CRE-like Sequence Research Articles

Phylogenetic tracing of midbrain-specific regulatory sequences suggests single origin of eubilaterian brains.

Conserved cis-regulatory elements (CREs) control Engrailed-, Pax2-, and dachshund-related gene expression networks directing the formation and function of corresponding midbrain circuits in arthropods and vertebrates. Polarized outgroup analyses of 31 sequenced metazoan genomes representing all animal clades reveal the emergence of Pax2- and dachshund-related CRE-like sequences in anthozoan Cnidaria. The full complement, including Engrailed-related CRE-like sequences, is only detectable in spiralians, ecdysozoans, and chordates that have a brain; they exhibit comparable genomic locations and extensive nucleotide identities that reveal the presence of a conserved core domain, all of which are absent in non-neural genes and, together, distinguish them from randomly assembled sequences. Their presence concurs with a genetic boundary separating the rostral from caudal nervous systems, demonstrated for the metameric brains of annelids, arthropods, and chordates and the asegmental cycloneuralian and urochordate brain. These findings suggest that gene regulatory networks for midbrain circuit formation evolved within the lineage that led to the common ancestor of protostomes and deuterostomes.

Prediction of designer-recombinases for DNA editing with generative deep learning

Site-specific tyrosine-type recombinases are effective tools for genome engineering, with the first engineered variants having demonstrated therapeutic potential. So far, adaptation to new DNA target site selectivity of designer-recombinases has been achieved mostly through iterative cycles of directed molecular evolution. While effective, directed molecular evolution methods are laborious and time consuming. Here we present RecGen (Recombinase Generator), an algorithm for the intelligent generation of designer-recombinases. We gather the sequence information of over one million Cre-like recombinase sequences evolved for 89 different target sites with which we train Conditional Variational Autoencoders for recombinase generation. Experimental validation demonstrates that the algorithm can predict recombinase sequences with activity on novel target-sites, indicating that RecGen is useful to accelerate the development of future designer-recombinases.

Transcription Factor CREB3L1 Regulates Vasopressin Gene Expression in the Rat Hypothalamus

Arginine vasopressin (AVP) is a neurohypophysial hormone regulating hydromineral homeostasis. Here we show that the mRNA encoding cAMP responsive element-binding protein-3 like-1 (CREB3L1), a transcription factor of the CREB/activating transcription factor (ATF) family, increases in expression in parallel with AVP expression in supraoptic nuclei (SONs) and paraventicular nuclei (PVNs) of dehydrated (DH) and salt-loaded (SL) rats, compared with euhydrated (EH) controls. In EH animals, CREB3L1 protein is expressed in glial cells, but only at a low level in SON and PVN neurons, whereas robust upregulation in AVP neurons accompanied DH and SL rats. Concomitantly, CREB3L1 is activated by cleavage, with the N-terminal domain translocating from the Golgi, via the cytosol, to the nucleus. We also show that CREB3L1 mRNA levels correlate with AVP transcription level in SONs and PVNs following sodium depletion, and as a consequence of diurnal rhythm in the suprachiasmatic nucleus. We tested the hypothesis that CREB3L1 activates AVP gene transcription. Both full-length and constitutively active forms of CREB3L1 (CREB3L1CA) induce the expression of rat AVP promoter-luciferase reporter constructs, whereas a dominant-negative mutant reduces expression. Rat AVP promoter deletion constructs revealed that CRE-like and G-box sequences in the region between -170 and -120 bp are important for CREB3L1 actions. Direct binding of CREB3L1 to the AVP promoter was shown by chromatin immunoprecipitation both in vitro and in the SON itself. Injection of a lentiviral vector expressing CREB3L1CA into rat SONs and PVNs resulted in increased AVP biosynthesis. We thus identify CREB3L1 as a regulator of AVP transcription in the rat hypothalamus.

Transcriptional regulation of human ferredoxin 1 in ovarian granulosa cells

Ferredoxin 1 (FDX1; adrenodoxin) is an iron–sulfur protein that is involved in various metabolic processes, including steroid hormone synthesis in mammalian tissues. We investigated the transcriptional regulation of FDX1 in ovarian granulosa cells. Previously, we reported that the NR5A family, including steroidogenic factor-1 (SF-1) and liver receptor homolog-1 could induce differentiation of human mesenchymal stem cells (hMSCs) into steroidogenic cells. A ChIP assay showed that SF-1 could bind to the FDX1 promoter in differentiated hMSCs. Luciferase reporter assays showed that transcription of FDX1 was synergistically activated by the NR5A family and 8Br-cAMP treatment through two SF-1 binding sites and a CRE-like sequence in a human ovarian granulosa cell line, KGN. Knockdown of FDX1 attenuated progesterone production in KGN cells. These results indicate transcription of FDX1 is regulated by the NR5A family and cAMP signaling, and participates in steroid hormone production in ovarian granulosa cells.

Involvement of transcription factor GATA-4 in regulation of CYP19 gene during folliculogenesis and luteinization in buffalo ovary

CYP19 gene encode aromatase, the key enzyme of estrogen biosynthesis, is regulated in species- and tissue-specific manner by alternate use of different promoters. Previously, we have reported the cloning and characterization of tissue-specific promoter and transcripts in buffalo ovary and placenta. In human and rat ovary, FSH induces the phosphorylation of transcription factor CREB (cAMP response element binding protein) through PKA pathway which binds to cAMP response element like sequence (CLS) in CYP19 gene ovarian promoter. However, in buffalo as well as in bovine, in silico analysis of ovary specific promoter sequence identified a single base pair deletion in CLS site and is designated as CLS-like sequence. To understand if CLS with a point mutation is still a functional cis-element and is involved in FSH stimulated transactivation of CYP19 gene in buffalo ovary, the present study was thus aimed to functionally characterize the role of buffalo CLS in CYP19 gene transactivation. We also studied the involvement of GATA-4, having consensus binding sites in CYP19 gene ovarian promoter in the vicinity of CLS during different stages of the buffalo estrus cycle. Reporter construct analyses and EMSA results showed that CLS is playing no significant role in CYP19 gene regulation in buffalo ovary. Real time absolute quantification of GATA-4 showed the differential expression of GATA-4 mRNA during folliculogenesis and luteinization with significantly higher transcript abundance in large follicle in comparison to other tissues. Western blot analysis of granulosa cells nuclear protein isolated from different stage of follicular development (small and large follicles) and differentiation (corpus luteum) showed that abundance of phosphorylated GATA-4 (Ser261) was significantly higher in granulosa cell nuclear protein of large follicles as compared to small follicles and corpora lutea. Interestingly, binding studies using ChIP showed significantly enhanced binding to the CYP19 gene promoter in large follicle which was seen to be declined in the luteal tissue. Similar results were obtained in the in vitro experiments as well. Finally, RNAi experiments were performed to validate the involvement of GATA-4 in CYP19 gene regulation. Results of RNAi showed that knockdown of GATA-4 mRNA significantly declined CYP19 gene mRNA as well as 17β-estradiol contents. In conclusion, result of the present study indicated that that in the absence of consensus CRE (cAMP response element); GATA-4 could be a downstream effector of cAMP/PKA pathway in regulation of CYP19 gene during folliculogenesis and luteinization.

Characterization of cAMP Response Element-Like Sequence in Gonadal Specific Aromatase Promoter Sequence ofXenopusEmbryos

The p450 aromatase gene has a tissue-specific promoter that is regulated by specific transcriptional factors. In rats and humans, a cAMP response element-like sequence (CLS) and an NR5A1/NR5A2 binding sequence have been identified as cis elements in the aromatase promoter; these cis elements mediate cAMP-induced expression in the ovaries and testes. CLS is recognized by a cAMP-responsive element binding protein (CREB) as the principal component. In this study, we performed a gel shift assay to analyze the proteins that interact with the cis-element in Xenopus aromatase. An electrophpretic mobility gel shift assay (EMSA) and matrix-associated laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) analysis of the proteins responsible for retarding the mobility of CLS revealed that ATF4 interacted in vitro with CLS in gonadal specific aromatase promoter sequence of Xenopus embryos. Although a significant difference was observed in aromatase mRNA expression between male and female gonads, no difference in the expression of ATF4 was observed between them at stage 50. With regard to aromatase expression in the gonad of Xenopus embryos, ATF4 might act in combination with multiple transcription factors as a trans-element of CLS in place of CREB.

Melatonin synthesis in retina: cAMP‐dependent transcriptional regulation of chicken arylalkylamineN‐acetyltransferase by a CRE‐like sequence and a TTATT repeat motif in the proximal promoter

Arylalkylamine N-acetyltransferase (AANAT) is the key regulatory enzyme controlling the daily rhythm of melatonin biosynthesis. In chicken retinal photoreceptor cells, Aanat transcription and AANAT activity are regulated in part by cAMP-dependent mechanisms. The purpose of this study was to identify regulatory elements within the chicken Aanat promoter responsible for cAMP-dependent induction. Photoreceptor-enriched retinal cell cultures were transfected with a luciferase reporter construct containing up to 4 kb of 5'-flanking region and the first exon of Aanat. Forskolin treatment stimulated luciferase activity driven by the ∼4 kb promoter construct and by all 5'-deletion constructs except the smallest, Aanat (-217 to +120)luc. Maximal basal and forskolin-stimulated expression levels were generated by the Aanat (-484 to +120)luc construct. This construct lacks a canonical cyclic AMP-response element (CRE), but contains two other potentially important elements in its sequence: an eight times TTATT repeat (TTATT₈) and a CRE-like sequence. Electrophoretic mobility shift assays, luciferase reporter assays, chromatin immunoprecipitation, and siRNA experiments provide evidence that these elements bind c-Fos, JunD, and CREB to enhance basal and forskolin-stimulated Aanat transcription. We propose that the CRE-like sequence and TTATT₈ elements in the 484 bp proximal promoter interact to mediate cAMP-dependent transcriptional regulation of Aanat.

BzpF is a CREB-like transcription factor that regulates spore maturation and stability in Dictyostelium

The cAMP response element-binding protein (CREB) is a highly conserved transcription factor that integrates signaling through the cAMP-dependent protein kinase A (PKA) in many eukaryotes. PKA plays a critical role in Dictyostelium development but no CREB homologue has been identified in this system. Here we show that Dictyostelium utilizes a CREB-like protein, BzpF, to integrate PKA signaling during late development. bzpF− mutants produce compromised spores, which are extremely unstable and germination defective. Previously, we have found that BzpF binds the canonical CRE motif in vitro. In this paper, we determined the DNA binding specificity of BzpF using protein binding microarray (PBM) and showed that the motif with the highest specificity is a CRE-like sequence. BzpF is necessary to activate the transcription of at least 15 PKA-regulated, late-developmental target genes whose promoters contain BzpF binding motifs. BzpF is sufficient to activate two of these genes. The comparison of RNA sequencing data between wild type and bzpF− mutant revealed that the mutant fails to express 205 genes, many of which encode cellulose-binding and sugar-binding proteins. We propose that BzpF is a CREB-like transcription factor that regulates spore maturation and stability in a PKA-related manner.

Mono-(2-ethylhexyl) phthalate induces NR4A subfamily and GIOT-1 gene expression, and suppresses CYP19 expression in human granulosa-like tumor cell line KGN

The mechanism for transcriptional suppression of CYP19 by mono-ethylhexyl phthalate (MEHP) in a human ovarian granulosa cell line (KGN) was investigated. It is known that the CYP19 gene transcript in KGN cells predominantly includes exon PII among the 11 alternate exon I sequences. MEHP was found to significantly suppress Forskolin (FSK)-induced CYP19 gene transcription, CYP19 promoter II activity and CYP19 enzyme activity in a dose-dependent manner. Promoter assays using 5′-deleted promoter II reporter constructs suggested that the region important for responsiveness to MEHP exposure includes a putative CRE-like sequence and an SF-1 (NR5A1)/LRH-1 (NR5A2) binding sequence. Meanwhile, MEHP exposure rapidly and transiently induced nuclear receptor 4A (NR4A) mRNA, and gradually and continuously induced gonadotropin-inducible ovarian transcription factor-1 (GIOT-1; ZNF461) mRNA in KGN cells. The ectopic expression of NR4As and GIOT-1 suppressed promoter II activity, while among the NR4As expressed, only Nur77 (NR4A1) secondarily induced GIOT-1 mRNA expression. Based on these results, we believe that induction of the Nur77-GIOT-1 system by MEHP is involved in the transcriptional suppression of the CYP19 gene, and GIOT-1 may attenuate the promoter II activity due to suppression of SF-1 and/or LRH-1 transactivation in KGN cells.

Regulation of endoplasmic reticulum stress response by a BBF2H7-mediated Sec23a pathway is essential for chondrogenesis

Many tissues have a specific signal transduction system for endoplasmic reticulum (ER) dysfunction; however, the mechanisms underlying the ER stress response in cartilage remain unclear. BBF2H7 (BBF2 human homologue on chromosome 7), an ER-resident basic leucine zipper transcription factor, is activated in response to ER stress and is highly expressed in chondrocytes. In this study, we generated Bbf2h7(-/-) mice to assess the in vivo function of BBF2H7. The mice showed severe chondrodysplasia and died by suffocation shortly after birth because of an immature chest cavity. The cartilage showed a lack of typical columnar structure in the proliferating zone and a decrease in the size of the hypertrophic zone, resulting in a significant reduction of extracellular matrix proteins. Interestingly, proliferating chondrocytes showed abnormally expanded ER, containing aggregated type II collagen (Col2) and cartilage oligomeric matrix protein (COMP). We identified Sec23a, which encodes a coat protein complex II component responsible for protein transport from the ER to the Golgi, as a target of BBF2H7, which directly bound to a CRE-like sequence in the promoter region of Sec23a to activate its transcription. When Sec23a was introduced to Bbf2h7(-/-) chondrocytes, the impaired transport and secretion of cartilage matrix proteins was totally restored, indicating that by activating protein secretion the BBF2H7-Sec23a pathway has a crucial role in chondrogenesis. Our findings provide a new link by which ER stress is converted to signalling for the activation of ER-to-Golgi trafficking.

Opposite Effects of Prolactin (Prl) on the Expression of Aromatase and Liver Receptor Homolog 1 (LRH-1) in Luteal Cells.

. effects are mediated in part by an increase in the expression of the estrogen receptors (2), which, in turn, are needed for estradiol action. Paradoxically, it has been shown that prolactin blocks the stimulation of estradiol production by FSH in granulosa cells (3, 4) and inhibits aromatase expression in the corpus luteum (5, 6). This inhibitory effect of Prl on estradiol production explains why low levels of aromatase expression are found in corpora lutea during early gestation when surges of prolactin are being released twice daily from the pituitary. However, there is no direct evidence indicating that pituitary prolactin in fact inhibits luteal aromatase at the beginning of pregnancy in the rat. In addition, the mechanism involved in the inhibition of aromatase expression by Prl is not known. In all species studied thus far the aromatase gene is controlled by at least two Figure 1Effect of the Inhibition of Prl Secretion on the Expression of Figure 4: Reversibility of the Inhibitory Effect of Prl. Luteal cells were cultured in the , promoters. We have demonstrated that the promoter located immediately upstream of the coding region, also known as the proximal promoter, controls the expression of aromatase in luteal cells (7, 8). The proximal promoter contains a cAMPresponsive element-like sequence (CLS) and two binding sites for members of the nuclear receptors 5A family of transcription factors (NR5a/SF-1 and NR5b/LRH-1) (9). This promoter also contains a response element for members of the GATA family of transcription factors and one AP-3 binding site. These binding sites are highly conserved between species. Of the factors that bind to these sites, LRH-1, SF-1, and GATA4 seem to play an important role in the regulation of aromatase in granulosa and luteal cells (Reviewed in 9). In this report we examined the regulation of aromatase by Prl in the corpus luteum Aromatase in the Corpus Luteum of Rats on Day 5 of Pregnancy. On day 4 of pregnancy, rats were treated with Prl or Vehicle 30 minutes before the administration of Ergocriptine (ERGO), an inhibitor of Prl secretion. Luteal aromatase expression was determined 24 hours after the administration of ERGO. Columns with different letter differ significantly. Figure 6: Expression of Aromatase, LRH-1, and SF-1 in the Rat Corpus Luteum during Pregnancy. absence (Control) or presence of Prl (1 ug/ml) for 10 days. On the 10th day, Prl was removed from the medium and the cells incubated for 12, 24, or 48 hours more. Aromatase expression was determined by RT-PCR. Columns with different letters differ significantly.

CAMP-dependent regulation of CYP19 gene in rabbit preovulatory granulosa cells and corpus luteum

Transcription of the CYP19 gene encoding the aromatase P450 enzyme in ovarian cells is under the control of the two gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH), via modulation of intracellular cyclic 3′,5′-adenosine monophosphate (cAMP) levels. Primary cultures of rabbit ovarian cells were used to identify the functional regions of the ovarian promoter (PII) that are responsive to the gonadotropic secondary messenger and to estradiol. Transfection experiments in granulosa and luteal cells with deleted constructs of the PII promoter show that the region between −274 and −193 bp is critical for cAMP-dependent transcriptional activity. A comparison of PII activities in granulosa and small luteal cells highlights a 50% decrease consecutive to the LH surge. Sequence analysis of the above mentioned region revealed the presence of a cAMP responsive element like sequence (CLS) and of a nuclear receptor element A (NREA). Binding of CREB to CLS has been shown using granulosa and luteal cells nuclear extracts. In addition, we identified the expression of NR5A1 (Steroidogenic Factor 1) and NR5A2 (Liver Receptor Homologue 1) in granulosa and luteal cells. However, the binding to NREA is observed only with granulosa cells nuclear extracts. Data suggest that the NR5A factors are not the main regulators of CYP19 gene, in contrast with the others genes of streroidogenesis enzymes, and additional sites may play an important role during the post-LH surge down-regulation of CYP19 transcription.

Phylogenetic prediction of cis-acting elements: a cre-like sequence in Norovirus genome?

BackgroundDiscrete RNA structures such as cis-acting replication elements (cre) in the coding region of RNA virus genomes create characteristic suppression of synonymous site variability (SSSV). Different phylogenetic methods have been developed to predict secondary structures in RNA viruses, for high-resolution thermodynamic scanning and for detecting SSSV. These approaches have been successfully in predicting cis-acting signals in different members of the family Picornaviridae and Caliciviridae. In order to gain insight into the identification of cis-acting signals in viruses whose mechanisms of replication are currently unknown, we performed a phylogenetic analysis of complete genome sequences from 49 Human Norovirus (NoV) strains.FindingsThe complete coding sequences of NoV ORF1 were obtained from the DDBJ database and aligned. Shannon entropy calculations and RNAalifold consensus RNA structure prediction identified a discrete, conserved, invariant sequence region with a characteristic AAACG cre motif at positions 240 through 291 of the RNA dependant RNA polymerase (RdRp) sequence (relative to strain [EMBL:EU794713]). This sequence region has a high probability to conform a stem-loop.ConclusionA new predicted stem-loop has been identified near the 5' end of the RdRp of Human NoV genome. This is the same location recently reported for Hepatovirus cre stem-loop.

Microarray transfection analysis of conserved genomic sequences from three immediate early genes

In an effort to define novel transcriptional regulatory elements, microarray cotransfection was used to functionally characterize conserved non-coding sequences (CNSs) of three immediate early genes: c- fos, JunB and EGR-1. Cotransfection of fluorescent CNS reporter constructs and expression vectors for constitutively active signaling proteins demonstrated that many of the CNSs alter both the basal and regulated expressions of reporter constructs, but the effects of these CNSs were usually specific for their homologous promoter. One CNS located in the first intron of the c- fos gene conferred regulation by cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG) and Raf. Mutagenesis and cotransfection experiments showed that PKA regulation of this c- fos intronic element was mediated by two adjacent CRE-like sequences and the transcription factor CREB. In the context of a reporter containing previously characterized regulatory elements, the novel intronic sequence contributed 50% of the transcriptional response to PKA. These studies suggest that microarray transfection studies may be useful in functional characterization of conserved genomic sequences on a larger scale.

The Species-Specific Differences in the cAMP Regulation of the Tissue-Type Plasminogen Activator Gene between Rat, Mouse and Human is Caused by a One-Nucleotide Substitution in the cAMP-Responsive Element of the Promoters

In rat ovarian cells tissue-type plasminogen activator (tPA) is induced by gonadotropins, by a cAMP-dependent pathway and the induction correlates with the time of follicle rupture in vivo. However, in mice, gonadotropins induce the related but distinct protease urokinase-type plasminogen activator (uPA). Comparison of rat, mouse and human tPA genes reveal that there is a species-specific difference in the promoter that could explain the difference in regulation of the tPA gene between these species. At the position where the rat promoter contains a consensus cAMP-responsive element (CRE), the mouse and human counterparts contains a CRE variant with a one-nucleotide substitution. Transient transfection experiments of rat glial and granulosa cells demonstrated that reporter constructs driven by rat but not mouse or human tPA promoters were efficiently induced by the cAMP-inducing agents forskolin or follicle-stimulating hormone. Following the conversion of the mouse and human CRE-like sequences to rat consensus CRE these promoters became cAMP responsive. In contrast the rat promoter, following conversion of the consensus CRE to the corresponding mouse and human CRE-like sequence, lost the ability to efficiently respond to cAMP. Deoxyribonuclease I footprinting analysis and electrophoretic mobility shift assays were used to examine interactions of nuclear factors with the consensus and variant CRE. Compared to rat CRE, the mouse and human CRE-like sequences had a drastically reduced binding affinity for a nuclear factor identified as the cAMP-responsive element binding protein. Thus the inability of the mouse and human tPA promoters to respond efficiently to forskolin and follicle-stimulation hormone seem to be due to the inability of these CRE-like sequences to efficiently bind transcription factor CRE binding protein.

The LacI/GalR family transcriptional regulator UriR negatively controls uridine utilization of Corynebacterium glutamicum by binding to catabolite-responsive element (cre)-like sequences

The Cg1547 protein of Corynebacterium glutamicum ATCC 13032 is a member of the LacI/GalR family of DNA-binding transcriptional regulators. A defined deletion in the cg1547 gene, now designated uriR (uridine utilization regulator), resulted in the mutant strain C. glutamicum KB1547. Comparison of gene expression levels in C. glutamicum KB1547 and the wild-type strain revealed enhanced expression of the uriR operon genes cg1546 (ribokinase), cg1545 (uridine transporter) and cg1543 (uridine-preferring nucleoside hydrolase). Gene expression of the uriR operon was stimulated by the presence of either uridine or ribose. Growth assays with C. glutamicum mutants showed that functional Cg1543 and Cg1545 proteins are essential for the utilization of uridine as the sole carbon source. Transcriptional regulation of the uriR operon is mediated by a 29 bp palindromic sequence composed of two catabolite-responsive element (cre)-like sequences and located in between the mapped -10 promoter region and the start codon of uriR. A similar cre sequence was detected in the upstream region of rbsK2 (cg2554), coding for a second ribokinase in C. glutamicum ATCC 13032. DNA band-shift assays with a streptavidin-tagged UriR protein and labelled oligonucleotides including the cre-like sequences of uriR and rbsK2 demonstrated the specific binding of the purified regulator in vitro. Whole-genome DNA microarray hybridizations comparing the gene expression in C. glutamicum KB1547 with that of the wild-type strain revealed that UriR is a pathway-specific repressor of genes involved in uridine utilization in C. glutamicum.

Distinct Chromatin Modulators Regulate the Formation of Accessible and Repressive Chromatin at the Fission Yeast Recombination Hotspotade6-M26

Histone acetyltransferases (HATs) and ATP-dependent chromatin remodeling factors (ADCRs) regulate transcription and recombination via alteration of local chromatin configuration. The ade6-M26 allele of Schizosaccharomyces pombe creates a meiotic recombination hotspot that requires a cAMP-responsive element (CRE)-like sequence M26, the Atf1/Pcr1 heterodimeric ATF/CREB transcription factor, the Gcn5 HAT, and the Snf22 SWI2/SNF2 family ADCR. Chromatin alteration occurs meiotically around M26, leading to the activation of meiotic recombination. We newly report the roles of other chromatin remodeling factors that function positively and negatively in chromatin alteration at M26: two CHD-1 family ADCRs (Hrp1 and Hrp3), a Spt-Ada-Gcn5 acetyltransferase component (Ada2), and a member of Moz-Ybf2/Sas3-Sas2-Tip60 family (Mst2). Ada2, Mst2, and Hrp3 are required for the full activation of chromatin changes around M26 and meiotic recombination. Acetylation of histone H3 around M26 is remarkably reduced in gcn5Delta, ada2Delta and snf22Delta, suggesting cooperative functions of these HAT complexes and Snf22. Conversely, Hrp1, another CHD-1 family ADCR, maintains repressive chromatin configuration at ade6-M26. Interestingly, transcriptional initiation site is shifted to a site around M26 from the original initiation sites, in couple with the histone acetylation and meiotic chromatin alteration induced around 3' region of M26, suggesting a collaboration between these chromatin modulators and the transcriptional machinery to form accessible chromatin. These HATs and ADCRs are also required for the regulation of transcription and chromatin structure around M26 in response to osmotic stress. Thus, we propose that multiple chromatin modulators regulate chromatin structure reversibly and participate in the regulation of both meiotic recombination and stress-induced transcription around CRE-like sequences.

Identification of regulatory elements in the Cyp19 proximal promoter in rat luteal cells

The cytochrome P450 aromatase (Cyp19) gene encodes an enzyme of crucial importance in the synthesis of estradiol. Estradiol is luteotropic in the rat. In this species, luteal Cyp19 expression increases progressively during pregnancy and falls before parturition. The mechanisms that control these changes are unknown. Using gel shift assays, we sought to identify the promoter regions that control Cyp19 expression in the rat corpus luteum (CL). The Cyp19 promoter contains a cAMP response element-like sequence (CLS), two nuclear receptor elements half sites (NREs), a GATA binding site, a Yin Yang-1 (YY1) response element, and an activation protein 3 (AP3) binding site. Nuclear extracts were obtained from CL of rats on days 4, 15, and 23 of pregnancy and from the ovaries of immature rats treated with vehicle or a hormone that induces Cyp19 expression in the follicles. CLS was active in immature ovaries but inactive in the CL of pregnant rats, whereas binding to NREs and GATA was observed in both tissues. YY1 was inactive in all samples tested. In the CL, AP3 binding was higher on day 15 of pregnancy when compared with day 4 and day 23 but it was absent in ovaries of immature rats, whereas luteinization increased AP3 binding activity. Mutation of the AP3 site blunted the stimulation of Cyp19 promoter activity in granulosa cells. Our results indicate that CLS is active only in follicles; whereas in the CL, binding to the GATA, NRE, and AP3 sites associates with changes in Cyp19 expression, suggesting that they control Cyp19 promoter activity in luteal cells.

Transcriptional Regulation of the Bovine Leukemia Virus Promoter by the Cyclic AMP-response Element Modulator τ Isoform

Bovine leukemia virus (BLV) expression is controlled at the transcriptional level through three Tax(BLV)-responsive elements (TxREs) responsive to the viral transactivator Tax(BLV). The cAMP-responsive element (CRE)-binding protein (CREB) has been shown to interact with CRE-like sequences present in the middle of each of these TxREs and to play critical transcriptional roles in both basal and Tax(BLV)-transactivated BLV promoter activity. In this study, we have investigated the potential involvement of the cAMP-response element modulator (CREM) in BLV transcriptional regulation, and we have demonstrated that CREM proteins were expressed in BLV-infected cells and bound to the three BLV TxREs in vitro. Chromatin immunoprecipitation assays using BLV-infected cell lines demonstrated in the context of chromatin that CREM proteins were recruited to the BLV promoter TxRE region in vivo. Functional studies, in the absence of Tax(BLV), indicated that ectopic CREMtau protein had a CRE-dependent stimulatory effect on BLV promoter transcriptional activity. Cross-link of the B-cell receptor potentiated CREMtau transactivation of the viral promoter. Further experiments supported the notion that this potentiation involved CREMtau Ser-117 phosphorylation and recruitment of CBP/p300 to the BLV promoter. Although CREB and Tax(BLV) synergistically transactivated the BLV promoter, CREMtau repressed this Tax(BLV)/CREB synergism, suggesting that a modulation of the level of Tax(BLV) transactivation through opposite actions of CREB and CREMtau could facilitate immune escape and allow tumor development.

PTHrP P3 promoter activity in breast cancer cell lines: Role of Ets1 and CBP (CREB binding protein)

Parathyroid hormone-related protein (PTHrP) is produced by many tumors including breast cancer. We have reported that Ets1 factor activates P3 PTHrP promoter in our model of tumorigenic breast cancer cell and not in pre- or non-tumorigenic cell lines, thus contributing to an increased PTHrP production. In this study, gel retardation assays revealed that Etsl and its promoter binding site (EBS) specifically formed complexes whose abundance correlates with Ets1 levels in the three cell lines. Coexpression of Etsl and CBP induced a synergistic activation of the P3 promoter only in the tumorigenic cell line. This synergism required the integrity of the EBS and was abrogated by E1A. All breast cancer cell lines showed high basal concentrations of phosphorylated CREB. Moreover a CRE-like sequence was also required for Ets1/CBP synergy and, finally, CREB expression was found to enhance the PTHrP P3 promoter activity. Thus a multipartite complex of transcription factors and coactivators seems to regulate PTHrP transcription and contribute to the alterations that promote tumorigenic behavior in breast epithelial cells.