Repression Of Promoter Activity Research Articles

The p53 tumor suppressor regulates AKR1B1 expression, a metastasis-promoting gene in breast cancer.

Alteration of metabolism in cancer cells is a central aspect of the mechanisms that sustain aggressive traits. Aldo-keto reductase 1 B1 (AKR1B1) catalyzes the reduction of several aldehydes to alcohols consuming NADPH. Nevertheless, the ability of AKR1B1 to reduce different substrates renders difficult to comprehensively ascertain its biological role. Recent evidence has implicated AKR1B1 in cancer; however, the mechanisms underlying its pro-oncogenic function remain largely unknown. In this work, we report that AKR1B1 expression is controlled by the p53 tumor suppressor. We found that breast cancer patients bearing wild-type TP53 have reduced AKR1B1 expression. In cancer cell lines, p53 reduced AKR1B1 mRNA and protein levels and repressed promoter activity in luciferase assays. Furthermore, chromatin immunoprecipitation assays indicated that p53 is recruited to the AKR1B1 promoter. We also observed that AKR1B1 overexpression promoted metastasis in the 4T1 orthotopic model of triple-negative breast cancer. Proteomic analysis of 4T1 cells overexpressing AKR1B1 showed that AKR1B1 exerts a marked effect on proteins related to metabolism, with a particular impact on mitochondrial function. This work provides novel insights on the link between the p53 pathway and metabolism in cancer cells and contributes to characterizing the alterations associated to the pathologic role of AKR1B1.

A metabolic regulatory network for the Caenorhabditis elegans intestine

SummaryMetabolic perturbations can affect gene expression, for instance to rewire metabolism. While numerous efforts have measured gene expression in response to individual metabolic perturbations, methods that determine all metabolic perturbations that affect the expression for a given gene or set of genes have not been available. Here, we use a gene-centered approach to derive a first-pass metabolic regulatory network for Caenorhabditis elegans by performing RNAi of more than 1,400 metabolic genes with a set of 19 promoter reporter strains that express a fluorescent protein in the animal’s intestine. We find that metabolic perturbations generally increase promoter activity, which contrasts with transcription factor (TF) RNAi, which tends to repress promoter activity. We identify several TFs that modulate promoter activity in response to perturbations of the electron transport chain and explore complex genetic interactions among metabolic pathways. This work provides a blueprint for a systems-level understanding of how metabolism affects gene expression.

MKRN3 inhibits the reproductive axis through actions in kisspeptin-expressing neurons

The identification of loss-of-function mutations in MKRN3 in patients with central precocious puberty in association with the decrease in MKRN3 expression in the medial basal hypothalamus of mice before the initiation of reproductive maturation suggests that MKRN3 is acting as a brake on gonadotropin-releasing hormone (GnRH) secretion during childhood. In the current study, we investigated the mechanism by which MKRN3 prevents premature manifestation of the pubertal process. We showed that, as in mice, MKRN3 expression is high in the hypothalamus of rats and nonhuman primates early in life, decreases as puberty approaches, and is independent of sex steroid hormones. We demonstrated that Mkrn3 is expressed in Kiss1 neurons of the mouse hypothalamic arcuate nucleus and that MKRN3 repressed promoter activity of human KISS1 and TAC3, 2 key stimulators of GnRH secretion. We further showed that MKRN3 has ubiquitinase activity, that this activity is reduced by MKRN3 mutations affecting the RING finger domain, and that these mutations compromised the ability of MKRN3 to repress KISS1 and TAC3 promoter activity. These results indicate that MKRN3 acts to prevent puberty initiation, at least in part, by repressing KISS1 and TAC3 transcription and that this action may involve an MKRN3-directed ubiquitination-mediated mechanism.

Cellular signaling crosstalk between Wnt signaling and gap junctions inbenzo[a]pyrene toxicity.

Gap junctional intercellular communication (GJIC) is considered a key biological mechanism to maintain homeostasis in cell differentiation and growth. In addition, as another major signaling pathway associated with cell proliferation and differentiation, Wnt/β-catenin signaling appears to trigger several cellular responses against injury. The purpose of the present study was to investigate the effects of a known toxic agent, benzo[a]pyrene (BaP), on the regulation and interaction between GJIC and Wnt/β-catenin signaling. BaP treatment resulted in GJIC inhibition and decreases the major GJIC protein connexin 43 (Cx43) in WB-F344 rat liver epithelial cells. We also found BaP-mediated downregulation of Wnt/β-catenin signaling related to the PI3K-Akt pathway. To identify the relationship between GJIC and Wnt/β-catenin signaling, we treated WB-F344 cells with the Wnt agonist CHIR99021 and found that it inhibited GJIC while causing a significant reduction in Cx43 expression at both the mRNA and protein levels, through the repression of promoter activity. This Wnt agonist-mediated GJIC inhibition was confirmed using a small interfering RNA directed against the Wnt antagonist Dact2, indicating that Wnt/β-catenin signaling negatively regulates GJIC. Despite the inverse correlation between Wnt/β-catenin signaling and Cx43 promoter activation as indicated by downregulation of β-catenin nuclear translocation and upregulation of Cx43 promoter activation involving HNF3β, BaP treatment decreased the Cx43 protein expression, which was associated with protein degradation, possibly through protein kinase C activation. In conclusion, our results revealed the mechanism of BaP-induced inhibition of GJIC and Wnt/β-catenin signaling. More importantly, linking Wnt/β-catenin signaling to Cx protein expression will have profound implications in understanding the relationships among different major signaling pathways associated with cell proliferation and differentiation in toxicity.

Characterization of a HBB -Locus Fetal Chromatin Domain Region during Adult Erythropoiesis

The hemoglobinopathies including sickle cell disease (SCD) and β-thalassemia are the most common genetic disorders in the world producing significant morbidity and mortality. Drug-mediated fetal hemoglobin (HbF) induction ameliorates the clinical severity of SCD and improves long-term survival. To define mechanisms of γ-globin regulation, previously we demonstrated the role of the Gγ-globin cAMP response element, in drug-mediated HbF induction (Sangerman et al., Blood 2006). Subsequently, data generated by the ENCODE project led us to investigate a region 4 kb upstream of Gγ-globin, which harbors a DNase hypersensitive site and enrichment of the histone 3 lysine 4, mono-methylated (H3K4Me1) enhancer mark, that we named the Fetal Chromatin Domain (FCD). Recently, a synthetic zinc finger DNA-binding domain targeting this region was shown to reduce γ-globin gene expression in K562 cells (Shen et al., Blood 128:320, 2016). Initially, we performed multispecies sequence alignments which revealed the FCD is located in a genomic region shared by Old World monkeys (humans and Baboons) with a delayed fetal to adult globin switch that occurs after birth. By contrast, the FCD region is absent in New World monkeys where the switch occurs during the fetal developmental stage. These findings support involvement of the FCD region in γ-globin regulation during hemoglobin switching.Subsequent studies were performed to define the functional role of the FCD during erythropoiesis. ENCODE long RNA-sequencing assay detected PolyA RNA species throughout the FCD region in K562 cells. Based on these findings, we designed two small-interference RNA molecules targeting the core FCD (siFCD). Both siFCD molecules were transfected into KU812 cells and individual γ-globin gene expression measured using RT-qPCR with gene-specific primers. The level of Gγ-globin mRNA increased by 64%, while Aγ-globin transcription was reduced 38%. These results suggest the FCD facilitates differential regulation of the γ-globin genes. To further characterize FCD function, a series of γ-globin promoter luciferase reporters were constructed with the sense or antisense 500-bp core FCD region, cloned downstream of the luciferase gene. In KU812 stable cell lines, the sense FCD construct enhanced Aγ-globin promoter activity 10-fold, whereas the antisense construct repressed promoter activity. By contrast, the FCD in both orientations silenced Gγ-globin promoter activity. These results support unidirectional gene-specific enhancer properties of the FCD. Subsequent pulldown assay with a 34-bp biotinylated FCD probe confirmed NFE2, BHLHE40 and cMyc binding, which was abolished by mutation of the E-box sequence (CACGTG) in the FCD core.To build on the KU812 cell data, we performed μChIP assay using primary erythroid progenitors generated from adult CD34+ stem cells (Zhu et al., Haematologica 2017). In our system, the γ-globin to β-globin gene switch occurred on day 4 in culture. Initially, we measured levels of acetylated histone H3 (AcH3), H3K4Me1, and TFIID binding in the β-globin locus control region hypersensitivity site 2 (HS2) and FCD. As expected, high AcH3 and H3K4Me1 marks occurred in HS2 consistent with its enhancer function. Similar patterns of histone marks and binding of TFIID, NFE2 and BHLHE40 occurred in HS2 and FCD region. It is known that HS2 participates in DNA looping to accomplish developmentally regulated globin gene expression during hemoglobin switching therefore chromosome conformation capture (3C) assay was performed in primary erythroid progenitors to determine if the FCD facilitates changes in chromatin structure. We observed strong long-range interactions between the FCD and HS2 with 4.4-fold maximal chromatin enrichment on day 5; likewise the FCD showed interactions with Aγ-globin (4.7-fold) and Gγ-globin (3.5-fold) by day 3 before switching occurred. Of note, low level interactions occurred between the FCD and β-globin gene at all time points tested.Collectively, these data support the FCD as a unidirectional enhancer element involved in γ-globin regulation during erythropoiesis. To further define mechanisms of FCD function, the effects of CrisprCas9-mediated deletions in the FCD on globin gene expression will be explored. DisclosuresNo relevant conflicts of interest to declare.

Krüppel-Like Factors 9 and 13 Block Axon Growth by Transcriptional Repression of Key Components of the cAMP Signaling Pathway

Krüppel-like factors (KLFs) are zinc finger transcription factors implicated in diverse biological processes, including differentiation of neural cells. The ability of mammalian neurons to elongate axons decreases during postnatal development in parallel with a decrease in cAMP, and increase in expression of several Klf genes. The paralogous KLFs 9 and 13 inhibit neurite outgrowth, and we hypothesized that their actions are mediated through repression of cAMP signaling. To test this we used the adult mouse hippocampus-derived cell line HT22 engineered to control expression of Klf9 or Klf13 with doxycycline, or made deficient for these Klfs by CRISPR/Cas9 genome editing. We also used primary hippocampal cells isolated from wild type, Klf9–/– and Klf13–/– mice. Forced expression of Klf9 or Klf13 in HT22 changed the mRNA levels of several genes involved with cAMP signaling; the predominant action was gene repression, and KLF13 influenced ∼4 times more genes than KLF9. KLF9 and KLF13 repressed promoter activity of the protein kinase a catalytic subunit alpha gene in transfection-reporter assays; KLF13, but not KLF9 repressed the calmodulin 3 promoter. Forskolin activation of a cAMP-dependent promoter was reduced after forced expression of Klf9 or Klf13, but was enhanced in Klf gene knockout cells. Forced expression of Klf9 or Klf13 blocked cAMP-dependent neurite outgrowth in HT22 cells, and axon growth in primary hippocampal neurons, while Klf gene knockout enhanced the effect of elevated cAMP. Taken together, our findings show that KLF9 and KLF13 inhibit neurite/axon growth in hippocampal neurons, in part, by inhibiting the cAMP signaling pathway.

MKRN3 inhibits the reproductive axis through actions in kisspeptin-expressing neurons.

The identification of loss-of-function mutations in MKRN3 in patients with central precocious puberty in association with the decrease in MKRN3 expression in the medial basal hypothalamus of mice before the initiation of reproductive maturation suggests that MKRN3 is acting as a brake on gonadotropin-releasing hormone (GnRH) secretion during childhood. In the current study, we investigated the mechanism by which MKRN3 prevents premature manifestation of the pubertal process. We showed that, as in mice, MKRN3 expression is high in the hypothalamus of rats and nonhuman primates early in life, decreases as puberty approaches, and is independent of sex steroid hormones. We demonstrated that Mkrn3 is expressed in Kiss1 neurons of the mouse hypothalamic arcuate nucleus and that MKRN3 repressed promoter activity of human KISS1 and TAC3, 2 key stimulators of GnRH secretion. We further showed that MKRN3 has ubiquitinase activity, that this activity is reduced by MKRN3 mutations affecting the RING finger domain, and that these mutations compromised the ability of MKRN3 to repress KISS1 and TAC3 promoter activity. These results indicate that MKRN3 acts to prevent puberty initiation, at least in part, by repressing KISS1 and TAC3 transcription and that this action may involve an MKRN3-directed ubiquitination-mediated mechanism.

Two functional variants at 6p21.1 were associated with lean mass

BackgroundLow lean body mass is the most important predictor of sarcopenia with strong genetic background. The aim of this study was to uncover genetic factors underlying lean mass development.Materials and methodsWe performed a genome-wide association study (GWAS) of fat-adjusted leg lean mass in the Framingham Heart Study (FHS, N = 6587), and replicated in the Women’s Health Initiative–African American sub-sample (WHI-AA, N = 847) and the Kansas City Osteoporosis Study (KCOS, N = 2219). We also cross-validated significant variants in the publicly available body mass index (BMI) summary results (N ~ 700,000). We then performed a series of functional investigations on the identified variants.ResultsFour correlated SNPs at 6p21.1 were identified at the genome-wide significance (GWS, α = 5.0 × 10−8) level in the discovery FHS sample (rs551145, rs524533, rs571770, and rs545970, p = 3.40–9.77 × 10−9), and were successfully replicated in both the WHI-AA and the KCOS samples (one-sided p = 1.61 × 10−3–0.04). They were further cross-validated by the large-scale BMI summary results (p = 7.0–9.8 × 10−3). Cis-eQTL analyses associated these SNPs with the NFKBIE gene expression. Electrophoresis mobility shift assay (EMSA) in mouse C2C12 myoblast cells implied that rs524533 and rs571770 were bound to an unknown transcription factor in an allelic specific manner, while rs551145 and rs545970 did not. Dual-luciferase reporter assay revealed that both rs524533 and rs571770 downregulated luciferase expression by repressing promoter activity. Moreover, the regulation pattern was allelic specific, strengthening the evidence towards their differential regulatory effects.ConclusionsThrough a large-scale GWAS followed by a series of functional investigations, we identified 2 correlated functional variants at 6p21.1 associated with leg lean mass. Our findings not only enhanced our understanding of molecular basis of lean mass development but also provided useful candidate genes for further functional studies.

EASI Transformation: An Efficient Transient Expression Method for Analyzing Gene Function in Catharanthus roseus Seedlings.

The Catharanthus roseus plant is the exclusive source of the valuable anticancer terpenoid indole alkaloids, vinblastine (VB) and vincristine (VC). The recent availability of transcriptome and genome resources for C. roseus necessitates a fast and reliable method for studying gene function. In this study, we developed an Agrobacterium-mediated transient expression method to enable the functional study of genes rapidly in planta, conserving the compartmentalization observed in the VB and VC pathway. We focused on (1) improving the transformation method (syringe versus vacuum agroinfiltration) and cultivation conditions (seedling age, Agrobacterium density, and time point of maximum transgene expression), (2) improving transformation efficiency through the constitutive expression of the virulence genes and suppressing RNA silencing mechanisms, and (3) improving the vector design by incorporating introns, quantitative and qualitative reporter genes (luciferase and GUS genes), and accounting for transformation heterogeneity across the tissue using an internal control. Of all the parameters tested, vacuum infiltration of young seedlings (10-day-old, harvested 3 days post-infection) resulted in the strongest increase in transgene expression, at 18 – 57 fold higher than either vacuum or syringe infiltration of other seedling ages. Endowing the A. tumefaciens strain with the mutated VirGN54D or silencing suppressors within the same plasmid as the reporter gene further increased expression by 2 – 10 fold. For accurate measurement of promoter transactivation or activity, we included an internal control to normalize the differences in plant mass and transformation efficiency. Including the normalization gene (Renilla luciferase) on the same plasmid as the reporter gene (firefly luciferase) consistently yielded a high signal and a high correlation between RLUC and FLUC. As proof of principle, we applied this approach to investigate the regulation of the CroSTR1 promoter with the well-known activator ORCA3 and repressor ZCT1. Our method demonstrated the quantitative assessment of both the activation and repression of promoter activity in C. roseus. Our efficient Agrobacterium-mediated seedling infiltration (EASI) protocol allows highly efficient, reproducible, and homogenous transformation of C. roseus cotyledons and provides a timely tool for the community to rapidly assess the function of genes in planta, particularly for investigating how transcription factors regulate terpenoid indole alkaloid biosynthesis.

Cooperative regulation of mouse aldose reductase (AKR1B3) gene transcription by Nrf2, TonEBP, and c-jun

Aldose reductase (AR), a member of aldo-keto reductase family, is the rate-limiting enzyme in the polyol pathway, and is known to play a key role in the pathogenesis of diabetic complications. AR also catalyzes the reduction of reactive aldehydes, thereby detoxifying endogenous as well as xenobiotic aldehydes in various tissues. The transcription of the AR gene was previously shown to be augmented by various stimuli including osmotic and oxidative stresses. A highly conserved region composed of an antioxidant response element (ARE), AP-1 site, and tonicity responsive enhancer (TonE) has been identified within the 5’-flanking region of the AR genes of humans, rats, and mice, which we designated as the multiple stress response region (MSRR). We previously showed that the transcription factor Nrf2 activated AR transcription via ARE within MSRR. In the present study, we examined the interactions among Nrf2, c-Jun, and the TonE-binding protein (TonEBP) in the regulation of AR gene transcription. In gene reporter assays using luciferase reporter constructs containing the MSRR of the mouse AR (AKR1B3) gene with HepG2 cells, the forced expression of Nrf2 or TonEBP significantly increased promoter activity. The synergistic augmentation of promoter activity was observed when Nrf2 and TonEBP were co-introduced. On the other hand, the co-expression of c-Jun repressed promoter activation by Nrf2 and TonEBP. The mutation of the AP-1 site within MSRR did not affect the repressive effects of c-Jun, while the introduction of truncated c-Jun proteins lacking the leucine zipper domain no longer suppressed Nrf2-or TonEBP-mediated transactivation, suggesting that c-Jun repressed promoter activity independently of the AP-1 site and that interactions with protein factors via the leucine zipper domain were necessary for its negative effects on Nrf2 and TonEBP. These results indicate that AR promoter activity is cooperatively regulated by multiple transcription factors via MSRR.

Identification of Morus notabilis MADS-box genes and elucidation of the roles of MnMADS33 during endodormancy

The MADS-box genes encode transcriptional regulators with various functions especially during floral development. A total of 54 putative Morus notabilis MADS-box genes (MnMADSs) were identified and phylogenetically classified as either type I (17 genes) or type II (37 genes). The detected genes included three FLOWERING LOCUS C-like (MnFLC-like) genes, MnMADS33, MnMADS50, and MnMADS7. MnFLC-like proteins could directly or indirectly repress promoter activity of the mulberry FLOWERING LOCUS T-like (MnFT) gene. Transgenic Arabidopsis thaliana overexpressing MnFLC-like genes exhibited delayed flowering and down-regulated expression of FT and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1). The gene expression analyses in floral bud indicated that MnMADS33 expression increased, while MnFT expression decreased during the induction of dormancy in response to cold conditions. Dormancy release resulted in the down-regulation of MnMADS33 expression and the up-regulation of MnFT expression. Furthermore, abscisic acid promoted the transcription of MnMADS33 and MnFT, although the expression level of MnFT gradually decreased. These results are consistent with the hypothesis that MnMADS33 negatively regulated the expression of MnFT to repress dormancy release and flowering in mulberry. This study may be relevant for future investigations regarding the effects of MnMADS genes on mulberry flowering development.

Salt induction and activation of MtlD, the key enzyme in the synthesis of the compatible solute mannitol in Acinetobacter baumannii.

Mannitol is the major compatible solute, next to glutamate, synthesized by the opportunistic human pathogen Acinetobacter baumannii under low water activities. The key enzyme for mannitol biosynthesis, MtlD, was identified. MtlD is highly similar to the bifunctional mannitol‐1‐phosphate dehydrogenase/phosphatase from Acinetobacter baylyi. After deletion of the mtlD gene from A. baumannii ATCC 19606T cells no longer accumulated mannitol and growth was completely impaired at high salt. Addition of glycine betaine restored growth, demonstrating that mannitol is an important compatible solute in the human pathogen. MtlD was heterologously produced and purified. Enzyme activity was strictly salt dependent. Highest stimulation was reached at 600 mmol/L NaCl. Addition of different sodium as well as potassium salts restored activity, with highest stimulations up to 41 U/mg protein by sodium glutamate. In contrast, an increase in osmolarity by addition of sugars did not restore activity. Regulation of mannitol synthesis was also assayed at the transcriptional level. Reporter gene assays revealed that expression of mtlD is strongly dependent on high osmolarity, not discriminating between different salts or sugars. The presence of glycine betaine or its precursor choline repressed promoter activation. These data indicate a dual regulation of mannitol production in A. baumannii, at the transcriptional and the enzymatic level, depending on high osmolarity.

The Kr\xfcppel-like factor 9 cistrome in mouse hippocampal neurons reveals predominant transcriptional repression via proximal promoter binding

BackgroundKrüppel-like factor 9 (Klf9) is a zinc finger transcription factor that functions in neural cell differentiation, but little is known about its genomic targets or mechanism of action in neurons.ResultsWe used the mouse hippocampus-derived neuronal cell line HT22 to identify genes regulated by Klf9, and we validated our findings in mouse hippocampus. We engineered HT22 cells to express a Klf9 transgene under control of the tetracycline repressor, and used RNA sequencing to identify genes modulated by Klf9. We found 217 genes repressed and 21 induced by Klf9. We also engineered HT22 cells to co-express biotin ligase and a Klf9 fusion protein containing an N-terminal biotin ligase recognition peptide. Using chromatin-streptavidin precipitation (ChSP) sequencing we identified 3,514 genomic regions where Klf9 associated. Seventy-five percent of these were within 1 kb of transcription start sites, and Klf9 associated in chromatin with 60% of the repressed genes. We analyzed the promoters of several repressed genes containing Klf9 ChSP peaks using transient transfection reporter assays and found that Klf9 repressed promoter activity, which was abolished after mutation of Sp/Klf-like motifs. Knockdown or knockout of Klf9 in HT22 cells caused dysregulation of Klf9 target genes. Chromatin immunoprecipitation assays showed that Klf9 associated in chromatin from mouse hippocampus with genes identified by ChSP sequencing on HT22 cells, and expression of Klf9 target genes was dysregulated in the hippocampus of neonatal Klf9-null mice. Gene ontology analysis revealed that Klf9 genomic targets include genes involved in cystokeletal remodeling, Wnt signaling and inflammation.ConclusionsWe have identified genomic targets of Klf9 in hippocampal neurons and created a foundation for future studies on how it functions in chromatin, and regulates neuronal morphology and survival across the lifespan.

Promoter regulatory domain identification of cassava starch synthase IIb gene in transgenic tobacco

Soluble starch synthase is a key enzyme in the starch biosynthesis pathway, and its enzyme activity significantly influences starch components in cassava storage root. However, studies on the regulation mechanism of soluble starch synthase gene are rare. In this study, we cloned the 5′ flanking sequence of the MeSSIIb gene and predicted the distribution of cis-elements. The region from −453 to −1 was considered the primary core promoter by the quantitative detection of GUS activity in transgenic tobacco plants containing 5′ truncated promoters fused with the GUS gene. Analysis results clarified that the region from −531 to −454 significantly repressed promoter activity. The region from −453 to −388 was a repressive domain of ethylene, and some unknown drought responsive cis-elements were located in the region from −387 to −1. These findings will provide useful information on the functional assay and transcriptional regulation mechanisms of the MeSSIIb gene.

Methyl‐CpG‐Binding Protein (MBD) Family: Epigenomic Read‐Outs Functions and Roles in Tumorigenesis and Psychiatric Diseases

Epigenetics is the study of the heritable changes on gene expression that are responsible for the regulation of development and that have an impact on several diseases. However, it is of equal importance to understand how epigenetic machinery works. DNA methylation is the most studied epigenetic mark and is generally associated with the regulation of gene expression through the repression of promoter activity and by affecting genome stability. Therefore, the ability of the cell to interpret correct methylation marks and/or the correct interpretation of methylation plays a role in many diseases. The major family of proteins that bind methylated DNA is the methyl-CpG binding domain proteins, or the MBDs. Here, we discuss the structure that makes these proteins a family, the main functions and interactions of all protein family members and their role in human disease such as psychiatric disorders and cancer.

The Leukemia Inhibitory Factor Receptor Gene Is a Direct Target of RUNX1.

Activation of cytokine signaling via the leukemia inhibitory factor receptor (LIFR) plays an integral role in hematopoiesis, osteogenesis, and placental development, along with mediating neurotrophic mechanisms. However, the regulatory control of the LIFR gene has remained largely unexplored. Here, we characterize the LIFR gene as a novel target of the RUNX1 transcription factor. The RUNX1 transcription factor is an essential regulator of hematopoiesis and is a frequent target of point mutations and chromosomal alterations in leukemia. RUNX1 regulates hematopoiesis through its control of genes important for hematopoietic cell growth, proliferation, and differentiation, including a number of cytokines and cytokine receptors. LIFR is regulated by two alternate promoters: a placental-specific and a ubiquitously active general promoter. We show that both of these promoters are regulated by RUNX1. However, in myeloid cells LIFR expression is driven solely by the general LIFR promoter with our data indicating that the placental promoter is epigenetically silenced in these cells. While RUNX1 activates the LIFR general promoter, the oncogenic RUNX1-ETO fusion protein generated by the t(8;21) translocation commonly associated with acute myeloid leukemia represses promoter activity. The data presented here establish LIFR as a transcriptional target of RUNX1 and suggest that disruption of RUNX1 activity in myeloid cells may result in altered LIFR signaling in these cells.

Methylation-dependent and independent regulatory regions in the Na,K-ATPase alpha4 (Atp1a4) gene may impact its testis-specific expression

The α4 Na,K-ATPase is a sperm-specific protein essential for sperm motility and fertility yet little is known about the mechanisms that regulate its expression in germ cells. Here, the potential involvement of DNA methylation in regulating the expression of this sperm-specific protein is explored. A single, intragenic CpG island (Mα4-CGI) was identified in the gene encoding the mouse α4 Na,K-ATPase (Atp1a4), which displayed reduced methylation in mouse sperm (cells that contain α4) compared to mouse kidney (tissue that lacks α4 expression). Unlike the intragenic CGI, the putative promoter (the −700 to +200 region relative to the transcriptional start site) of Atp1a4 did not show differential methylation between kidney and sperm nevertheless it did drive methylation-dependent reporter gene expression in the male germ cell line GC-1spg. Furthermore, treatment of GC-1spg cells with 5-aza2-deoxycytidine led to upregulation of the α4 transcript and decreased methylation of both the Atp1a4 promoter and the Mα4-CGI. In addition, Atp1a4 expression in mouse embryonic stem cells deficient in DNA methyltransferases suggests that both maintenance and de novo methylation are involved in regulating its expression. In an attempt to define the regulatory function of the Mα4-CGI, possible roles of the Mα4-CGI in regulating Atp1a4 expression via methylation-dependent transcriptional elongation inhibition in somatic cells and via its ability to repress promoter activity in germ cells were uncovered. In all, our data suggests that both the promoter and the intragenic CGI could combine to provide multiple modes of regulation for optimizing the Atp1a4 expression level in a cell type-specific manner.

Expression of xCT and activity of system xc− are regulated by NRF2 in human breast cancer cells in response to oxidative stress

Cancer cells adapt to high levels of oxidative stress in order to survive and proliferate by activating key transcription factors. One such master regulator, the redox sensitive transcription factor NF E2 Related Factor 2 (NRF2), controls the expression of cellular defense genes including those encoding intracellular redox-balancing proteins involved in glutathione (GSH) synthesis. Under basal conditions, Kelch-like ECH-associated protein 1 (KEAP1) targets NRF2 for ubiquitination. In response to oxidative stress, NRF2 dissociates from KEAP1, entering the nucleus and binding to the antioxidant response element (ARE) in the promoter of its target genes. Elevated reactive oxygen species (ROS) production may deplete GSH levels within cancer cells. System xc−, an antiporter that exports glutamate while importing cystine to be converted into cysteine for GSH synthesis, is upregulated in cancer cells in response to oxidative stress. Here, we provided evidence that the expression of xCT, the light chain subunit of system xc−, is regulated by NRF2 in representative human breast cancer cells. Hydrogen peroxide (H2O2) treatment increased nuclear translocation of NRF2, also increasing levels of xCT mRNA and protein and extracellular glutamate release. Overexpression of NRF2 up-regulated the activity of the xCT promoter, which contains a proximal ARE. In contrast, overexpression of KEAP1 repressed promoter activity and decreased xCT protein levels, while siRNA knockdown of KEAP1 up-regulated xCT protein levels and transporter activity. These results demonstrate the importance of the KEAP1/NRF2 pathway in balancing oxidative stress in breast cancer cells through system xc−. We have previously shown that xCT is upregulated in various cancer cell lines under oxidative stress. In the current investigation, we focused on MCF-7 cells as a model for mechanistic studies.

Control of transcriptional repression of the vitellogenin receptor gene in largemouth bass (Micropterus salmoides) by select estrogen receptors isotypes.

The vitellogenin receptor (Vtgr) plays an important role in fish reproduction. This receptor functions to incorporate vitellogenin (Vtg), a macromolecule synthesized and released from the liver in the bloodstream, into oocytes where it is processed into yolk. Although studies have focused on the functional role of Vtgr in fish, the mechanistic control of this gene is still unexplored. Here we report the identification and analysis of the first piscine 5' regulatory region of the vtgr gene which was cloned from largemouth bass (Micropterus salmoides). Using this putative promoter sequence, we investigated a role for hormones, including insulin and 17β-estradiol (E2), in transcriptional regulation through cell-based reporter assays. No effect of insulin was observed, however, E2 was able to repress transcriptional activity of the vtgr promoter through select estrogen receptor subtypes, Esr1 and Esr2a but not Esr2b. Electrophoretic mobility shift assay demonstrated that Esr1 likely interacts with the vtgr promoter region through half ERE and/or SP1 sites, in part. Finally we also show that ethinylestradiol (EE2), but not bisphenol-A (BPA), represses promoter activity similarly to E2. These results reveal for the first time that the Esr1 isoform may play an inhibitory role in the expression of LMB vtgr mRNA under the influence of E2, and potent estrogens such as EE2. In addition, this new evidence suggests that vtgr may be a target of select endocrine disrupting compounds through environmental exposures.

KLFII Epigenetically Regulates Glycodelin-A, a Marker of Endometrial Biology Via Histone-Modifying Chromatin Mechanisms

Endometrial biology is characterized by programmed proliferation and differentiation that is synchronous with ovarian folliculogenesis to maximize the chance of pregnancy. Glycodelin-A, an endometrial secretory protein, promotes pregnancy mostly through immunomodulatory mechanisms. Glycodelin-A is repressed during the proliferative and early secretory phase and activated thereafter. Progesterone activates glycodelin via the Sp1 (Specificity Protein 1) transactivator. We identify a novel role for Kruppel-like transcription factor 11 (KLF11) as a glycodelin-A repressor. Although KLF11 bound 2 distinct regulatory elements, it regulated glycodelin promoter activity differentially through each element. Whereas KLF11 weakly activated glycodelin promoter activity via a region that also bound Sp1, the dominant effect of KLF11 was repression of promoter activity, messenger RNA (mRNA), and protein expression via a novel, specific binding element. KLF11 mediated this repression by recruiting the SIN3/histone deacetylase (HDAC) corepressor complex to the glycodelin promoter. KLF11 may solely, or by competing with Sp1, repress glycodelin-A levels and thereby influence its role in the endometrium.