We further characterized the putative promoter regions of Emi1 and Emi2 genes, and these could be regulated by activators E2F and E2F1-E2F4, respectively. Finally, PLK1-PLK4, Emi1, and Emi2 mRNA expression levels in human adult, fetal tissues, and several cell lines indicated that each gene has a unique expression pattern but is uniquely expressed in common tissues and cells such as the testes and thymus. Collectively, these results indicate that E2F can integrate G(1)/S and G(2)/M to oscillate the cell cycle by regulating mitotic genes PLK and Emi, leading to determination of the cell fate.

Puma is a novel target of soy isoflavone genistein but is dispensable for genistein-induced cell fate determination.

Here, we attempted to identify novel target genes of genistein in human A549 cells. Using analysis of proteins related to cell cycle and apoptotic pathways, we confirmed an elevated level of p53 accompanying p21 Waf1/Cip1 protein in genistein-treated or genistin-treated A549 and WI-38 cells, but not in HeLa cells. In addition, a p53-upregulated modulator of apoptosis (Puma) protein accumulated significantly in genistein-treated A549 and WI-38 cells, but not in genistin-treated or beta-estradiol-treated cells, though the growth of any ingredient-treated cells was severely inhibited. Intriguingly, the caspase-3 activity of genistein-treated A549 cells, in which Puma or p53 expression was knocked-down by RNA interference (RNAi), remained unaltered compared to that in cells transfected with irrelevant RNAi. These results raise a concern that molecular targets identified by powerful omic approaches may not necessarily represent key molecules responsible for given cellular phenotypes and thus must be verified by conclusive assays.

Transcriptional regulation of an evolutionary conserved intergenic region of CDT2-INTS7.

BACKGROUND: In the mammalian genome, a substantial number of gene pairs (approximately 10%) are arranged head-to-head on opposite strands within 1,000 base pairs, and separated by a bidirectional promoter(s) that generally drives the co-expression of both genes and results in functional coupling. The significance of unique genomic configuration remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: Here we report on the identification of an intergenic region of non-homologous genes, CDT2, a regulator of DNA replication, and an integrator complex subunit 7 (INTS7), an interactor of the largest subunit of RNA polymerase II. The CDT2-INTS7 intergenic region is 246 and 245 base pairs long in human and mouse respectively and is evolutionary well-conserved among several mammalian species. By measuring the luciferase activity in A549 cells, the intergenic human sequence was shown to be able to drive the reporter gene expression in either direction and notably, among transcription factors E2F, E2F1 approximately E2F4, but not E2F5 and E2F6, this sequence clearly up-regulated the reporter gene expression exclusively in the direction of the CDT2 gene. In contrast, B-Myb, c-Myb, and p53 down-regulated the reporter gene expression in the transcriptional direction of the INTS7 gene. Overexpression of E2F1 by adenoviral-mediated gene transfer resulted in an increased CDT2, but not INTS7, mRNA level. Real-time polymerase transcription (RT-PCR) analyses of the expression pattern for CDT2 and INTS7 mRNA in human adult and fetal tissues and cell lines revealed that transcription of these two genes are asymmetrically regulated. Moreover, the abundance of mRNA between mouse and rat tissues was similar, but these patterns were quite different from the results obtained from human tissues. CONCLUSIONS/SIGNIFICANCE: These findings add a unique example and help to understand the mechanistic insights into the regulation of gene expression through an evolutionary conserved intergenic region of the mammalian genome.

Systems biology-based identification of crosstalk between E2F transcription factors and the Fanconi anemia pathway.

Fanconi anemia (FA) is an autosomal recessive disorder characterized by congenital abnormalities, bone marrow failure, chromosome fragility, and cancer susceptibility. At least eleven members of the FA gene family have been identified using complementation experiments. Ubiquitin-proteasome has been shown to be a key regulator of FA proteins and their involvement in the repair of DNA damage. Here, we identified a novel functional link between the FA/BRCA pathway and E2F-mediated cell cycle regulome. In silico mining of a transcriptome database and promoter analyses revealed that a significant number of FA gene members were regulated by E2F transcription factors, known to be pivotal regulators of cell cycle progression - as previously described for BRCA1. Our findings suggest that E2Fs partly determine cell fate through the FA/BRCA pathway.

A combined computational and experimental study on the structure-regulation relationships of putative mammalian DNA replication initiator GINS.

GINS, a heterotetramer of SLD5, PSF1, PSF2, and PSF3 proteins, is an emerging chromatin factor recognized to be involved in the initiation and elongation step of DNA replication. Although the yeast and Xenopus GINS genes are well documented, their orthologous genes in higher eukaryotes are not fully characterized. In this study, we report the genomic structure and transcriptional regulation of mammalian GINS genes. Serum stimulation increased the GINS mRNA levels in human cells. Reporter gene assay using putative GINS promoter sequences revealed that the expression of mammalian GINS is regulated by 17beta-Estradiol-stimulated estrogen receptor alpha, and human PSF3 acts as a gene responsive to transcription factor E2F1. The goal of this study is to present the current data so as to encourage further work in the field of GINS gene regulation and functions in mammalian cells.