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To identify regulatory elements in the proximal human ATP-binding cassette transporter A1 (hABCA1) gene promoter we transfected RAW cells with plasmids containing mutations in the E-box, AP1, and liver X receptor (LXR) elements as well as the two Sp1 motifs. Point mutations in either Sp1 site or in the AP1 site had only a minor effect whereas mutation of the LXR element decreased promoter activity. In contrast, mutation or deletion of the E-box motif caused a 3-fold increase in transcriptional activity under basal conditions. Gel shift and DNaseI footprint analysis showed binding of a protein or protein complex to this region. Preincubation of nuclear extracts with antibodies established that USF1, USF2, and fos related antigen (Fra) 2 bind to DNA sequences in the human ABCA1 promoter that contains the intact E-box but not the mutant or deleted E-box. Co-transfection of USF1 and USF2 enhanced, but Fra2 repressed, ABCA1 promoter activity. Thus, a complex consisting of USF1, USF2, and Fra2 binds the E-box motif 147 bp upstream of the transcriptional start site and facilitates repression of the human ABCA1 promoter. These combined studies identify a novel site in the human ABCA1 promoter involved in the regulation of ABCA1 gene expression.—Yang, X-P., L. A. Freeman, C. L. Knapper, M. J. A. Amar, A. Remaley, H. B. Brewer, Jr., and S. Santamarina-Fojo. The E-box motif in the proximal ABCA1 promoter mediates transcriptional repression of the ABCA1 gene.

[Display omitted] • Regulation of the transcription factor MYC:MAX is crucial for fundamental cellular processes. • The disordered MAX N-terminus binds to the MYC:MAX DBD in a DNA-competitive manner. • The MAX N-terminus decreases affinity to DNA and accelerates binding kinetics. • Phosphorylation further enhances the effects of the MAX N-terminus. • Intramolecular interactions enable MYC:MAX to rapidly scan DNA for optimal promoter sites. The intrinsically disordered protein MYC belongs to the family of basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factors (TFs). In complex with its cognate binding partner MAX, MYC preferentially binds to E-Box promotor sequences where it controls fundamental cellular processes such as cell cycle progression, metabolism, and apoptosis. Intramolecular regulation of MYC:MAX has not yet been investigated in detail. In this work, we use Nuclear Magnetic Resonance (NMR) spectroscopy to identify and map interactions between the disordered MAX N-terminus and the MYC:MAX DNA binding domain (DBD). We find that this binding event is mainly driven by electrostatic interactions and that it is competitive with DNA binding. Using NMR spectroscopy and Surface Plasmon Resonance (SPR), we demonstrate that the MAX N-terminus serves to accelerate DNA binding kinetics of MYC:MAX and MAX:MAX dimers, while it simultaneously provides specificity for E-Box DNA. We also establish that these effects are further enhanced by Casein Kinase 2-mediated phosphorylation of two serine residues in the MAX N-terminus. Our work provides new insights how bHLH-LZ TFs are regulated by intramolecular interactions between disordered regions and the folded DNA binding domain. [ABSTRACT FROM AUTHOR]

Injury triggers a genetic program that induces gene expression for regeneration. Recent studies have identified regeneration-response enhancers (RREs); however, it remains unclear whether a common mechanism operates in these RREs. We identified three RREs from the zebrafish fn1b promoter by searching for conserved sequences within the surrounding genomic regions of regeneration-induced genes and performed a transgenic assay for regeneration response. Two regions contained in the transposons displayed RRE activity when combined with the -0.7 kb fn1b promoter. Another non-transposon element functioned as a stand-alone enhancer in combination with a minimum promoter. By searching for transcription factor-binding motifs and validation by transgenic assays, we revealed that the cooperation of E-box and activator protein 1 motifs is necessary and sufficient for regenerative response. Such RREs respond to variety of tissue injuries, including those in the zebrafish heart and Xenopus limb buds. Our findings suggest that the fidelity of regeneration response is ensured by the two signals evoked by tissue injuries. It is speculated that a large pool of potential enhancers in the genome has helped shape the regenerative capacities during evolution., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

A hallmark of liver fibrosis is the activation of hepatic stellate cells (HSC), which results in their production of fibrotic molecules, a process that is largely regulated by connective tissue growth factor (CCN2). CCN2 is increasingly expressed during HSC activation because of diminished expression of microRNA-214 (miR-214), a product of dynamin 3 opposite strand (DNM3os) that directly suppresses CCN2 mRNA. We show that an E-box in the miR-214 promoter binds the basic helix-loop-helix transcription factor, Twist1, which drives miR-214 expression and results in CCN2 suppression. Twist1 expression was suppressed in HSC of fibrotic livers or in cultured HSC undergoing activation in vitro or after treatment with ethanol. Furthermore, Twist1 decreasingly interacted with DNM3os as HSC underwent activation in vitro. Nanovesicular exosomes secreted by quiescent but not activated HSC contained high levels of Twist1, thus reflecting the suppression of cellular Twist1 during HSC activation. Exosomal Twist1 was intercellularly shuttled between HSC and stimulated expression of miR-214 in the recipient cells, causing expression of CCN2 and its downstream effectors to be suppressed. Additionally, the miR-214 E-box in HSC was also regulated by hepatocyte-derived exosomes, showing that functional transfer of exosomal Twist1 occurs between different cell types. Finally, the levels of Twist1, miR-214, or CCN2 in circulating exosomes from fibrotic mice reflected fibrosis-induced changes in the liver itself, highlighting the potential utility of these and other constituents in serum exosomes as novel circulating bio-markers for liver fibrosis. These findings reveal a unique function for cellular or exosomal Twist1 in CCN2-dependent fibrogenesis. [ABSTRACT FROM AUTHOR]

The MYC transcription factor plays a crucial role in the regulation of cell cycle progression, apoptosis, angiogenesis, and cellular transformation. Due to its oncogenic activities and overexpression in a majority of human cancers, it is an interesting target for novel drug therapies. MYC binding to the E-box (5′- CACGTGT-3′) sequence at gene promoters contributes to more than 4000 MYC-dependent transcripts. Owing to its importance in MYC regulation, we designed a novel sequence-specific DNA-binding pyrrole-imidazole ( PI) polyamide, Myc-5, that recognizes the E-box consensus sequence. Bioinformatics analysis revealed that the Myc-5 binding sequence appeared in 5′- MYC binding E-box sequences at the eIF4G1, CCND1, and CDK4 gene promoters. Furthermore, Ch IP coupled with detection by quantitative PCR indicated that Myc-5 has the ability to inhibit MYC binding at the target gene promoters and thus cause downregulation at the mRNA level and protein expression of its target genes in human Burkitt's lymphoma model cell line, P493.6, carrying an inducible MYC repression system and the K562 (human chronic myelogenous leukemia) cell line. Single i.v. injection of Myc-5 at 7.5 mg/kg dose caused significant tumor growth inhibition in a MYC-dependent tumor xenograft model without evidence of toxicity. We report here a compelling rationale for the identification of a PI polyamide that inhibits a part of E-box-mediated MYC downstream gene expression and is a model for showing that phenotype-associated MYC downstream gene targets consequently inhibit MYC-dependent tumor growth. [ABSTRACT FROM AUTHOR]

Taxus cell suspension culture is a sustainable technology for the industrial production of paclitaxel (Taxol®), a highly modified diterpene anti-cancer agent. The methyl jasmonate (MJ)-mediated paclitaxel biosynthetic pathway is not fully characterized, making metabolic engineering efforts difficult. Here, promoters of seven genes (TASY, T5αH, DBAT, DBBT, PAM, BAPT, and DBTNBT), encoding enzymes of the paclitaxel biosynthetic pathway were isolated and used to drive MJ-inducible expression of a GUS reporter construct in transiently transformed Taxus cells, showing that elicitation of paclitaxel production by MJ is regulated at least in part at the level of transcription. The paclitaxel biosynthetic pathway promoters contained a large number of E-box sites (CANNTG), similar to the binding sites for the key MJ-inducible transcription factor AtMYC2 from Arabidopsis thaliana. Three MJ-inducible MYC transcription factors similar to AtMYC2 (TcJAMYC1, TcJAMYC2, and TcJAMYC4) were identified in Taxus. Transcriptional regulation of paclitaxel biosynthetic pathway promoters by transient over expression of TcJAMYC transcription factors indicated a negative rather than positive regulatory role of TcJAMYCs on paclitaxel biosynthetic gene expression. [ABSTRACT FROM AUTHOR]

Differentiated embryo chondrocyte 2 (DEC2/Sharp-1/Bhlhe41), a basic helix-loop-helix (bHLH) transcription factor, has been shown to regulate the transcription of target genes by binding to their E-box elements. We identified a possible DEC2-response element (consensus E-box: CACGTG) in the promoter region of Twist1. Forced expression of DEC2 significantly repressed Twist1 promoter activity under normoxia and under hypoxia as assessed by a luciferase reporter assay. In addition, over-expression of DEC2 repressed Twist1 mRNA expression assessed by quantitative real-time PCR. Site-directed mutagenesis studies showed that mutagenesis of the consensus E-box sequence eliminated the ability of DEC2 to reduce the Twist1 promoter activity. Chromatin immunoprecipitation (ChIP) assays confirmed that the DEC2-mediated repression is primarily achieved by binding to the E-box in the Twist1 promoter. Knockdown of DEC2 by siRNA significantly attenuated the repression of Twist1 expression. DEC2 and Twist1 exhibit inversed protein expression patterns during development of mouse tongue embryo tissue. Given the fact that DEC2 protein is emerging as an important regulator in a vast array of cellular events, including cell differentiation, maturation of lymphocytes and the molecular clock, our study elucidates an important mechanism by which DEC2 regulates cellular function by modulating the expression of Twist1. [ABSTRACT FROM AUTHOR]

Gene amplification and/or overexpression of the transcription factor c-MYC, which binds to the E-box sequence (5′-CACGTG-3′), has been observed in many human tumors. In this study, we have designed 5 pyrrole–imidazole (PI) polyamides recognizing E-box, and found that, among them, Myc-6 significantly suppresses malignant phenotypes of human osteosarcoma MG63 cells both in vitro and in vivo. Intriguingly, knockdown of the putative Myc-6 target MALAT1 encoding long noncoding RNA remarkably impaired cell growth of MG63 cells. Collectively, our present findings strongly suggest that Myc-6 exerts its tumor-suppressive ability at least in part through the specific down-regulation of MALAT1 . [ABSTRACT FROM AUTHOR]

Renal function and blood pressure exhibit a circadian pattern of variation, but the molecular mechanism underlying this circadian regulation is not fully understood. We have previously shown that the circadian clock protein Per1 positively regulates the basal and aldosterone-mediated expression of the alpha subunit of the renal epithelial sodium channel (αENaC). The mechanism of this regulation has not been determined however. To further elucidate the mechanism of Mineralocorticoid Receptor (MR) and Per1 action, site-directed mutagenesis, DNA pull-down assays and chromatin immunoprecipitation methods were used to investigate the coordinate regulation of αENaC by Per1 and MR. Mutation of two circadian response E-boxes in the human αENaC promoter abolished both basal and aldosterone-mediated promoter activity. DNA pull down assays demonstrated the interaction of both MR and Per1 with the E-boxes from the αENaC promoter. These observations were corroborated by chromatin immunopreciptation experiments showing increased occupancy of MR and Per1 on an E-box of the αENaC promoter in the presence of aldosterone. This is the first report of an aldosterone-mediated increase in Per1 on a target gene promoter. Taken together, these results demonstrate the novel finding that Per1 and MR mediate the aldosterone response of αENaC through DNA/protein interaction in renal collecting duct cells.

Based on their extracellular expression and targeting of the clock gene Bmal1, miR‐142‐3p and miR‐494 were analyzed for evidence of vesicle‐mediated communication between cells and intracellular functional activity. Our studies demonstrate that: miR‐142‐3p + miR‐494 overexpression decreases endogenous BMAL1 levels, increases the period of Per2 oscillations, and increases extracellular miR‐142‐3p/miR‐494 abundance in conditioned medium; miRNA‐enriched medium increases intracellular expression of miR‐142‐3p and represses Bmal1 3′‐UTR activity in naïve cells; and inhibitors of vesicular trafficking modulate intercellular communication of these miRNAs and ensemble Per2 rhythms. Thus, miR‐142‐3p and miR‐494 may function as cis‐ and trans‐acting signals contributing to local temporal coordination of cell‐autonomous circadian clocks.MiR‐142‐3p and miR‐494 repress endogenous BMAL1 levels and modulate ensemble rhythms. MiR‐142‐3p is communicated between cells by vesicular trafficking. Inhibition of vesicular trafficking disrupts miRNA communication. Inhibition of vesicular trafficking alters ensemble clock gene rhythms. MiRNAs may function as trans‐acting signals coordinating local time among cells. [ABSTRACT FROM AUTHOR]

Melanin synthesis is a complex phenomenon which involves about 192 known gene products. Among them, MITF is a key transcription factor for tyrosinase, Trp1 and Trp2 proteins, which are essential for melanin biosynthesis. Thus, intervening inhibitor for the MITF-E-box complex formation can downregulate melanin synthesis. The focus of the present study is to develop a surface plasmon resonance-based system to screen the MITF-E-box complex inhibitor. The standardization of the MITF and E-box binding assay was calibrated for kinetics and specificity, in the presence of a pre-incubated 22 mer sequence containing mutated E-box (CTTGAG) along MITF. The binding assay with C17 was optimized and the steady-state kinetics was evaluated. C17 was identified as inhibitor to MITF-E-box, by virtual screening followed by in vitro assessment and EMSA assay. The k and k were found to be 5.5 × 103 M s and 0.0014 s, respectively, while the steady-state association constant ( K) was 3.928 × 106 M. The resonance variations after inhibition were quantified and analyzed to develop the standard method for screening of microphthalmia transcription factor-E-box binding inhibitor. [ABSTRACT FROM AUTHOR]

Gastrin and its precursors act as growth factors for the normal and neoplastic gastrointestinal mucosa. As the hypoxia mimetic cobalt chloride upregulates the gastrin gene, the effect of other metal ions on gastrin promoter activity was investigated. Gastrin mRNA was measured by real-time PCR, gastrin peptides by RIA, and gastrin promoter activity by dual-luciferase reporter assay. Exposure to Zn2+ ions increased gastrin mRNA concentrations in the human gastric adenocarcinoma cell line AGS in a dose-dependent manner, with a maximum stimulation of 55±14-fold at 100 mM (P<0.05). Significant stimulation was also observed with Cd2+ and Cu2+, but not with Ca2+, Mg2+, Ni2+, or Fe3+ ions. Activation of MAPK and phosphatidylinositol 3-kinase pathways is necessary but not sufficient for gastrin induction by Zn2C. Deletional mutation of the gastrin promoter identified an 11 bp DNA sequence, which contained an E-box motif, as necessary for Zn2+-dependent gastrin induction. The fact that E-box binding transcription factors play a crucial role in the epithelial-mesenchymal transition (EMT), together with our observation that Zn2+ ions upregulate the gastrin gene in AGS cells by an E-box-dependent mechanism, suggests that Zn2+ ions may induce an EMT, and that gastrin may be involved in the transition. [ABSTRACT FROM AUTHOR]

The basic helix-loop-helix (bHLH) family members DEC1 and DEC2 function as transcription factors by directly binding to class B E-box region in the proximal promoter of target genes, and have recently been reported to play important roles in many human physiological and pathological processes such as cancer. However, it is a great challenge to quantitatively analyze the binding affinity and selectivity of sequence-specific interaction in DEC1/DEC2-E-box recognition since there are numerous potential competitive DNA binders of the transcription factors in cellular context. In the present study, we describe a novel model peptide approach to fast and reliably characterize the interaction behavior of DEC1 and DEC2 with DNA. In the procedure, a series of peptides that mimic the DNA recognition helices of DEC1 and DEC2 as well as other bHLH proteins are derived, and their binding potencies to cognate E-box and noncognate DNA decoys are evaluated using a residue-level affinity predictor built upon a distinct set of structure-solved, affinity-known protein-DNA complexes. By systematically examining the affinity distribution profiles of different peptides binding to the E-box and to a large number of decoys it is found that single-point mutations on peptide key residues such as H6A and R14A can significantly reduce both the affinity and selectivity of DEC1 and DEC2 toward E-box, but the mutations do not substantially influence their binding capability to most decoys, and a fraction (~22 %) of DNA decoys exhibit stronger interaction potency with DEC peptide as compared to E-box, suggesting that the DEC1 and DEC2 possess a moderate specificity that allows the transcription factors to bind, and regulate, a number of potential target genes. [ABSTRACT FROM AUTHOR]

Renal function and blood pressure (BP) exhibit a circadian pattern of variation, but the molecular mechanism underlying this circadian regulation is not fully understood. We have previously shown that the circadian clock protein Per1 positively regulates the basal and aldosterone-mediated expression of the alpha subunit of the renal epithelial sodium channel (αENaC). The mechanism of this regulation has not been determined however. To further elucidate the mechanism of mineralocorticoid receptor (MR) and Per1 action, site-directed mutagenesis, DNA pull-down assays and chromatin immunoprecipitation (ChIP) methods were used to investigate the coordinate regulation of αENaC by Per1 and MR. Mutation of two circadian response E-boxes in the human αENaC promoter abolished both basal and aldosterone-mediated promoter activity. DNA pull down assays demonstrated the interaction of both MR and Per1 with the E-boxes from the αENaC promoter. These observations were corroborated by ChIP experiments showing increased occupancy of MR and Per1 on an E-box of the αENaC promoter in the presence of aldosterone. This is the first report of an aldosterone-mediated increase in Per1 on a target gene promoter. Taken together, these results demonstrate the novel finding that Per1 and MR mediate the aldosterone response of αENaC through DNA/protein interaction in renal collecting duct cells. [ABSTRACT FROM AUTHOR]

Abstract: The circadian clock co-ordinates physiology and behavior with the day/night cycle. It consists of a transcriptional–translational feedback loop that generates self-sustained oscillations in transcriptional activity with a roughly 24h period via E-box enhancer elements. Numerous in vivo aspects of core clock feedback loop function are still incompletely understood, including its maturation during development, tissue-specific activity and perturbation in disease states. Zebrafish are promising models for biomedical research due to their high regenerative capacity and suitability for in vivo drug screens, and transgenic zebrafish lines are valuable tools to study transcriptional activity in vivo during development. To monitor the activity of the core clock feedback loop in vivo, we created a transgenic zebrafish line expressing a luciferase reporter gene under the regulation of a minimal promoter and four E-boxes. This Tg(4xE-box:Luc) line shows robust oscillating reporter gene expression both under light-dark cycles and upon release into constant darkness. Luciferase activity starts to oscillate during the first days of development, indicating that the core clock loop is already functional at an early stage. To test whether the Tg(4xE-box:Luc) line could be used in drug screens aimed at identifying compounds that target the circadian clock in vivo, we examined drug effects on circadian period. We were readily able to detect period changes as low as 0.7h upon treatment with the period-lengthening drugs lithium chloride and longdaysin in an assay set-up suitable for large-scale screens. Reporter gene mRNA expression is also detected in the adult brain and reveals differential clock activity across the brain, overlapping with endogenous clock gene expression. Notably, core clock activity is strongly correlated with brain regions where neurogenesis takes place and can be detected in several types of neural progenitors. Our results demonstrate that the Tg(4xE-box:Luc) line is an excellent tool for studying the regulation of the circadian clock and its maturation in vivo and in real time. Furthermore, it is highly suitable for in vivo screens targeting the core clock mechanism that take into account the complexity of an intact organism. Finally, it allows mapping of clock activity in the brain of a vertebrate model organism with prominent adult neurogenesis and high regeneration capacity. [Copyright &y& Elsevier]

Highlights: [•] We examined the effects of NAD(P)H on transcription factors for circadian. [•] NAD(P)H enhances the DNA-binding activity of NPAS2/BMAL1, but not BMAL1/BMAL1. [•] The N-terminal 1–61 amino acids of NPAS2 are sufficient to sense NAD(P)H. [•] NAD(P)+, 2′,5′-ADP, and nicotinamide do not affect the DNA-binding activity. [ABSTRACT FROM AUTHOR]

The expression of the gene encoding myostatin ( MSTN), the product of which is a negative regulator of skeletal muscle growth and development in mammals, is regulated by many cis-regulatory elements, including enhancer box (E-box) motifs. While E-box motif mutants of MSTN exhibit altered expression of myostatin in many animal models, the phenotypes of these mutations in chicken are not investigated. In this study, we cloned and sequenced the full encoded DNA sequence of MSTN gene and its upstream promoter region in Wenshang Luhua chicken breed. After analysis of the sequence, 13 E-box motifs were identified in the MSTN promoter region, which were denoted by E1 to E13 according to their positions in the region. Although many single nucleotide polymorphisms (SNPs) were revealed in the MSTN promoter region, only two SNPs were in the E-boxes, i.e., the first nucleotide of the E3 and the fifth nucleotide of E4. The effects of these two polymorphisms on the expression of MSTN gene were explored both with MSTN-GFP reporter constructs in vitro and real-time PCR in vivo. The results suggested that the E-boxes in the chicken MSTN promoter region are involved in the regulation of myostatin expression and the polymorphisms in E3 and E4 altered the expression of myostatin. [ABSTRACT FROM AUTHOR]

Abstract: Pod-1/Tcf21 is expressed at epithelial-mesenchymal interaction sites during development of many organs. Different approaches have demonstrated that Pod-1 transcriptionally inhibits Sf-1/NR5A1 during gonadal development. Disruption of Sf-1 can lead to disorders of adrenal development, while increased dosage of SF-1 has been related to increased adrenal cell proliferation and tumorigenesis. In this study, we analyzed whether POD-1 overexpression inhibits the endogenous Sf-1 expression in human and mouse adrenocortical tumor cells. Cells were transiently transfected with luciferase reporter gene under the control of Sf-1 promoter and with an expression vector encoding Pod-1. Pod-1 construct inhibited the transcription of the Sf1/Luc reporter gene in a dose-dependent manner in mouse Y-1 adrenocortical carcinoma (ACC) cells, and inhibited endogenous SF-1 expression in the human H295R and ACC-T36 adrenocortical carcinoma cells. These results were validated by chromatin immunoprecipitation assay with POD-1-transfected H295R cells using primers specific to E-box sequence in SF-1 promoter region, indicating that POD-1 binds to the SF-1 E-box promoter. Moreover, POD-1 over-expression resulted in a decrease in expression of the SF-1 target gene, StAR (Steroidogenic Acute Regulatory Protein). Lastly, while the induced expression of POD-1 did not affect the cell viability of H295R/POD-1 or ACC-T36/POD-1 cells, the most significantly enriched KEGG pathways for genes negatively correlated to POD-1/TCF21 in 33 human ACCs were those associated with cell cycle genes. [Copyright &y& Elsevier]

Abstract: The basic helix–loop–helix family of proteins, which function as transcription factors, have been intensively studied in plants and animals. However, the molecular mechanism of these factors contributing to stress tolerance is unknown. Here, we report on the overexpression of OrbHLH001 from Dongxiang wild rice (Oryza rufipogon) conferring salt tolerance in transgenic rice plants. The expression of OrbHLH001 was tissue specific, mainly in phloem tissues throughout the plant. Ion assay with the scanning ion-selective electrode technique showed that NaCl stress has a greater influence on Na+ efflux and K+ influx in OrbHLH001-overexpressed plants than the wild type. OrbHLH001 protein can induce the expression of OsAKT1 to regulate the Na+/K+ ratio in OrbHLH001-overexpressed plants by specifically binding to an E-box motif in the promoter region of OsAKT1. The mechanism may have potential use in rice molecular breeding. [Copyright &y& Elsevier]

Members of the Snail/Gfi-1 domain family of zinc finger proteins are known to recognize the E-box sequence CANNTG, such as that found in the promoter of E-cadherin, however, no studies have shown that the internal 'NN' dinucleotides can play a role in different binding affinities. We show via gel shift assays that only the sequences CACCTG and CAGGTG can be recognized more strongly by the SNAG-ZFP members such as Slug, Smuc, Snail, and Scratch while the other combinations of the internal nucleotides were bound weakly. All 16 possible dinucleotide combinations were tested by competition EMSAs to determine their relative binding affinities. The K value for the best-binding sequences was approximately 1.25 × 10 M, while the other interactions were less effective. Our study has shown for the first time how different internal dinucleotide combinations of the E-box can be recognized differently by different transcription factors and also sheds light into how this transcription factor binding site may participate in DNA-protein interactions. [ABSTRACT FROM AUTHOR]

Disturbances of sleep-wake rhythms are an important problem in Alzheimer's disease (AD). Circadian rhythms are regulated by clock genes. Transforming growth factor-beta (TGF-β) is overexpressed in neurons in AD and is the only cytokine that is increased in cerebrospinal fluid (CSF). Our data show that TGF-β2 inhibits the expression of the clock genes Period ( Per) 1, Per2, and Rev-erbα, and of the clock-controlled genes D-site albumin promoter binding protein ( Dbp) and thyrotroph embryonic factor ( Tef). However, our results showed that TGF-β2 did not alter the expression of brain and muscle Arnt-like protein-1 ( Bmal1). The concentrations of TGF-β2 in the CSF of 2 of 16 AD patients and of 1 of 7 patients with mild cognitive impairment were in the dose range required to suppress the expression of clock genes. TGF-β2-induced dysregulation of clock genes may alter neuronal pathways, which may be causally related to abnormal sleep-wake rhythms in AD patients. [ABSTRACT FROM AUTHOR]

Abstract: Testis-specific tandemly repeated Stellate genes are part of the Ste–Su(Ste) genetic system required for male fertility in Drosophila melanogaster. Stellate genes encode a functional homolog of the β-subunit of protein kinase CK2. Derepression of Stellate results in their over-expression, meiotic disturbances and male sterility. Stellate genes are represented by clustered copies in the X chromosome and carry promoters shared with another X-chromosome cluster, βNACtes genes, encoding putative β-subunits of the nascent polypeptide-associated complex. Using Electrophoretic Mobility Shift Assay, we revealed in the Stellate promoter three cis-acting elements, E-boxes, the loss of which greatly diminished the reporter gene expression in Drosophila testes. We identified that these E-boxes were recognized by helix–loop–helix protein, dUSF (Drosophila ortholog of mammalian USF) in testis nuclear extract. All three E-boxes were preserved in the promoters of both euchromatic and heterochromatic Stellate clusters. Two analogous E-boxes were detected in the promoters of 5′-copies of the duplicated βNACtes gene pairs, whereas the 3′-copies lacked these sites but possessed a new binding site for a testis protein distinct from dUSF. Here we characterized a new type of testis-specific core promoter and identified dUSF as its interacting transcription factor. [Copyright &y& Elsevier]

Circadian E-boxes in the promoters of mPer1, mPer2, mCry1 and mDBP play a key role in regulating rhythmic gene expression by recruiting the BMAL1/CLOCK heterodimer. However, each of these circadian E-boxes seems different from one another. In the mouse liver, BMAL1/CLOCK binds to E-boxes in the mPer1 and mPer2 promoters constantly but binds with dynamic daily changes to mCry1 and mDBP promoters. It is unclear whether DNA methylation of these circadian E-boxes and the surrounding CpG islands would affect protein/DNA interactions and lead to distinct binding features. In the present study, bisulfite sequencing analysis indicated that all E-boxes and their surrounding CpG islands were free from methylation, suggesting that DNA methylation does not determine distinct binding features. The methylation status of the E-box in the MAP kinase phosphatase 1 (MKP1) promoter was also examined. Our results indicated that DNA methylation does not regulate the tissue-specific clock control of MKP1. [ABSTRACT FROM AUTHOR]

Abstract: Melanocortin 4 receptor (Mc4r/MC4R) is a G-Protein coupled receptor that is expressed in the hypothalamus and implicated in body weight control. Mutations in MC4R are the most frequent cause of monogenetic forms of human obesity. Despite its importance, the MC4R signaling pathways and transcriptional regulation underlying the melanocortin pathway are far from being fully understood. The transcription factor nescient helix-loop-helix 2 (Nhlh2) influences the melanocortin pathway through transcriptional regulation of prohormone convertase I, which influences the production of melanocortin peptides. In the present study, Nhlh2’s role as a transcriptional regulator of Mc4r has been demonstrated. Nhlh2 knockout mice have reduced hypothalamic expression of Mc4r mRNA, suggesting that it could be a direct or indirect transcriptional regulator of the Mc4r promoter. To demonstrate direct transcriptional regulation, chromatin immunoprecipitation and electrophoretic gel shift assays show that Nhlh2 binds to the E-Boxes located at −551, −366 and +54 on the Mc4r promoter. Leptin-induced transactivation of the Mc4r promoter is significantly higher in the presence of exogenously added Nhlh2. siRNA knockdown of Nhlh2 leads to significantly reduced endogenous Mc4r mRNA expression levels in N29/2 cell line. Transactivation using promoters with mutations in each of the E-Boxes results in significantly reduced transactivation efficiency compared to the WT Mc4r promoter, suggesting that Nhlh2 regulates Mc4r transcription through these sites. Findings from these studies, combined with previous work implicating Nhlh2 as a transcriptional regulator of both the Mc4r gene and the melanocortin pathway, suggest that Nhlh2’s transcriptional activity directly influences the human and rodent body weight control pathways. [Copyright &y& Elsevier]

Abstract: Clusterin is a stress-response protein that is involved in diverse biological processes, including cell proliferation, apoptosis, tissue differentiation, inflammation, and lipid transport. Its expression is upregulated in a broad spectrum of diverse pathological states. Clusterin was recently reported to be associated with diabetes, metabolic syndrome, and their sequelae. However, the regulation of clusterin expression by metabolic signals was not addressed. In this study we evaluated the effects of glucose on hepatic clusterin expression. Interestingly, high glucose concentrations significantly increased clusterin expression in primary hepatocytes and hepatoma cell lines, but the conventional promoter region of the clusterin gene did not respond to glucose stimulation. In contrast, the first intronic region was transcriptionally activated by high glucose concentrations. We then defined a glucose response element (GlRE) of the clusterin gene, showing that it consists of two E-box motifs separated by five nucleotides and resembles carbohydrate response element (ChoRE). Unexpectedly, however, these E-box motifs were not activated by ChoRE binding protein (ChREBP), but were activated by sterol regulatory element binding protein-1c (SREBP-1c). Furthermore, we found that glucose induced recruitment of SREBP-1c to the E-box of the clusterin gene intronic region. Taken together, these results suggest that clusterin expression is increased by glucose stimulation, and SREBP-1c plays a crucial role in the metabolic regulation of clusterin. [Copyright &y& Elsevier]

Abstract: Proneural genes encode bHLH transcription factors that are key regulator of neurogenesis in both vertebrates and invertebrates. How these transcription factors regulate targets required for neural determination and/or specification is beginning to be understood. In this study, we show that zebrafish deltaA is a transcriptional target of proneural factors. Using a combination of transient and stable transgenic reporters, we show that regulation of deltaA by one such proneural factor, Ngn1, requires three clustered E-box binding sites that act in a non-redundant manner. Furthermore, we show that as for other proneural targets, members of the different proneural families regulate deltaA expression via distinct cis-regulatory modules (CRMs). Interestingly, however, while the deltaA CRM regulated by a second proneural factor, Ascl1, has been conserved between delta genes of different species, we show that the Ngn1 CRM has not. These results suggest that evolutionary constraints on the mechanism by which Ngn1 regulates gene expression appear less strict than for Ascl1. [ABSTRACT FROM AUTHOR]

The circadian transcription of the tim gene is tightly regulated by the protein complex dCLK/CYC, which directly interacts with a series of closely spaced E-box and E-box—like elements in the Drosophila timeless promoter. The tim promoter from D. melanogaster has been studied in detail both in tissue cultures and in living flies yet has never been investigated in other species. This article presents a detailed functional analysis of the tim promoter from the drosophilid fly, Chymomyza costata, in Drosophila tissue cultures. A comparison of tim promoters from wt and npd-mutants confirmed that the 1855 bp deletion in the latter removes crucial regulatory cis-elements as well as the minimal promoter, being subsequently responsible for the lack of tim mRNA expression. Deletion and substitution mutations of the wt tim promoter showed that the region containing the canonical E-box, TER-box, and 2 incomplete E-box sequences is essential for CLK/CYC-mediated expression, while the PERR element appears to be a repressor in S2 cells. Furthermore, the expression of the circadian genes timeless, period , vrille, and doubletime was quantified in C. costata adults. Striking differences were found in expression profiles for tim, per, and vri between wild-type and npd-mutant individuals. [ABSTRACT FROM PUBLISHER]