CACCC Sites Research Articles

ERK5/HDAC5-mediated, resveratrol-, and pterostilbene-induced expression of MnSOD in human endothelial cells.

Mitochondrial oxidative stress is closely correlated with numerous cardiovascular diseases. Manganese superoxide dismutase (MnSOD) is an important antioxidant enzyme in mitochondria. Although polyphenols can induce the expression of MnSOD, their corresponding mechanisms remains unclear. In this study, we tested the hypothesis that resveratrol and pterostilbene can activate the expression of MnSOD through an AMPK-ERK5/HDAC5-KLF2 pathway. Our results revealed that two stilbenes reduced mitochondrial superoxide-free radicals, and endothelial cell senescence, and increased the mRNA expression of several genes related to mitochondrial function, including MnSOD. Moreover, two stilbenes upregulated the activation of human MnSOD promoter luciferase reporter gene and protein level in human umbilical vein endothelial cells. Similarly, two stilbenes also stimulated LKB1, AMPKα, extracellular-signal related kinase 5 (ERK5) phosphorylation, and histone acetylase 5 (HDAC5) and Kruppel-like factor 2 (KLF2) expression. The knockdown of AMP-activated protein kinase (AMPK), ERK5, and HDAC5 by using short-hairpin RNA blocked pterostilbene-induced phosphorylation of their downstream signaling proteins and the expression of KLF2. Furthermore, using a chromatin immunoprecipitation-PCR detection method, we found that resveratrol and pterostilbene promoted KLF2 binding to CACCC sites of the human MnSOD promoter. Resveratrol and pterostilbene can activate MnSOD expression through ERK5/HDAC5 pathway, thus alleviating mitochondrial oxidative stress in endothelial cells that relates to cardiovascular disease.

Zebrafish globin switching occurs in two developmental stages and is controlled by the LCR

Globin gene switching is a complex, highly regulated process allowing expression of distinct globin genes at specific developmental stages. Here, for the first time, we have characterized all of the zebrafish globins based on the completed genomic sequence. Two distinct chromosomal loci, termed major (chromosome 3) and minor (chromosome 12), harbor the globin genes containing α/β pairs in a 5′–3′ to 3′–5′ orientation. Both these loci share synteny with the mammalian α-globin locus. Zebrafish globin expression was assayed during development and demonstrated two globin switches, similar to human development. A conserved regulatory element, the locus control region (LCR), was revealed by analyzing DNase I hypersensitive sites, H3K4 trimethylation marks and GATA1 binding sites. Surprisingly, the position of these sites with relation to the globin genes is evolutionarily conserved, despite a lack of overall sequence conservation. Motifs within the zebrafish LCR include CACCC, GATA, and NFE2 sites, suggesting functional interactions with known transcription factors but not the same LCR architecture. Functional homology to the mammalian α-LCR MCS-R2 region was confirmed by robust and specific reporter expression in erythrocytes of transgenic zebrafish. Our studies provide a comprehensive characterization of the zebrafish globin loci and clarify the regulation of globin switching.

A Network of Krüppel-like Factors (Klfs): Klf8 IS REPRESSED BY Klf3 AND ACTIVATED BY Klf1 IN VIVO

Transcription factors of the Sp/Klf (Krüppel-like factor) family regulate biological processes such as hematopoiesis, adipogenesis, and stem cell maintenance. Here we show that Bklf or Klf3 (Basic Krüppel-like factor) represses the Klf8 (Krüppel-like Factor 8) gene in vivo. Conversely, Eklf or Klf1 (Erythroid Krüppel-like factor) activates the Klf8 gene. Klf8 is driven by two promoters, both of which contain multiple CACCC sites. Klf3 can repress Klf1-mediated activation of both promoters. Chromatin immunoprecipitation experiments confirm that Klf3 occupies both Klf8 promoters in vivo. Interestingly, in Klf3 knock-out tissue Klf1 gains access, binds, and activates both Klf8 promoters. These results demonstrate direct competition between activating and repressing Klfs in vivo. Together with previous evidence that Klf1 directly activates the Klf3 gene, the results reveal an elaborate network of cross-talk within the Klf family. The recognition of cross-regulation and potential redundancy between Klf family members is critical to the interpretation of various Klf knock-out mice and the understanding of individual Klfs in particular contexts.

Erythroid Kruppel-Like Factor Regulates E2F4 and the G1 Cdk Inhibitor, p18.

Erythroid Kruppel-Like Factor (EKLF) is a zinc finger transcription factor which is essential for β-globin gene expression. Knockout mice die from anemia at E15, but restoration of globin chain imbalance does not rescue anemia or increase survival. Cell lines derived from EKLF null mice undergo proliferation arrest upon reactivation of a conditional EKLF-ER fusion protein, suggesting a role in cell cycle control. A transcriptional profiling experiment comparing the global gene expression in EKLF null and wild type fetal liver identified many differentially expressed genes, a number of which function in G1 and at the G1/S checkpoint of the cell cycle. The Cyclin dependent kinase (Cdk) inhibitor, p18, and the S phase transcription factor E2F4 were both found to be significantly down regulated in EKLF null mice and this result was confirmed by real-time PCR. Interestingly, E2F4 knockout mice have a similar phenotype to EKLF knockout mice. Bioinformatic searches of the p18 and E2F4 genes shows that each contains phylogenetically conserved CACC box motifs capable of binding EKLF within longer regions of conservation in promoter and intron regions. The p18 gene contains two conserved CACCC sites upstream of the start of transcription, which are required for EKLF dependent promoter activity in luciferase reporter assays. The transcription factor E2F4 contains a conserved EKLF-binding CACC site within an intron that is closely associated with two conserved GATA1 binding sites. We show by a chromatin immunoprecipitation (ChIP) assays that the E2F4 intron and p18 promoter are occupied by EKLF in vivo. Together, these results suggest that EKLF is likely to directly regulate expression of key cell cycle genes in vivo to drive the switch from proliferation to differentiation of erythrocytes. The loss of EKLF is likely to result in aberrant proliferation and predisposition to leukemia.

Transcriptional Activation of the Human Inducible Nitric-oxide Synthase Promoter by Krüppel-like Factor 6

Nitric oxide is a ubiquitous free radical that plays a key role in a broad spectrum of signaling pathways in physiological and pathophysiological processes. We have explored the transcriptional regulation of inducible nitric-oxide synthase (iNOS) by Krüppel-like factor 6 (KLF6), an Sp1-like zinc finger transcription factor. Study of serial deletion constructs of the iNOS promoter revealed that the proximal 0.63-kb region can support a 3-6-fold reporter activity similar to that of the full-length 16-kb promoter. Within the 0.63-kb region, we identified two CACCC sites (-164 to -168 and -261 to -265) that bound KLF6 in both electrophoretic mobility shift and chromatin immunoprecipitation assays. Mutation of both these sites abrogated the KLF6-induced enhancement of the 0.63-kb iNOS promoter activity. The binding of KLF6 to the iNOS promoter was significantly increased in Jurkat cells, primary T lymphocytes, and COS-7 cells subjected to NaCN-induced hypoxia, heat shock, serum starvation, and phorbol 12-myristate 13-acetate/ ionophore stimulation. Furthermore, in KLF6-transfected and NaCN-treated COS-7 cells, there was a 3-4-fold increase in the expression of the endogenous iNOS mRNA and protein that correlated with increased production of nitric oxide. These findings indicate that KLF6 is a potential transactivator of the human iNOS promoter in diverse pathophysiological conditions.

Structural and functional cross-talk between a distant enhancer and the epsilon-globin gene promoter shows interdependence of the two elements in chromatin.

We investigated the requirements for enhancer-promoter communication by using the human beta-globin locus control region (LCR) DNase I-hypersensitive site 2 (HS2) enhancer and the epsilon-globin gene in chromatinized minichromosomes in erythroid cells. Activation of globin genes during development is accompanied by localized alterations of chromatin structure, and CACCC binding factors and GATA-1, which interact with both globin promoters and the LCR, are believed to be critical for globin gene transcription activation. We found that an HS2 element mutated in its GATA motif failed to remodel the epsilon-globin promoter or activate transcription yet HS2 nuclease accessibility did not change. Accessibility and transcription were reduced at promoters with mutated GATA-1 or CACCC sites. Strikingly, these mutations also resulted in reduced accessibility at HS2. In the absence of a globin gene, HS2 is similarly resistant to nuclease digestion. In contrast to observations in Saccharomyces cerevisiae, HS2-dependent promoter remodeling was diminished when we mutated the TATA box, crippling transcription. This mutation also reduced HS2 accessibility. The results indicate that the epsilon-globin promoter and HS2 interact both structurally and functionally and that both upstream activators and the basal transcription apparatus contribute to the interaction. Further, at least in this instance, transcription activation and promoter remodeling by a distant enhancer are not separable.

The Flavobacterium okeanokoites adenine-N6-specific DNA-methyltransferase M.FokI is a tandem enzyme of two independent domains with very different kinetic properties.

The Flavobacterium okeanokoites adenine-N6-specific DNA-methyltransferase, M.FokI, modifies both adenine residues within its asymmetric recognition sequence 5'-GGATG/CATCC-3'. It is a fusion protein comprising two independent enzymes. We have cloned, overexpressed and purified full-length M.FokI as well as both individual domains and analyzed their kinetics of DNA methylation using unmethylated and hemimethylated oligodeoxynucleotide substrates. Our data show that both domains of M.FokI methylate DNA independently of each other but cooperate in DNA binding. In agreement to former studies, the N-terminal domain of M.FokI modifies the upper strand of the recognition sequence (GGATG). It strongly prefers hemimethylated (5'-GGATG/CmATCC-3'; mA = N6-methyladenosine) over unmethylated substrates. In contrast, the C-terminal domain prefers unmethylated DNA substrates. Surprisingly, in addition to methylating the lower strand of the recognition sequence (CATCC), M.FokI-(335-647) also modifies the upper strand (GGATG), albeit with a lower activity. In addition, methylation was detected at CACCC sites, but not at sites in which a central AT dinucleotide is flanked by at least one A x T or T x A base pair. These results suggests that M.FokI-(335-647) either has a very degenerate specificity for (G/C)AT(G/C) and sites similar to CATCC or GGATG or, alternatively, that it has a dual specificity for CATCC and GGATG.

Estrogen receptor diminishes DNA-binding activities of chicken GATA-1 and CACCC-binding proteins.

The estrogen receptor (ER) repressed erythroid differentiation and erythroid-specific gene expression. In this study, we investigated the effect of ER alpha (referred to throughout as ER) on DNA-binding activities of transcription factors involved in regulating the expression of erythroid-specific genes, and, in particular, the histone H5 gene. Using electrophoretic mobility shift assays, we found that in the presence of rabbit reticulocyte lysate, human ER reduced the binding activities of chicken immature erythrocyte nuclear extracted proteins to GATA and CACCC sites in the H5 promoter and enhancer. In contrast, the binding activities of NF1 and Sp1 were not affected by ER. Binding of ER to an estrogen response element was enhanced by addition of rabbit reticulocyte lysate. This lysate was also necessary for ER to diminish the DNA-binding activity of GATA-1. These results suggest that additional factor(s) are necessary for full ER function. Both GATA-1 and CACCC-binding proteins are critical for the developmentally regulated expression of erythroid-specific genes. We hypothesize that interference in DNA-binding activities of GATA-1 and CACCC-binding proteins is the mechanism by which the ER inhibits regulation of these genes.

Properties of the mouse α‐globin HS‐26: Relationship to HS‐40, the major enhancer of human α‐globin gene expression

HS-26, the mouse homologue of HS-40, is the major regulatory element of the mouse α-globin gene locus. Like HS-40, HS-26 is located within an intron of a house-keeping gene; comparison of the nucleotide sequences of HS-26 and HS-40 reveals conservation of the sequences and positions of several DNA binding motifs in the 5′ regions of both elements (3 GATA, 2 NFE-2, and 1 CACCC sites) and the absence in HS-26 of three CACCC sites and one GATA site that are present in the 3′ region of HS-40, suggesting that the two elements might not be identical. We report here that when HS-26 is linked to a 1.5 kb Psti human α-globin gene fragment, it has a weak enhancer activity in induced MEL cells and in transgenic embryos, and it does not have any detectable activity in adult transgenic mice. This suggests that HS-26 does not have Locus Control Region (LCR) activity but can act as an enhancer during the embryonic life when integrated at a permissive locus. To further test the importance of HS-26 at its natural locus, we have generated embryonic stem cells and chimeric animals in which 350 bp containing HS-26 have been replaced by a neomycin resistance gene by homologous recombination. The sizes of the chimeras' red cells were then estimated by measuring forward scattering on a FacsScan apparatus in hypotonic conditions. This revealed that a fraction of the chimeric animals' red cells were smaller than normal mouse red cells and were very similar to cells from mice heterozygous for α-thalassemia. Density gradient analysis also suggested the presence of thalassemic cells. These results indicated that despite its lack of LCR activity, HS-26 is important for the regulation of the mouse α-globin gene locus. Am. J. Hematol. 54:30–39, 1997 © 1997 Wiley-Liss, Inc.

Characterization of the Promoter Region of Human BCL-6 Gene

The BCL-6 gene encodes a zinc finger-transcription factor and frequently is rearranged in diffuse large cell lymphoma. We have reported here the isolation of the 5′-flanking region of the human BCL-6 gene. Nucleotides sequence of 1.5 Kb promoter region contains TATA box but no CAAT box and a number of potential regulatory elements including CACCC, E-box, and GATA-1 sites. Studies of the promoter activity showed the first 657 nucleotides of the 5′-flanking region significantly drove transcription of the luciferase reporter gene in human Burkitt′s lymphoma cell line.

PURINE NUCLEOTIDE MODULATION OF COMPLEX ASSEMBLY BETWEEN THE DROSOPHILA CACCC AND DRE, BINDING-PROTEINS IN RAS2 REGULATION

Previous characterisation of the Drosophila ras2/rop bidirectional promoter regulatory mechanism revealed two DNA-binding protein factors. These were named DCF (Drosophila CACCC-binding Factor) and DREF (Drosophila Replication Related Element-binding Factor) respectively. A major protein complex consisting of these two transcription factors specifically binds the CACCC and DRE sites. In the present study we show that limited trypsin digestion of the major complex dissociates DCF and DREF, in the active conformation, able to bind the CACCC and DRE motifs. In addition, we show that DNA-binding activity of the DREF/DCF heterodimer is specifically inhibited by the presence of purine nucleotides, while that of the individual factors remains unaltered.

Transcriptional Role of a Conserved GATA-1 Site in the Human ε-Globin Gene Promoter

The epsilon-globin gene is the first of the human beta-like globin genes to be expressed during development. We have analyzed protein-DNA interactions in the epsilon-globin promoter region by DNase I footprinting and electrophoretic mobility shift experiments using nuclear extracts from K562 human erythroid cells and from nonerythroid HeLa cells. A restricted set of ubiquitous proteins, including Sp1, bound to regions of the promoter including the CACCC and CCAAT sites. Three interactions, at positions -213, -165, and +3 relative to the transcription start site, were erythroid specific and corresponded to binding of GATA-1, a transcription factor highly restricted to the erythroid lineage. Interestingly, the GATA-1 site at -165 has been conserved in the promoters of 10 mammalian embryonic globin genes. Point mutations demonstrate that GATA-1 binding to this site is necessary for interaction with an erythroid-specific enhancer but that in the absence of an enhancer, GATA-1 does not increase transcription.

Transcriptional role of a conserved GATA-1 site in the human epsilon-globin gene promoter.

The epsilon-globin gene is the first of the human beta-like globin genes to be expressed during development. We have analyzed protein-DNA interactions in the epsilon-globin promoter region by DNase I footprinting and electrophoretic mobility shift experiments using nuclear extracts from K562 human erythroid cells and from nonerythroid HeLa cells. A restricted set of ubiquitous proteins, including Sp1, bound to regions of the promoter including the CACCC and CCAAT sites. Three interactions, at positions -213, -165, and +3 relative to the transcription start site, were erythroid specific and corresponded to binding of GATA-1, a transcription factor highly restricted to the erythroid lineage. Interestingly, the GATA-1 site at -165 has been conserved in the promoters of 10 mammalian embryonic globin genes. Point mutations demonstrate that GATA-1 binding to this site is necessary for interaction with an erythroid-specific enhancer but that in the absence of an enhancer, GATA-1 does not increase transcription.