Identification of an evolutionarily conserved cis-regulatory element controlling the Peg3 imprinted domain.

The mammalian Peg3 domain harbors more than 20 evolutionarily conserved regions (ECRs) that are spread over the 250-kb genomic interval. The majority of these ECRs are marked with two histone modifications, H3K4me1 and H3K27ac, suggesting potential roles as distant regulatory elements for the transcription of the nearby imprinted genes. In the current study, the chromatin conformation capture (3C) method was utilized to detect potential interactions of these ECRs with the imprinted genes. According to the results, one region, ECR18, located 200-kb upstream of Peg3 interacts with the two promoter regions of Peg3 and Zim2. The observed interaction is most prominent in brain, but was also detected in testis. Histone modification and DNA methylation on ECR18 show no allele bias, implying that this region is likely functional on both alleles. In vitro assays also reveal ECR18 as a potential enhancer or repressor for the promoter of Peg3. Overall, these results indicate that the promoters of several imprinted genes in the Peg3 domain interact with one evolutionarily conserved region, ECR18, and further suggest that ECR18 may play key roles in the transcription and imprinting control of the Peg3 domain as a distant regulatory element.

DNA-binding motif and target genes of the imprinted transcription factor PEG3.

The Peg3 gene is expressed only from the paternally inherited allele located on proximal mouse chromosome 7. The PEG3 protein encoded by this imprinted gene is predicted to bind DNA based on its multiple zinc finger motifs and nuclear localization. In the current study, we demonstrated PEG3's DNA-binding ability by characterizing its binding motif and target genes. We successfully identified target regions bound by PEG3 from mouse brain extracts using chromatin immunoprecipitation analysis. PEG3 was demonstrated to bind these candidate regions through the consensus DNA-binding motif AGTnnCnnnTGGCT. In vitro promoter assays established that PEG3 controls the expression of a given gene through this motif. Consistent with these observations, the transcriptional levels of a subset of the target genes are also affected in a mutant mouse model with reduced levels of PEG3 protein. Overall, these results confirm PEG3 as a DNA-binding protein controlling specific target genes that are involved in distinct cellular functions.

NAC1 is an actin-binding protein that is essential for effective cytokinesis in cancer cells.

NAC1 is a transcriptional corepressor protein that is essential to sustain cancer cell proliferation and migration. However, the underlying molecular mechanisms of NAC1 function in cancer cells remain unknown. In this study, we show that NAC1 functions as an actin monomer-binding protein. The conserved BTB protein interaction domain in NAC1 is the minimal region for actin binding. Disrupting NAC1 complex function by dominant-negative or siRNA strategies reduced cell retraction and abscission during late-stage cytokinesis, causing multinucleation in cancer cells. In Nac1-deficient murine fibroblasts, restoring NAC1 expression was sufficient to partially avert multinucleation. We found that siRNA-mediated silencing of the actin-binding protein profilin-1 in cancer cells caused a similar multinucleation phenotype and that NAC1 modulated the binding of actin to profillin-1. Taken together, our results indicate that the NAC1/actin/profilin-1 complex is crucial for cancer cell cytokinesis, with a variety of important biologic and clinical implications.

Identification of PBX1 target genes in cancer cells by global mapping of PBX1 binding sites.

PBX1 is a TALE homeodomain transcription factor involved in organogenesis and tumorigenesis. Although it has been shown that ovarian, breast, and melanoma cancer cells depend on PBX1 for cell growth and survival, the molecular mechanism of how PBX1 promotes tumorigenesis remains unclear. Here, we applied an integrated approach by overlapping PBX1 ChIP-chip targets with the PBX1-regulated transcriptome in ovarian cancer cells to identify genes whose transcription was directly regulated by PBX1. We further determined if PBX1 target genes identified in ovarian cancer cells were co-overexpressed with PBX1 in carcinoma tissues. By analyzing TCGA gene expression microarray datasets from ovarian serous carcinomas, we found co-upregulation of PBX1 and a significant number of its direct target genes. Among the PBX1 target genes, a homeodomain protein MEOX1 whose DNA binding motif was enriched in PBX1-immunoprecipicated DNA sequences was selected for functional analysis. We demonstrated that MEOX1 protein interacts with PBX1 protein and inhibition of MEOX1 yields a similar growth inhibitory phenotype as PBX1 suppression. Furthermore, ectopically expressed MEOX1 functionally rescued the PBX1-withdrawn effect, suggesting MEOX1 mediates the cellular growth signal of PBX1. These results demonstrate that MEOX1 is a critical target gene and cofactor of PBX1 in ovarian cancers.

Defining NOTCH3 target genes in ovarian cancer.

NOTCH3 gene amplification plays an important role in the progression of many ovarian and breast cancers, but the targets of NOTCH3 signaling are unclear. Here, we report the use of an integrated systems biology approach to identify direct target genes for NOTCH3. Transcriptome analysis showed that suppression of NOTCH signaling in ovarian and breast cancer cells led to downregulation of genes in pathways involved in cell-cycle regulation and nucleotide metabolism. Chromatin immunoprecipitation (ChIP)-on-chip analysis defined promoter target sequences, including a new CSL binding motif (N1) in addition to the canonical CSL binding motif, that were occupied by the NOTCH3/CSL transcription complex. Integration of transcriptome and ChIP-on-chip data showed that the ChIP target genes overlapped significantly with the NOTCH-regulated transcriptome in ovarian cancer cells. From the set of genes identified, we showed that the mitotic apparatus organizing protein DLGAP5 (HURP/DLG7) was a critical target. Both the N1 motif and the canonical CSL binding motif were essential to activate DLGAP5 transcription. DLGAP5 silencing in cancer cells suppressed tumorigenicity and inhibited cellular proliferation by arresting the cell cycle at the G(2)-M phase. In contrast, enforced expression of DLGAP5 partially counteracted the growth inhibitory effects of a pharmacologic or RNA interference-mediated NOTCH inhibition in cancer cells. Our findings define direct target genes of NOTCH3 and highlight the role of DLGAP5 in mediating the function of NOTCH3.

Oncogenic potential of yin yang 1 mediated through control of imprinted genes.

The transcription factor Yin Yang (YY) 1 is one of the most evolutionarily well-conserved DNA binding proteins that is ubiquitously expressed among different tissue types. YY1 functions as a critical regulator for a diverse set of genes, making its role in the cancerous environment elusive. Recent studies have demonstrated that clusters of YY1 binding sites are overrepresented in imprinted gene loci. These clustered binding sites may function as a molecular rheostat with respect to YY1 protein levels. YY1 levels were documented to be altered in various tumor tissues in conjunction with the transcriptional levels of the imprinted genes it regulates. This review highlights the unexplored mechanism through which fluctuations in YY1 protein levels alter the transcriptional status of imprinted genes containing clustered YY1 binding sites, which potentially could affect cancer development and/or progression.

Protein or amino acid deprivation differentially regulates the hepatic forkhead box protein A (FOXA) genes through an activating transcription factor-4-independent pathway.

The FOXA (forkhead box A) proteins (FOXA1, FOXA2, and FOXA3) play a critical role in the development of the liver, and they also regulate metabolism in adult hepatic tissue. The liver responds to changes in nutrient availability by initiating a number of stress signaling pathways. The present studies demonstrated that in mouse dams fed a low-protein diet hepatic expression of FOXA2 and FOXA3 messenger RNA, but not FOXA1, was induced. Conversely, fetal liver did not exhibit this regulation. Amino acid deprivation of HepG2 hepatoma cells also enhanced transcription from the FOXA2 and FOXA3 genes. In contrast, endoplasmic reticulum stress inhibited the expression of FOXA1, only slightly induced FOXA2, and had no effect on FOXA3. The FOXA2 and FOXA3 messenger RNA induction by amino acid deprivation did not require activating transcription factor 4, a critical component of the conventional amino acid response (AAR) pathway, but their induction was partially dependent on CCAAT/enhancer-binding protein beta. Simultaneous knockdown of both FOXA2 and FOXA3 by small interfering RNA did not affect the activation of other amino acid responsive genes, suggesting that the FOXA proteins are not required for the known AAR pathway. Collectively, the results document that the hepatic FOXA family of genes are differentially regulated by amino acid availability.

Activated transcription via mammalian amino acid response elements does not require enhanced recruitment of the Mediator complex.

It is unclear whether Mediator complex in yeast is necessary for all RNA polymerase II (Pol II) transcription or if it is limited to genes activated by environmental stress. In mammals, amino acid limitation induces SNAT2 transcription through ATF4 binding at an amino acid response element. ATF4 is the functional counterpart to the yeast amino acid-dependent regulator GCN4 and GCN4 recruits Mediator during transcriptional activation. Consistent with enhanced SNAT2 transcription activity, the present data demonstrate that amino acid limitation increased SNAT2 promoter association of the general transcription factors that make up the preinitiation complex, including Pol II, but there was no increase in Mediator recruitment. Furthermore, siRNA knockdown of eight Mediator subunits caused no significant decrease in SNAT2 transcription. The estrogen-dependent pS2 gene was used as a positive control for both the ChIP and the siRNA approaches and the data demonstrated the requirement for Mediator recruitment. These results document that activation of the SNAT2 gene by the mammalian amino acid response pathway occurs independently of enhanced Mediator recruitment.

MEK signaling is required for phosphorylation of eIF2alpha following amino acid limitation of HepG2 human hepatoma cells.

The mammalian amino acid response (AAR) pathway is up-regulated by protein or amino acid depletion. This pathway involves detection of uncharged tRNA by the GCN2 kinase, phosphorylation of the translation initiation factor eIF2alpha (eukaryotic initiation factor 2alpha), and, through subsequent translational control, enhanced de novo synthesis of the transcription factor ATF4. The present studies demonstrate that inhibition of MEK activation in HepG2 human hepatoma cells by PD98059 or U0126 blocked the increased phosphorylation of eIF2alpha and ATF4 synthesis triggered by amino acid limitation, showing that the AAR requires activation of the MEK-ERK pathway. Inhibitors of the JNK or p38 MAPK pathways were ineffective. Consequently, inhibition of MEK activation blocked transcriptional induction of ATF4 target genes, but the induction was rescued by overexpression of ATF4 protein. Furthermore, the enhanced ERK phosphorylation following amino acid deprivation required GCN2 kinase activity and eIF2alpha phosphorylation. Inhibition of protein phosphatase 1 action on phospho-eIF2alpha by knockdown of GADD34 did not block the sensitivity to PD98059, suggesting that MEK functions to enhance GCN2-dependent eIF2alpha phosphorylation rather than suppressing dephosphorylation. Collectively, these results document a critical interdependence between the MEK-ERK MAPK signaling pathway and the amino acid stress-activated pathway.

Deprivation of protein or amino acid induces C/EBPbeta synthesis and binding to amino acid response elements, but its action is not an absolute requirement for enhanced transcription.

A nutrient stress signalling pathway is triggered in response to protein or amino acid deprivation, namely the AAR (amino acid response), and previous studies have shown that C/EBPbeta (CCAAT/enhancer-binding protein beta) expression is up-regulated following activation of the AAR. DNA-binding studies, both in vitro and in vivo, have revealed increased C/EBPbeta association with AARE (AAR element) sequences in AAR target genes, but its role is still unresolved. The present results show that in HepG2 human hepatoma cells, the total amount of C/EBPbeta protein, both the activating [LAP* and LAP (liver-enriched activating protein)] and inhibitory [LIP (liver-enriched inhibitory)] isoforms, was increased in histidine-deprived cells. Immunoblotting of subcellular fractions and immunostaining revealed that most of the C/EBPbeta was located in the nucleus. Consistent with these observations, amino acid limitation caused an increase in C/EBPbeta DNA-binding activity in nuclear extracts and chromatin immunoprecipitation revealed an increase in C/EBPbeta binding to the AARE region in vivo, but at a time when transcription from the target gene was declining. A constant fraction of the basal and increased C/EBPbeta protein was phosphorylated on Thr(235) and the phospho-C/EBPbeta did bind to an AARE. Induction of AARE-enhanced transcription was slightly greater in C/EBPbeta-deficient MEFs (mouse embryonic fibroblasts) or C/EBPbeta siRNA (small interfering RNA)-treated HepG2 cells compared with the corresponding control cells. Transient expression of LAP*, LAP or LIP in C/EBPbeta-deficient fibroblasts caused suppression of increased transcription from an AARE-driven reporter gene. Collectively, the results demonstrate that C/EBPbeta is not required for transcriptional activation by the AAR pathway but, when present, acts in concert with ATF3 (activating transcription factor 3) to suppress transcription during the latter stages of the response.

Activation of the ATF3 gene through a co-ordinated amino acid-sensing response programme that controls transcriptional regulation of responsive genes following amino acid limitation.

Expression of ATF3 (activating transcription factor 3) is induced by a variety of environmental stress conditions, including nutrient limitation. In the present study, we demonstrate that the increase in ATF3 mRNA content following amino acid limitation of human HepG2 hepatoma cells is dependent on transcriptional activation of the ATF3 gene, through a highly co-ordinated amino acid-responsive programme of transcription factor synthesis and action. Studies using transient over-expression and knockout fibroblasts showed that several ATF and C/EBP (CCAAT/enhancer-binding protein) family members contribute to ATF3 regulation. Promoter analysis showed that a C/EBP-ATF composite site at -23 to -15 bp relative to the transcription start site of the ATF3 gene functions as an AARE (amino acid response element). Chromatin immunoprecipitation demonstrated that amino acid limitation increased ATF4, ATF3, and C/EBPbeta binding to the ATF3 promoter, but the kinetics of each was markedly different. Immediately following histidine removal, there was a rapid increase in histone H3 acetylation prior to an enhancement in ATF4 binding and in histone H4 acetylation. These latter changes closely paralleled the initial increase in RNA pol II (RNA polymerase II) binding to the promoter and in the transcription rate from the ATF3 gene. The increase in ATF3 and C/EBPbeta binding was considerably slower and more closely correlated with a decline in transcription rate. A comparison of the recruitment patterns between ATF and C/EBP transcription factors and RNA polymerase II at the AARE of several amino acid-responsive genes revealed that a highly co-ordinated response programme controls the transcriptional activation of these genes following amino acid limitation.

Characterization of the amino acid response element within the human sodium-coupled neutral amino acid transporter 2 (SNAT2) System A transporter gene.

The neutral amino acid transport activity, System A, is enhanced by amino acid limitation of mammalian cells. Of the three gene products that encode System A activity, the one that exhibits this regulation is SNAT2 (sodium-coupled neutral amino acid transporter 2). Fibroblasts that are deficient in the amino acid response pathway exhibited little or no induction of SNAT2 mRNA. Synthesis of SNAT2 mRNA increased within 1-2 h after amino acid removal from HepG2 human hepatoma cells. The amino acid responsive SNAT2 genomic element that mediates the regulation has been localized to the first intron. Increased binding of selected members of the ATF (activating transcription factor) and C/EBP (CCAAT/enhancer-binding protein) families to the intronic enhancer was established both in vitro and in vivo. In contrast, there was no significant association of these factors with the SNAT2 promoter. Expression of exogenous individual ATF and C/EBP proteins documented that specific family members are associated with either activation or repression of SNAT2 transcription. Chromatin immunoprecipitation analysis established in vivo that amino acid deprivation led to increased RNA polymerase II recruitment to the SNAT2 promoter.