Negative Regulation Of Gene Transcription Research Articles

Abstract 2605: LOXL3 knock out affects pathways which involve cytoskeleton regulation, proliferation and apoptosis in glioblastoma cells

Abstract Lysyl oxidase-like 3 (LOXL3), belonging to the lysyl oxidase family of copper-dependent amino oxidase, is responsible for the crosslinking of collagen and elastin. In tumors, the presence of LOXL3 has been associated with genomic stability, cell proliferation, and metastasis. In silico analysis has shown that glioblastoma was among the tumors with the highest LOXL3 expression levels. Moreover, LOXL3 expression increased with malignancy grade amongst diffusely infiltrative astrocytomas (from grade 2 to 4). In the present work, CRISPR-Cas9 gene editing was used to knock out LOXL3 in the human glioma U87MG cell line to evaluate the role of LOXL3 in glioblastoma. Analyses of protein expression level and genomic mutation mediated by CRISPR-Cas9 by two different sgRNA (denominated sgRNA1 and 2) were performed and the knock out efficiency and genome editing were confirmed. Transcriptome analysis of these cells revealed a downregulation of genes that coded for proteins involved in microtubule organization, cell division, and vesicle trafficking. On the other hand, upregulated genes when LOXL3 was knocked out in U87MG cells were involved in actin cytoskeleton, negative regulation of gene transcription, including chromatin remodeling, and cell-cell adhesion. Functional in vitro assays showed that LOXL3 knocked out cells presented decreased cell proliferation due to longer cell cycles with increased G1 and G2/M checkpoints compared to control cells. Additionally, LOXL3 knocked out U87MG cells presented a higher percentage of cells in the early phase of apoptosis with an increment after temozolomide treatment. Our results suggest an important role of LOXL3 in glioblastoma possibly mediated by cytoskeleton regulation, affecting cell proliferation, cell cycle, and cell death. These results reinforced the importance of LOXL3 in tumors, especially in glioblastoma, highlighting it as a potential therapeutic target. Citation Format: Talita S. Laurentino, Roseli S. Soares, Antonio M. Lerario, Suely K. Marie, Sueli Mieko Oba-Shinjo. LOXL3 knock out affects pathways which involve cytoskeleton regulation, proliferation and apoptosis in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2605.

Profiling of Human Molecular Pathways Affected by Retrotransposons at the Level of Regulation by Transcription Factor Proteins.

Endogenous retroviruses and retrotransposons also termed retroelements (REs) are mobile genetic elements that were active until recently in human genome evolution. REs regulate gene expression by actively reshaping chromatin structure or by directly providing transcription factor binding sites (TFBSs). We aimed to identify molecular processes most deeply impacted by the REs in human cells at the level of TFBS regulation. By using ENCODE data, we identified ~2 million TFBS overlapping with putatively regulation-competent human REs located in 5-kb gene promoter neighborhood (~17% of all TFBS in promoter neighborhoods; ~9% of all RE-linked TFBS). Most of REs hosting TFBS were highly diverged repeats, and for the evolutionary young (0–8% diverged) elements we identified only ~7% of all RE-linked TFBS. The gene-specific distributions of RE-linked TFBS generally correlated with the distributions for all TFBS. However, several groups of molecular processes were highly enriched in the RE-linked TFBS regulation. They were strongly connected with the immunity and response to pathogens, with the negative regulation of gene transcription, ubiquitination, and protein degradation, extracellular matrix organization, regulation of STAT signaling, fatty acids metabolism, regulation of GTPase activity, protein targeting to Golgi, regulation of cell division and differentiation, development and functioning of perception organs and reproductive system. By contrast, the processes most weakly affected by the REs were linked with the conservative aspects of embryo development. We also identified differences in the regulation features by the younger and older fractions of the REs. The regulation by the older fraction of the REs was linked mainly with the immunity, cell adhesion, cAMP, IGF1R, Notch, Wnt, and integrin signaling, neuronal development, chondroitin sulfate and heparin metabolism, and endocytosis. The younger REs regulate other aspects of immunity, cell cycle progression and apoptosis, PDGF, TGF beta, EGFR, and p38 signaling, transcriptional repression, structure of nuclear lumen, catabolism of phospholipids, and heterocyclic molecules, insulin and AMPK signaling, retrograde Golgi-ER transport, and estrogen signaling. The immunity-linked pathways were highly represented in both categories, but their functional roles were different and did not overlap. Our results point to the most quickly evolving molecular pathways in the recent and ancient evolution of human genome.

Epigenetic up-regulation of ribosome biogenesis and more aggressive phenotype triggered by the lack of the histone demethylase JHDM1B in mammary epithelial cells.

The alterations of ribosome biogenesis and protein synthesis play a direct role in the development of tumors. The accessibility and transcription of ribosomal genes is controlled at several levels, with their epigenetic regulation being one of the most important. Here we explored the JmjC domain-containing histone demethylase 1B (JHDM1B) function in the epigenetic control of rDNA transcription. Since JHDM1B is a negative regulator of gene transcription, we focused on the effects induced by JHDM1B knock-down (KD). We studied the consequences of stable inducible JHDM1B silencing in cell lines derived from transformed and untransformed mammary epithelial cells. In these cellular models, prolonged JHDM1B downregulation triggered a surge of 45S pre-rRNA transcription and processing, associated with a re-modulation of the H3K36me2 levels at rDNA loci and with changes in DNA methylation of specific CpG sites in rDNA genes. We also found that after JHDM1B KD, cells showed a higher ribosome content: which were engaged in mRNA translation. JHDM1B KD and the consequent stimulation of ribosomes biogenesis conferred more aggressive features to the tested cellular models, which acquired a greater clonogenic, staminal and invasive potential. Taken together, these data indicate that the reduction of JHDM1B leads to a more aggressive cellular phenotype in mammary gland cells, by virtue of its negative regulatory activity on ribosome biogenesis.

Abstract A15: Epigenetic up-regulation of ribosome biogenesis and more aggressive phenotype triggered by the lack of the histone demethylase JHDM1B in mammary epithelial cells

Abstract Alterations of ribosome biogenesis and protein synthesis play a direct role in the development and the behavior of tumors. The accessibility and transcription of ribosomal genes are controlled at several levels, with their epigenetic regulation being one of the most important. Here we explored the JmjC domain-containing histone demethylase 1B (JHDM1B) function in the epigenetic control of rDNA transcription. Since JHDM1B is a negative regulator of gene transcription, we focused on the effects induced by its downregulation. We studied the consequences of inducible JHDM1B knock-down (KD) in cell lines derived from transformed (MDA-MB-231) and untransformed (MCF-10A) mammary epithelial cells. In these cellular models, sustained JHDM1B down-regulation triggered an increase in 45S pre-rRNA transcription and processing, associated with a re-modulation of the H3K36me2 levels at the rDNA loci. By means of the EpiTYPER assay, we observed that JHDM1B KD reproducibly induced quantitative changes in DNA methylation of specific CpG sites in rDNA genes. By polysomal profile analysis, we also found that JHDM1B KD induced an higher content of ribosomes in both the pre-polysomal and polysomal fractions indicating an increase in global protein synthesis. We then investigated in vitro and in vivo the behavior of cells after JHDM1B KD and the associated stimulation of ribosomes biogenesis. JHDM1B down-regulation conferred more aggressive features to the tested cellular models, which acquired a greater clonogenic and invasive potential in vitro. The study of tumors generated in nude Balb/c mice after xenografting MDA-MB-231 cells, indicated that JHDM1B KD triggered an incremental (although not statistically significant) trend in tumor growth. In addition, the selective nucleolar staining of sections from these tumors revealed a significant increase in the average nucleolar area after JHDM1B KD. Taken together, these data indicate that the reduction of JHDM1B leads to an increase in ribosome production and to a more aggressive cellular behavior in mammary gland cells. Citation Format: Alice Galbiati, Marianna Penzo, Maria Giulia Bacalini, Carmine Onofrillo, Ania Naila Guerrieri, Paolo Garagnani, Claudio Franceschi, Davide Treré, Lorenzo Montanaro. Epigenetic up-regulation of ribosome biogenesis and more aggressive phenotype triggered by the lack of the histone demethylase JHDM1B in mammary epithelial cells. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A15.

Menin Epigenetically Represses Hedgehog Signaling in MEN1 Tumor Syndrome

Multiple endocrine neoplasia type 1 (MEN1) is an inherited tumor syndrome that includes susceptibility to pancreatic islet tumors. This syndrome results from mutations in the MEN1 gene, encoding menin. Although menin acts as an oncogenic cofactor for mixed lineage leukemia (MLL) fusion protein-mediated histone H3 lysine 4 methylation, the precise basis for how menin suppresses gene expression and proliferation of pancreatic beta cells remains poorly understood. Here, we show that menin ablation enhances Hedgehog signaling, a proproliferative and oncogenic pathway, in murine pancreatic islets. Menin directly interacts with protein arginine methyltransferase 5 (PRMT5), a negative regulator of gene transcription. Menin recruits PRMT5 to the promoter of the Gas1 gene, a crucial factor for binding of Sonic Hedgehog (Shh) ligand to its receptor PTCH1 and subsequent activation of the Hedgehog signaling pathway, increases repressive histone arginine symmetric dimethylation (H4R3m2s), and suppresses Gas1 expression. Notably, MEN1 disease-related menin mutants have reduced binding to PRMT5, and fail to impart the repressive H4R3m2s mark at the Gas1 promoter, resulting in its elevated expression. Pharmacologic inhibition of Hedgehog signaling significantly reduces proliferation of insulinoma cells, and expression of Hedgehog signaling targets including Ptch1, in MEN1 tumors of mice. These findings uncover a novel link between menin and Hedgehog signaling whereby menin/PRMT5 epigenetically suppresses Hedgehog signaling, revealing it as a target for treating MEN1 tumors.

EHMT1 Protein Binds to Nuclear Factor-κB p50 and Represses Gene Expression

Transcriptional homeostasis relies on the balance between positive and negative regulation of gene transcription. Methylation of histone H3 lysine 9 (H3K9) is commonly correlated with gene repression. Here, we report that a euchromatic H3K9 methyltransferase, EHMT1, functions as a negative regulator in both the NF-κB- and type I interferon-mediated gene induction pathways. EHMT1 catalyzes H3K9 methylation at promoters of NF-κB target genes. Moreover, EHMT1 interacts with p50, and, surprisingly, p50 appears to repress the expression of type I interferon genes and genes activated by type I interferons by recruiting EHMT1 to catalyze H3K9 methylation at their promoter regions. Silencing the expression of EHMT1 by RNA interference enhances expression of a subset NF-κB-regulated genes, augments interferon production, and augments antiviral immunity.

Control of Cognition and Adaptive Behavior by the GLP/G9a Epigenetic Suppressor Complex

The genetic basis of cognition and behavioral adaptation to the environment remains poorly understood. Here we demonstrate that the histone methyltransferase complex GLP/G9a controls cognition and adaptive responses in a region-specific fashion in the adult brain. Using conditional mutagenesis in mice, we show that postnatal, neuron-specific deficiency of GLP/G9a leads to derepression of numerous nonneuronal and neuron progenitor genes in adult neurons. This transcriptional alteration is associated with complex behavioral abnormalities, including defects in learning, motivation, and environmental adaptation. The behavioral changes triggered by GLP/G9a deficiency are similar to key symptoms of the human 9q34 mental retardation syndrome that is associated with structural alterations of the GLP/EHMT1 gene. The likely causal role of GLP/G9a in mental retardation in mice and humans suggests a key role for the GLP/G9a-controlled histone H3K9 dimethylation in regulation of brain function through maintenance of the transcriptional homeostasis in adult neurons.

Osteopenia in transgenic mice with osteoblast-targeted expression of the inducible cAMP early repressor

ICER is a member of the CREM family of basic leucine zipper transcription factors that acts as a dominant negative regulator of gene transcription. Four different isoforms of ICER (I, Iγ, II and IIγ) are transcribed from the P2 promoter of the Crem gene. We previously found that each of the ICER isoforms is induced by parathyroid hormone in osteoblasts. The goal of the present study was to assess the function of ICER in bone by overexpressing ICER in osteoblasts of transgenic mice. ICER I and ICER II cDNAs, each containing an N-terminal FLAG epitope tag, were cloned downstream of a fragment containing 3.6 kb of the rat Col1a1 promoter and most of the rat Col1a1 first intron to produce pOBCol3.6-ICER I and pOBCol3.6-ICER II transgenes, respectively. Multiple lines of mice were generated bearing the ICER I and ICER II transgenes. At 8 weeks of age, ICER I and ICER II transgenic mice had lower body weights and decreased bone mineral density of femurs and vertebrae. Further studies were done with ICER I transgenic mice, which had greatly reduced trabecular bone volume and a markedly decreased bone formation rate in femurs. Osteoblast differentiation and osteocalcin expression were reduced in ex vivo bone marrow cultures from ICER I transgenic mice. ICER I antagonized the activity of ATF4 at its consensus DNA binding site in the osteocalcin promoter in vitro. Thus, transgenic mice with osteoblast-targeted overexpression of ICER exhibited osteopenia caused primarily by reduced bone formation. We speculate that ICER regulates the activity and/or expression of ATF/CREB factors required for normal bone formation.

DNA Methylation and the Expression of IL-4 and IFN-γ Promoter Genes in Patients with Bronchial Asthma

Although CpG methylation is thought to be a negative regulator of gene transcription, its relationship with cytokine expression remains unclear. Interleukin (IL)-4 and interferon (IFN)-gamma are major cytokines that affect the differentiation of naïve CD4+ T lymphocytes into the Th1 and Th2 lineage. We used bisulfite deoxyribonucleic acid modification and sequencing to examine the relationship between CpG methylation and IL-4 and IFN-gamma gene expression before and after allergen stimulation in human CD4+ T lymphocytes from sensitized hosts. In naïve cells, the CpGs in the promoter regions were methylated largely in both the IL-4 and IFN-gamma genes. After Dermatophagoides pteronyssinus/Dermatophagoides farinae stimulation, the degree of unmethylation in the IL-4 gene increased in cells from patients with bronchial asthma. After phytohemagglutinin stimulation, the degree of unmethylation increased in cells from nonallergic control subjects. The concentration of IL-4 was strongly correlated with the degree of unmethylation in the patient group. These data suggest that CpGs located at -80 of the IL-4 gene and at -295, -186, and +122 of the IFN-gamma gene have regulatory activities important for cytokine expression. We conclude that the stimulation of CD4+ T lymphocytes causes considerable increases in the degree of unmethylation and that in sensitized hosts, the extent of unmethylation correlates with the concentration of IL-4.

Bypassing Cellular Senescence by Ezh2.

In this study mouse embryonic fibroblasts (MEFs) and hematopoietic stem cells (HSCs) were used to identify genes involved in the process of cellular aging. MEFs are able to undergo a distinct number of population doublings, after which they enter a state of irreversible growth arrest termed replicative senescence. Previous studies of others and us have shown that aging is regulated by an intrinsic genetic program. In order to identify candidate genes involved in senescence we assessed gene expression profiles of proliferating and senescent MEFs. Interestingly, a significant number of genes that was higher expressed in proliferating vs. senescent MEFs, are involved in epigenetic regulation of gene transcription by modulating histone methylation and deacetylation. The top candidate gene was Enhancer of zeste homolog 2 (Ezh2), a Polycomb group protein (PcG), involved in histone methylation and deacetylation and known to function as a negative regulator of gene transcription. It has been reported that Ezh2 is essential during development, since Ezh2-deficient mice die early during embryonic development. Ezh2 expression was rapidly downregulated during senescence in primary MEFs, but was readily detectable in spontaneously transformed cells. Retroviral overexpression of Ezh2 in primary MEFs resulted in bypassing the senescence program. In addition, overexpression of Ezh2 in spontaneously transformed MEFs increased proliferation rates. We found that Ezh2 is highly expressed in purified Lin−Sca-1+c-kit+ (LSK) HSCs. In agreement with MEF studies, terminal differentiation of stem cells in vitro resulted in rapid downregulation of Ezh2 expression. Furthermore, overexpression of Ezh2 in purified HSCs results in increased proliferation rate in vitro. Moreover, HSCs transduced with Ezh2 were enriched for CFU-GM activity. Ongoing studies will address whether overexpression of Ezh2 in stem cells will affect their self-renewal potential in vivo. In an extensive genetic screen we identified genes that correlate with Ezh2 expression in stem cells, potential targets or partners of Ezh2, among others Top2a, MKi67, Pcna, and Eed. These data were deposited in an on-line resource (www.WebQTL.org), to uncover genetic networks that are involved in regulating the process of aging.

Dlx3 transcriptional regulation of osteoblast differentiation: temporal recruitment of Msx2, Dlx3, and Dlx5 homeodomain proteins to chromatin of the osteocalcin gene.

Genetic studies show that Msx2 and Dlx5 homeodomain (HD) proteins support skeletal development, but null mutation of the closely related Dlx3 gene results in early embryonic lethality. Here we find that expression of Dlx3 in the mouse embryo is associated with new bone formation and regulation of osteoblast differentiation. Dlx3 is expressed in osteoblasts, and overexpression of Dlx3 in osteoprogenitor cells promotes, while specific knock-down of Dlx3 by RNA interference inhibits, induction of osteogenic markers. We characterized gene regulation by Dlx3 in relation to that of Msx2 and Dlx5 during osteoblast differentiation. Chromatin immunoprecipitation assays revealed a molecular switch in HD protein association with the bone-specific osteocalcin (OC) gene. The transcriptionally repressed OC gene was occupied by Msx2 in proliferating osteoblasts, while Dlx3, Dlx5, and Runx2 were recruited postproliferatively to initiate transcription. Dlx5 occupancy increased over Dlx3 in mature osteoblasts at the mineralization stage of differentiation, coincident with increased RNA polymerase II occupancy. Dlx3 protein-DNA interactions stimulated OC promoter activity, while Dlx3-Runx2 protein-protein interaction reduced Runx2-mediated transcription. Deletion analysis showed that the Dlx3 interacting domain of Runx2 is from amino acids 376 to 432, which also include the transcriptionally active subnuclear targeting sequence (376 to 432). Thus, we provide cellular and molecular evidence for Dlx3 in regulating osteoprogenitor cell differentiation and for both positive and negative regulation of gene transcription. We propose that multiple HD proteins in osteoblasts constitute a regulatory network that mediates development of the bone phenotype through the sequential association of distinct HD proteins with promoter regulatory elements.

“Antiparallel” DNA Loop in Gal Repressosome Visualized by Atomic Force Microscopy

DNA looping is often involved in positive and negative regulation of gene transcription in both prokaryotes and eukaryotes. The transcription of the gal operon of Escherichia coli from two overlapping promoters P1 and P2 is negatively regulated via Gal repressosome assembly. It involves binding of two dimeric Gal repressor proteins (GalR) to two operators, O E and O I , flanking the two promoters, and formation of 113 bp DNA loop due to tetramerization of the two bound GalR dimers. The process requires negatively supercoiled DNA and the presence of the histone-like protein HU. Previous modeling of the repressosome based on evaluation of DNA elastic energy suggested a mutual antiparallel, rather than parallel, orientation of the two gal operators in an under-twisted DNA loop. To visualize the Gal loop by atomic force microscopy (AFM), plasmid DNA molecules were constructed with increased distance between the two operators. The AFM results demonstrated the formation of an antiparallel DNA loop in the Gal repressosome consistent with our earlier hypothesis. Importantly, the overall shape of the GalR mediated loop proved to be indistinguishable from that in the chimerical loop of the same size containing two lac operators (instead of two gal operators) and formed by LacI. In addition, a possibility of the gal operon repression mediated by GalR in the absence of HU was shown in the new DNA constructs. Implications of these findings for the DNA structural organization in bacterial nucleoid are discussed.

Regulation of oct-1 gene transcription is different in lymphoid and non-lymphoid cells

Transcription factor Oct-1 is expressed in all eukaryotic cells acting as a positive or negative regulator of gene transcription and DNA replication. Being a ubiquitous nuclear protein, Oct-1 also takes part in the regulation of tissue-specific gene expression. In this paper, we have found that human oct-1 gene is regulated by two promoters, located in OTF-1 locus upstream of 1U and 1L exons, respectively. The DNA region preceding U exon has a pattern typical of the constitutive gene promoters. The 5′-region upstream of 1L–exon is AT-rich, contains no TATA box, but has two octamer sequences targeted by Oct-1 and Oct-2 proteins. Analysis of promoter activity is carried out by transfection of recombinant plasmids in non-lymphoid HEK293 and lymphoid Raji cells. In non-lymphoid cells, efficiency of transcription from the 1U promoter several times exceeded that from the 1L promoter. The 1U promoter activity is little increased in the presence of an external enhancer. A different expression pattern was observed if the same constructs were transfected into lymphoid Raji cells. In this case, the level of transcription from the 1L promoter (the L–2 fragment, containing a proximal octamer site) in the presence of the enhancer was significantly higher than that of any fragments containing 1U promoter. It was shown that distal regions of both 1U and 1L were capable of silencing activity. In Raji cells, the enhancer completely overcomes the activity of U silencer, but only partly overcomes that of L silencer. Our data on tissue-specific features of 1L promoter and interaction of both oct-1 promoters with enhancer and silencers in different cell types point to a fine tissue-specific regulation of the oct-1 gene expression, especially in lymphoid cells.

IGF-I isoforms and ageing skeletal muscle: an 'unresponsive' hypertrophy agent?

IGF-I isoforms and ageing skeletal muscle: an 'unresponsive' hypertrophy agent?

The zinc finger transcription factor Zfp60 is a negative regulator of cartilage differentiation.

The differentiation of many mesenchyme-derived cells, including cells that form bone and cartilage, is regulated at the level of gene transcription, but many of the factors involved in this regulation remain to be identified. In this study, a modified RNA fingerprinting technique was used to identify the KRAB domain zinc finger transcription factor Zfp60 as a candidate regulator of cell differentiation in mouse calvaria primary cultures. The highest expression of Zfp60 mRNA in vivo was found between embryonic day 11 (E11) and E15 during mouse embryonic development, coinciding with stages of active organ formation. The expression of Zfp60 mRNA and protein was analyzed further in mouse embryos during skeletal development. The most prominent expression was found in prehypertrophic chondrocytes, where it coincides with the expression of key regulators of chondrocyte maturation, Indian hedgehog (Ihh), and the parathyroid hormone-related peptide (PTHrP) receptor. Zfp60 mRNA was also found transiently expressed during chondrogenesis of C1 cells in vitro, preceding collagen type X expression and cellular hypertrophy. Overexpression of Zfp60 inhibited cartilage differentiation in the chondrogenic ATDC5 cell line. These results suggest a role for Zfp60 as a negative regulator of gene transcription, specifically during the development and/or differentiation of chondrocytes.

Altered Cellular Distribution of Tuberin and Glucocorticoid Receptor in Sporadic Fundic Gland Polyps

Gastric fundic gland polyps (FGPs) are considered hamartomas, and various gastrointestinal hamartomas are associated with tuberous sclerosis complex (TSC). The aim of this study was to investigate a possible link between TSC proteins (hamartin and tuberin) and sporadic FGPs. We examined 33 sporadic FGPs and 26 biopsies of normal fundic mucosa by immunohistochemistry. Nuclear immunoreactivity for tuberin was dramatically reduced or lost in most sporadic FGPs, and tuberin unexpectedly accumulated in the cytoplasm in oxyntic glands. About 18% (6/33) of FGPs were immunopositive in an average of 1.7% of oxyntic cell nuclei, compared with 77% (20/26) of controls in an average of 24.4% of oxyntic cell nuclei (P < .01). No change in hamartin was noted. We further examined the tuberin-associated proteins glucocorticoid receptor (GCR) and p27. Nuclear immunoreactivity for GCR was lost in most sporadic FGPs, but p27 distribution was normal. Sporadic FGPs had a low frequency of staining for Ki-67 except for some cells from cystic components, which is consistent with their slow growth. Our results are consistent with the hypothesis that tuberin may play an important role in pathogenesis of sporadic FGPs. First, an altered cellular localization of tuberin may lead to the deregulation of cell proliferation by interrupting its interaction with hamartin. Second, altered cellular localization of tuberin may preclude its negative regulation of gene transcription mediated by GCR.

Fibrates increase human REV-ERBalpha expression in liver via a novel peroxisome proliferator-activated receptor response element.

Fibrates are widely used hypolipidemic drugs that act by modulating the expression of genes involved in lipid and lipoprotein metabolism. Whereas the activation of gene transcription by fibrates occurs via the nuclear receptor peroxisome proliferator-activated receptor-alpha (PPARalpha) interacting with response elements consisting of a direct repeat of the AGGTCA motif spaced by one nucleotide (DR1), the mechanisms of negative gene regulation by fibrates and PPARalpha are largely unknown. In the present study, we demonstrate that fibrates induce the expression of the nuclear receptor Rev-erbalpha, a negative regulator of gene transcription. Fibrates increase Rev-erbalpha mRNA levels both in primary human hepatocytes and in HepG2 hepatoblastoma cells. In HepG2 cells, fibrates furthermore induce Rev-erbalpha protein synthesis rates. Transfection studies with reporter constructs driven by the human Rev-erbalpha promoter revealed that fibrates induce Rev-erbalpha expression at the transcriptional level via PPARalpha. Site-directed mutagenesis experiments identified a PPAR response element that coincides with the previously identified Rev-erbalpha negative autoregulatory Rev-DR2 element. Electromobility shift assay experiments indicated that PPARalpha binds as heterodimer with 9-cis-retinoic acid receptor to a subset of DR2 elements 5' flanked by an A/T-rich sequence such as in the Rev-DR2. PPARalpha and Rev-erbalpha bind with similar affinities to the Rev-DR2 site. In conclusion, these data demonstrate human Rev-erbalpha as a PPARalpha target gene and identify a subset of DR2 sites as novel PPARalpha response elements. Finally, the PPARalpha and Rev-erbalpha signaling pathways cross-talk through competition for binding to those response elements.

Characterization of a response element in the 5'-flanking region of the avian (chicken) PTH gene that mediates negative regulation of gene transcription by 1,25-dihydroxyvitamin D3 and binds the vitamin D3 receptor.

Analysis of the 5'-flanking region of the avian (chicken) PTH (cPTH) gene has revealed a DNA segment between -74 and -60 that is analogous to the consensus sequence for the vitamin D3 response element (VDRE). The DNA segment consists of two imperfect direct repeats, GGGTCA and GGGTGT, which are separated by a 3-bp spacer. The functionality of the putative VDRE was verified by transfection studies in opossum kidney cells using plasmid constructs that contained various regions of the cPTH gene 5'-flanking sequence and promoter fused to the gene for chloramphenicol acetyl transferase (CAT). Likewise, negative regulation of gene transcription by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] was detected when the cPTH VDRE was inserted immediately upstream from truncated forms of the cPTH or the SV40 early promoter. Using gel mobility shift assays, the cPTH VDRE was compared with the human osteocalcin (hOC) VDRE, which activates gene transcription in the presence of 1,25-(OH)2D3. With a partially purified nuclear extract from dog intestine, the two VDREs produced gel shift patterns that were remarkably similar, with the exception that the binding affinity for the hOC sequence was notably greater. Both VDREs produced two major bound complexes (B1 and B2), which could be completely abolished by the addition of an excess of unlabeled hOC VDRE or a monoclonal antibody specific for the VDR protein. Furthermore, similar protein:DNA complexes were observed when either the cPTH or hOC VDRE were incubated with a mixture of purified preparations of recombinant VDR and retinoid X receptor alpha proteins. Ethylation interference analysis showed that the base contacts made by complexes B1 and B2 with the cPTH VDRE were essentially the same and were restricted primarily to the two half-site sequences.

Characterization of a response element in the 5'-flanking region of the avian (chicken) PTH gene that mediates negative regulation of gene transcription by 1,25-dihydroxyvitamin D3 and binds the vitamin D3 receptor

Characterization of a response element in the 5'-flanking region of the avian (chicken) PTH gene that mediates negative regulation of gene transcription by 1,25-dihydroxyvitamin D3 and binds the vitamin D3 receptor

Homeodomain protein MHox and MADS protein myocyte enhancer-binding factor-2 converge on a common element in the muscle creatine kinase enhancer

MHox is a mesoderm-specific homeodomain protein that binds an A/T-rich element that is essential for activity of the muscle creatine kinase (MCK) enhancer. The MHox binding site also binds the ubiquitous homeodomain protein Oct-1 as well as myocyte enhancer-binding factor-2 (MEF2), which belongs to the MADS superfamily of transactivators. To determine which of these proteins activates MCK transcription through the A/T element, we mutated this sequence such that it would selectively bind MHox, MEF2, or Oct-1 and tested the activities of the mutant enhancers in skeletal muscle cells. These mutant enhancers revealed that only MEF2 is able to activate the MCK enhancer through the A/T element. The convergence of homeodomain and MADS proteins on the A/T element in the MCK enhancer provides a mechanism through which a single DNA sequence can mediate positive and negative regulation of gene transcription and is reminiscent of the roles of these two classes of transcription factors in the control of other cell-specific genes.