Distal Promoter Sequences Research Articles

Transcriptional regulation of the mouse EphA4, Ephrin-B2 and Ephrin-A3 genes by the circadian clock machinery

ABSTRACT Circadian rhythms originate from molecular feedback loops. In mammals, the transcription factors CLOCK and BMAL1 act on regulatory elements (i.e. E-boxes) to shape biological functions in a rhythmic manner. The EPHA4 receptor and its ligands Ephrins (EFN) are cell adhesion molecules regulating neurotransmission and neuronal morphology. Previous studies showed the presence of E-boxes in the genes of EphA4 and specific Ephrins, and that EphA4 knockout mice have an altered circadian rhythm of locomotor activity. We thus hypothesized that the core clock machinery regulates the gene expression of EphA4, EfnB2 and EfnA3. CLOCK and BMAL1 (or NPAS2 and BMAL2) were found to have transcriptional activity on distal and proximal regions of EphA4, EfnB2 and EfnA3 putative promoters. A constitutively active form of glycogen synthase kinase 3β (GSK3β; a negative regulator of CLOCK and BMAL1) blocked the transcriptional induction. Mutating the E-boxes of EphA4 distal promoter sequence reduced transcriptional induction. EPHA4 and EFNB2 protein levels did not show circadian variations in the mouse suprachiasmatic nucleus or prefrontal cortex. The findings uncover that core circadian transcription factors can regulate the gene expression of elements of the Eph/Ephrin system, which might contribute to circadian rhythmicity in biological processes in the brain or peripheral tissues.

Conserved structure of the NPR1 gene distal promoter isolated from a chili pepper (Capsicum annuum L.) in West Sumatera

Background: The non-expressor of pathogenesis related gene 1 (NPR1) protein is one of the key regulators in the systemic acquired resistance plant defense system. The cis-acting elements of its distal promoter gene are characterized by salicylic acid inducing elements such as the W-box, RAV1AAT and ASF1, accompanied by enhancer and silencer elements. This study was aimed to isolate and characterize the distal promoter sequence of the NPR1 gene (PD_CbNPR1) from the chili pepper (Capsicum annuum L.) genotype Berangkai, a local genotype known to produce large yields, but is susceptible to viral infection. Elucidating its sequence structure will open a broad range of possibilities to engineer the NPR1 gene expression which is important to improve chili pepper resistant. Methods: PCR-based cloning combined with a primer walking strategy was applied in this study. The BioEdit tool was used to edit the sequence and verify sequence integrity, while homology analysis was conducted with BLASTn searching. Identification of a cis-acting element was detected by PLACE, PlantCare, and PlantPAN. Results: Isolation of the complete distal promoter sequence of PD_CbNPR1 produced a fragment 5,950 bp in size. BLASTn search analysis indicated that PD_CbNPR1 sequence is highly conserved (99% identity) showing only a single nucleotide polymorphism (SNP) (base substitution) compared with its reference sequence. Analysis using PLACE tools successfully identified nine cis-acting elements containing a W-box, WLE1, RAV1AAT, TATA-box, CAAT-box, GARE and GT1 with multi repeats and diverse motives, as well as enhancer and silencer elements, which is characterized by a CCAAT-box and GAGAAATT pattern, respectively. Conclusion: The distal promoter of the NPR1 gene is highly conserved, showing only one SNP caused by one base substitution event.

Partial isolation of distal promoter sequence of the NPR1 gene from local chilli Pepper [Capsicum annuum L.] genotype berangkai

Understanding of NPR1gene expression is a crucial aspect in improvement of chilli pepper resistant. The NPR1 gene is known to confer broad spectrum resistance toward many phytopathogens by activating SARmechanism. For that reason, characterization of the NPR1-gene promotor is necessary. The distal promoter of the NPR1-gene in general is characterized by the presence of enhancer, silencer, and cis-acting elements, located 5,950 bp from the ATG starting point. Therefore this study is focused to the isolateion of the distal promoter sequence of the NPR1 gene from a local Capsicum annul L. Genotype Berangkai. Specific primer pairs designatedas PD_CbNPR1-F1 [FR] and PD_CbNPR1-F2 [FR] were designed to amplify the whole targeted segments. However, only 1,873 bp in total [31.5 %] of sequences could successfully be elucidated from the first round, covering 970 and 993 bp from its upstream and downstream segments respectively. Hhomology analysis using BLAST tool successfully confirmed 99 and 100 % homology with its reference sequence derived from cv. Zunla-1. These result indicated that the NPR1 distal promoter sequence is successfully isolated from chilli pepper [Capsicum annuum L] genotype Berangkai. However, ffurther analysis using primer walking strategy should be undertaken in order to identify the entire sequence of its promoter region.

Conserved structure of the NPR1 gene distal promoter isolated from a chili pepper (Capsicum annuum L.) in West Sumatera

Background: The non-expressor of pathogenesis related gene 1 (NPR1) protein is one of the key regulators in the systemic acquired resistance plant defence system. The cis-acting elements of its distal promoter gene are characterized by salicylic acid inducing elements such as the W-box, RAV1AAT and ASF1, accompanied with enhancer and silencer elements. This study was aimed to isolate and characterize the distal promoter sequence of the NPR1 gene (PD_CbNPR1) from the chili pepper (Capsicum annuum L.) genotype Berangkai, a local genotype known to produce large yields, but is susceptible to viral infection. Elucidating its sequence structure will open a broad range of possibilities to engineer the NPR1 gene expression which is important to improve chili pepper resistant. Methods: PCR-based cloning combined with a primer walking strategy was applied in this study. The BioEdit tool was used to edit the sequence and verify sequence integrity, while homology analysis was conducted with BLASTn searching. Identification of a cis-acting element was detected by PLACE. Results: Isolation of the complete distal promoter sequence of PD_CbNPR1 produced a fragment 5,950 bp in size. BLASTn search analysis indicated that PD_CbNPR1 sequence is highly conserved (99% homology) showing only a single nucleotide polymorphism (SNP) (base substitution) compared with its reference sequence. Analysis using PLACE tools successfully identified nine cis-acting elements containing a W-box, WLE1, RAV1AAT, TATA-box, CAAT-box, GARE and GT1 with multi repeats and diverse motives, as well as enhancer and silencer elements, which is characterized by a CCAAT-box and GAGAAATT pattern, respectively. Conclusion: The distal promoter of the NPR1 gene is highly conserved, showing only one SNP caused by one base substitution event.

Gonadotrope-Specific Expression and Regulation of Ovine Follicle Stimulating Hormone Beta: Transgenic and Adenoviral Approaches Using Primary Murine Gonadotropes

The beta subunit of follicle stimulating hormone (FSHB) is expressed specifically in pituitary gonadotropes in vertebrates. Transgenic mouse studies have shown that enhancers in the proximal promoter between −172/−1 bp of the ovine FSHB gene are required for gonadotrope expression of ovine FSHB. These enhancers are associated with regulation by activins and gonadotropin releasing hormone (GnRH). Additional distal promoter sequence between −4741/−750 bp is also required for expression. New transgenic studies presented here focus on this distal region and narrow it to 1116 bp between −1866/−750 bp. In addition, adenoviral constructs were produced to identify these critical distal sequences using purified primary mouse gonadotropes as an in vitro model system. The adenoviral constructs contained −2871 bp, −750 bp or −232 bp of the ovine FSHB promoter. They all showed gonadotrope-specific regulation since they were induced only in purified primary gonadotropes by activin A (50 ng/ml) and inhibited by GnRH (100 nM) in the presence of activin (except −232FSHBLuc). However, basal expression of all three viral constructs (in the presence of follistatin to block cellular induction by activin) was relatively high in pituitary non-gonadotropes as well as gonadotropes. Thus, gonadotrope-specific regulation associated with the proximal promoter was observed as expected, but the model was blind to distal promoter elements between −2871/−750 necessary for gonadotrope-specific expression of ovine FSHB in vivo. The new adenoviral-based in vitro technique did detect, however, a novel GnRH response element between −750 bp and −232 bp of the ovine FSHB promoter. We conclude that adenoviral-based studies in primary gonadotropes can adequately recognize regulatory elements on the ovine FSHB promoter associated with gonadotrope-specific regulation/expression, but that more physiologically based techniques, such as transgenic studies, will be needed to identify sequences between −1866/−750 bp of the ovine FSHB promoter that are also required for tissue/cell specific expression in vivo.

284 IDENTIFICATION AND CHARACTERIZATION OF THE 5′-FLANKING REGION OF THREE MOUSE MATERNAL GENES (HISTONE H1OO, NUCLEOPLASMIN 2, AND ZYGOTE ARREST 1): TRANSCRIPTIONAL ACTIVITY IN MOUSE OOCYTES

Maternal transcripts that accumulate during oocyte growth are involved in the meiotic maturation, the initiation of the first mitosis, and the later pre-implantation development. Although the conserved E-box sequences in promoter region of some maternal genes (for example, Zp3 and Gdf-9), which are important in regulating gene transcripts as binding sites of transcriptional factors, may play a role in the oocyte-specific expression in ovary, the molecular mechanism that regulates the expression of the maternal genes is still not known. In this study, we have focused on the transcriptional activity of promoter regions to clarify the molecular mechanism of transcriptional regulation of these maternal genes [Histone H1oo (H1oo), Nucleoplasmin 2 (Npm2), and Zygote arrest 1 (Zar1)]. First, we observed the expression of firefly luciferase expression vectors under promoter regions of 3 maternal genes in oocytes isolated from 10- to 12-day-old mice, which is mainly NSN-type, transcriptionally active form (Bouniol-Baly et al. 1999 Biol. Reprod. 60, 580–587). Transcriptional activities of H1oo (-3975), Npm2 (-2610), and Zar1 (-5187) promoters were detected in oocytes, the relative luciferase activities being an average of 70, 130, and 12, respectively. On the other hand, these promoter activities were not detected in embryos at the 2-cell stage. Furthermore, deletion analysis of promoter elements (E-boxes) of H1oo and Npm2 was done by microinjecting deletion constructs into oocytes. In the H1oo promoter, deletion of sequences between -3975 and -72 bp from the transcription start site resulted in one-third of the level obtained in the full H1oo (-3975) promoter. In addition, deletion of sequence -68 bp resulted in no detection of luciferase activity. These findings indicate that the putative distal promoter sequences exist at the 52-flanking region (-3975 to -759) of the H1oo gene and that the region (-314 to -68) including the E-box region (-72) may be required for high-level transcriptional activity of the H1oo promoter. In the Npm2 promoter, deletion of sequences between -2610 and -180 bp from the transcription start site resulted in one-third of the level of wild-type activity of the Npm2 (-2610) promoter. In addition, deletion of sequence -101 bp resulted in no detection of luciferase activity. These findings also indicate that 3 putative distal promoter sequences exist at the 52-flanking region (-2610 to -210) of the Npm2 gene and that the region (-210 to -101) that includes the E-box region (-180) is crucial for high-level transcriptional activity of the Npm2 promoter. In conclusion, the E-box may be a key regulatory region for the expression of two of the maternal genes (H1oo and Npm2) examined. Currently, we are attempting to identify the transcriptional factor binding sites by DNase I footprint analysis and gel mobility shift assay.

Transcriptional Regulation of Mouse MARCKS Promoter in Immortalized Hippocampal Cells

Mouse MARCKS is a prominent myristoylated alanine-rich C kinase substrate implicated in brain development, calcium/calmodulin signaling, and membrane cytoskeletal restructuring, and is developmentally regulated in a cell- and tissue-specific fashion. In this study, transcriptional regulation of mouse MARCKS promoter in the neuronally derived immortalized hippocampal cells (HN33) was examined for a portion of 5′-flanking genomic sequence from −993 to +1 relative to the translation start site. Transfection experiments carried out in this neural cell line identified, for the first time, that the distal promoter segment from −993 to −713 plays a crucial role as an enhancer/activator element in the up-regulation of the basal transcription activity driven by MARCKS core promoter sequence. Motif analyses revealed at least 12 overlapping potential transcription factor binding sites in this region, among which a prominent GA-rich sequence centered at −765 has been shown to be functionally important in the binding of Sp1 protein-like complex. Deletion of the GA-rich segment significantly reduced the MARCKS promoter activity. Further, competitive EMSA indicated two additional sites within the −993/−713 that may also interact with Sp1 protein, demonstrating that the activator function of −993/−713 is under control of multiple Sp1 transcription factors. Unlike the distal promoter sequence, the proximal core promoter sequence (−649/−438) contains a GC-rich box and a Z-DNA-forming segment and is critical to basal transcription. The deletion of −649/−438 segment has been shown to drastically impair the promoter activity even in the presence of −993/−713, suggesting that its presence is also important to the function of −993/−713. These data emphasize that the synergistic interaction between distal and proximal promoter sequences is indispensable for the optimal MARCKS promoter function in the immortalized hippocampal cells. The discovery of the activator function of the MARCKS distal promoter region, and its potential interaction with multiple Sp proteins may provide a new clue to the understanding of Macs transcriptional regulation in brain.

Promoter structure and gene expression of the mouse inositol 1,4,5-trisphosphate receptor type 3 gene

Inositol 1,4,5-trisphosphate receptor type 3 (IP 3R3) is a ubiquitously expressed IP 3R gene in the IP 3R gene family. We identified an upstream region of the mouse IP 3R3 genomic DNA. Transcription start points for the IP 3 R3 gene were found to be located mainly at four sites between nucleotide position −325 and −285 relative to the first ATG codon. The major start point was mapped around −325. Transcription promotion ability was detected between −325 and −285 in an IP 3R3 proximal promoter sequence. The promoter had no TATA-box but was highly GC-rich and contained two putative Sp1-binding sites. There was no sequence similarity between promoter regions of IP 3 R3 and IP 3 R2, another ubiquitous gene, except for GC-boxes. By using a series of 5′-truncation versions and a transient luciferase assay, we detected multiple common and cell-type-dependent regulatory regions within the distal promoter sequence downstream from −4.0 kb that function positively or negatively. The IP 3 R3 gene was highly transcribed in the kidney, spleen, heart, and skeletal muscle, and this tissue distribution pattern was nearly complementary to that of IP 3R2. We found that IP 3R3 gene expression was repressed in retinoic acid-treated and neural differentiated P19 mouse embryonic carcinoma cells.

Analysis of the promoter of the cudA gene reveals novel mechanisms of Dictyostelium cell type differentiation.

The cudA gene encodes a nuclear protein that is essential for normal multicellular development. At the slug stage cudA is expressed in the prespore cells and in a sub-region of the prestalk zone. We show that cap site distal promoter sequences direct cudA expression in prespore cells, while proximal sequences direct expression in the prestalk sub-region. The promoter domain that directs prespore-specific transcription consists of a positively acting region, that has the potential to direct expression in all cells within the slug, and a negatively acting region that prevents expression in the prestalk cells. Dd-STATa is the STAT protein that regulates commitment to stalk cell gene expression, where it is known to function as a transcriptional repressor. We show that Dd-STATa binds in vitro to the positively acting part of the prespore domain of the cudA promoter. However, Dd-STATa cannot be utilised for this purpose in vivo, because analysis of a Dd-STATa null mutant strain shows that Dd-STATa is not necessary for cudA transcription in prespore cells. In contrast, the part of the cudA promoter that directs prestalk-specific expression contains a binding site for Dd-STATa that is essential for its biological activity. Dd-STATa appears therefore to serve as a direct activator of cudA transcription in prestalk cells, while a protein with a DNA binding specificity highly related to that of Dd-STATa is utilised to activate cudA transcription in prespore cells.

Full Activity From Human β-Globin Locus Control Region Transgenes Requires 5′HS1, Distal β-Globin Promoter, and 3′ β-Globin Sequences

The locus control region (LCR) activates high-level human β-globin transgene expression. LCR cassettes composed of 5′HS2-4 linked to the 815 bp β-globin proximal promoter do not express fully. Here, we show that LCR (5′HS2-4) β-globin transgenes that also contain either 5′HS1 or the distal promoter fail to express fully in single- and low-copy transgenic mice. In contrast, full expression is obtained in the presence of both 5′HS1 and the distal promoter. Nine factor binding sites were identified in 5′HS1, using in vitro DNaseI footprint and gel retardation assays, and these include a strong Sp1/Sp3 site, four GATA-1 sites, and two sites that encompass an ACTAAC motif. LCR (5′HS1-4) β-globin transgene constructs with the distal promoter deleted or replaced by spacer DNA show that specific distal promoter sequences are required for full expression. An LCR (5′HS1-4) transgene construct with truncated downstream β-globin gene sequences indicates that 3′ sequences also play an important role. These results show that full expression of the β-globin gene directed by the LCR requires 5′HS1, the distal β-globin promoter, and 3′ sequences, and has implications for gene therapy construct design and models of LCR activation.

Full Activity From Human β-Globin Locus Control Region Transgenes Requires 5′HS1, Distal β-Globin Promoter, and 3′ β-Globin Sequences

AbstractThe locus control region (LCR) activates high-level human β-globin transgene expression. LCR cassettes composed of 5′HS2-4 linked to the 815 bp β-globin proximal promoter do not express fully. Here, we show that LCR (5′HS2-4) β-globin transgenes that also contain either 5′HS1 or the distal promoter fail to express fully in single- and low-copy transgenic mice. In contrast, full expression is obtained in the presence of both 5′HS1 and the distal promoter. Nine factor binding sites were identified in 5′HS1, using in vitro DNaseI footprint and gel retardation assays, and these include a strong Sp1/Sp3 site, four GATA-1 sites, and two sites that encompass an ACTAAC motif. LCR (5′HS1-4) β-globin transgene constructs with the distal promoter deleted or replaced by spacer DNA show that specific distal promoter sequences are required for full expression. An LCR (5′HS1-4) transgene construct with truncated downstream β-globin gene sequences indicates that 3′ sequences also play an important role. These results show that full expression of the β-globin gene directed by the LCR requires 5′HS1, the distal β-globin promoter, and 3′ sequences, and has implications for gene therapy construct design and models of LCR activation.

Cellular and viral trans-acting factors modulate N-myc2 promoter activity in woodchuck liver tumors.

Activation of the N-myc2 oncogene by integration of woodchuck hepatitis virus (WHV) DNA is a central event in woodchuck liver oncogenesis. In this study, we have evaluated the influence of several cellular and viral trans-acting factors and mediators of inflammation on N-myc2 promoter activity in hepatoma cell lines. Ets oncoproteins, including Ets1, Ets2 and PEA3 efficiently activated a chimeric N-myc2 promoter/luciferase reporter gene. By electrophoretic mobility shift assays, we show that Etsl and Ets2 proteins can efficiently bind two consensus Ets sites located within a 59 bp sequence upstream of the N-myc2 transcription start site. Site-directed mutagenesis of these Ets-binding motifs abolished transactivation of the N-myc2 promoter by Ets proteins. Addition of interleukin-6 (IL-6) induced a weak but reproducible activation of the N-myc2 promoter, while IL-1 was ineffective. We further show that the N-myc2 promoter can be transactivated by the hepadna-virus X protein, and that distal promoter sequences are required for both IL-6 and X responsiveness. Similar effects of these factors were observed in the context of the N-myc2 promoter activated by WHV cis-regulatory elements. In view of the high-level expression of the N-myc2 oncogene in most woodchuck liver tumors, the Ets oncoproteins, inflammation-associated cytokine IL-6 and the viral X transactivator might play important roles in hepadnavirus-associated tumorigenesis.

Differential Induction of HNF-3 Transcription Factors during Neuronal Differentiation

We have investigated the regulation of transcription factors HNF-3α and HNF-3β during the retinoic acid-mediated differentiation of mouse P19 cells. Retinoic acid treatment converts P19 stem cells into neurons and astrocytes and we have clearly shown that gene expression of both HNF-3α and HNF-3β is activated during this process. HNF-3α transcription was detected 2 h after addition of retinoic acid and took place in the absence ofde novoprotein synthesis. This suggests that HNF-3α is a primary target for retinoic acid action. HNF-3α induction displays a biphasic profile and HNF-3α mRNA reaches maximal levels at 2 and 6 days postdifferentiation. Additional experiments strongly suggest that the second peak is due to HNF-3α induction in postmitotic neurons. P19 stem cells, on the other hand, do not contain any detectable HNF-3α mRNA. According to our studies, the retinoic acid-mediated induction of HNF-3α occurs at the level of transcriptional initiation and is conferred by distal promoter sequences. In comparison to HNF-3α, HNF-3β induction is a subsequent event and detectable levels of HNF-3β mRNA materialize approximately 1 day after addition of retinoic acid to P19 stem cells. Time course studies firmly demonstrate that HNF-3β mRNA peaks at about 2 days postdifferentiation and then declines to virtually unreadable levels. This temporal pattern is consistent with HNF-3β being a secondary target for retinoic acid. In analogy to HNF-3α, HNF-3β activation also takes place at the level of transcriptional initiation. Recent studies implicate HNF-3α and HNF-3β in early mammalian neurogenesis. The detection of HNF-3α/β activation during P19 cell differentiation provides us with a convenient cell culture system to elucidate the induction mechanism and the precise role of both transcriptional regulators in the formation of neuronal cells.

Transcriptional Induction of Collagenase-1 in Differentiated Monocyte-like (U937) Cells is Regulated by AP-1 and an Upstream C/EBP-β Site

In this report, we demonstrate that the AP-1 site and a distal promoter element regulate transcriptional induction of collagenase-1 during monocytic differentiation. Chloramphenicol acetyltransferase expression constructs containing regions of the human collagenase-1 promoter were stably or transiently transfected into U937 cells, and reporter activity was assessed at various times after the onset of phorbol 12-myristate 13-acetate (PMA)-mediated differentiation. Rapid and strong induction of promoter activity was lost in constructs with a mutant AP-1 element; however, at 16-96 h post-PMA, the mutant collagenase-1 promoter displayed AP-1 independent PMA-mediated transactivation. The AP-1 mutant constructs also showed delayed transcriptional activation in PMA-treated fibroblasts. Western and supershift analyses indicated that functional Jun and Fos proteins were present in nuclear extracts of PMA-differentiated U937 cells. Promoter deletion constructs demonstrated the potential role of distal promoter sequences in regulating collagenase-1 transcription. In particular, Western, supershift, and promoter deletion analyses suggested a role for CCAAT/enhancer-binding protein-beta (C/EBP-beta) binding site between -2010 and -1954 in regulating transcription of collagenase-1 in monocytic cells. Our findings suggest that distinct regulatory elements, acting somewhat independently of each other, control expression of collagenase-1. In addition, our data suggests that the rapid PMA-mediated induction of collagenase-1 transcription is controlled by a mechanism distinct from that regulating the sustained expression of this proteinase in activated macrophages.

Structure of the distal human gonadotropin releasing hormone (hGnrh) gene promoter and functional analysis in Gt1-7 neuronal cells

To assess potential species-specific expression of gonadotropin releasing hormone (GnRH), the distal human (h) GnRH promoter was cloned, characterized and tested in gene transfer studies. The nucleotide sequence of approximately 3.8 kb of 5'-flanking region was determined. Homology to the rat (r) GnRH sequence was observed in the proximal promoter region between -551 h (-424 r) and the transcriptional start site and within multiple distal promoter regions. In contrast, there was little similarity in the sequences between -1131/-551 h and -1031/-424 r. A deletion panel of 5'-flanking hGnRH promoter constructs was made and tested in transient transfection assays in GnRH-producing mouse GT1-7 neuronal cells. The largest hGnRH promoter construct (-3832/+5 h) exhibited high levels of reporter activity, similar to that observed with the largest rGnRH construct (-3026/+116 r). However, in contrast to the rat gene, deletion of distal promoter sequences of the hGnRH promoter to -1971, -1131 or -551 did not result in a decrease in luciferase reporter activity. Further truncation to -350 resulted in a 3-fold decrease in luciferase activity. There was no preferential use of the putative upstream hGnRH start site in neuronal cells. DNase I protection assays showed unique protection patterns with nuclear extracts from GT1-7 and Gn10 neuronal cells and the hGnRH and rGnRH promoter fragments. These data suggest the presence of different cis-acting elements and transacting factors that mediate species-specific neuronal GnRH expression.

Ca 2+/calmodulin kinase types II and IV regulate c- fos transcription in the AtT20 corticotroph cell line

In the corticotroph cell line AtT20, Ca 2+ stimulates c- fos mRNA and nuclear c- fos protein accumulation. We show that Ca 2+ stimulates transcription of a chloramphenicol-acetyl-transferase reporter gene driven by the c- fos promoter. This effect is mimicked by both constitutively active Ca 2+ calmodulin -dependent protein kinase types II (CamK II) and IV (CamK IV). The effects of CamK IV are synergistic with those obtained by cAMP stimulation, whereas CamK II-dependent transcription is not further stimulated. Deletion of the distal promoter sequence revealed that CamK IV, and to a lesser extent CamK II, activate a cis-acting element(s) located between −99 and −53 bp prior to the transcription initiation site. Moreover, CamK II further stimulates transcription through the fos AP1-like element. CamK IV has a lower effect on this element but represses the serum response element. The latter observation might account for the lower transcriptional effects of CamK IV on the full length c- fos promoter.

Methylation status and chromatin structure of an early response gene (ornithine decarboxylase) in resting and stimulated NIH-3T3 fibroblasts.

The early response gene ornithine decarboxylase (odc) is indispensable for normal and malignant cell growth. Although DNA methylation is generally associated with chromatin condensation and gene inactivation, the odc gene is heavily methylated at CCGG-sequences in animal cell lines. In this work we analyzed the chromatin structure and the DNA methylation status at the CpG-rich promoter sequences at the odc locus in mouse 3T3 fibroblasts. We show that the proximal promoter region of the odc locus is not hypermethylated, while the distal promoter sequences appear to have a few methylated CCGG-sites and display methylation polymorphism. Furthermore, it was found that the 5' promoter region of odc is constitutively more sensitive to micrococcal nuclease than the coding and 3' regions of the odc gene. Stimulation of the cells with serum resulted in an appearance of a DNase I sensitive site at the promoter region. The chromatin structure of the mid-coding and 3' regions of the odc gene also underwent structural changes that were accompanied by the rapid accumulation of odc mRNA. Such changes were not detected in the chromatin structure of glyceraldehyde-3-phosphate dehydrogenase (gadph) gene, whose expression remains invariant upon serum stimulation. These data suggest that the chromatin structure may play an important role in the rapid transcriptional activation of odc and other immediate early genes during serum stimulation.

Compensatory effect of distal promoter sequences on the basal expression of a microinjected 70-kilodalton heat shock protein gene after the midblastula transition of Xenopus laevis embryogenesis

The promoter sequences involved in the basal expression of a human 70-kilodalton heat shock protein (HSP70) gene during Xenopus embryogenesis were analyzed by microinjection of mutant promoters of a HSP70--chloramphenicol acetyltransferase fusion gene into fertilized eggs and following their expression during early development. While deletion of the HSP70 gene promoter to--100 base pairs (bp) did not affect basal transcription in postmidblastula stage embryos, linker-scanner mutations in the CCAAT and purine box elements blocked expression. However, extension of the 5' boundary to--188 bp restored full wild-type expression to these mutants. These results suggest that multiple redundant cis-acting regulatory elements present in the human HSP70 gene promoter can function during Xenopus embryogenesis.

Developmental regulation of lck gene expression in T lymphocytes.

In the mouse and human, mRNA transcripts encoding the lymphocyte-specific protein tyrosine kinase p56lck are derived from two separate promoters resulting in heterogeneity in the 5' untranslated region sequence. The proximal promoter lies just 5' to the coding region for the gene and is active only in thymocytes. In contrast, the distal promoter lies 34 kilobases (kb) 5' in the human, and is active both in thymocytes and mature peripheral T cells. As previously reported, transgenic mice bearing functional proximal promoter sequence juxtaposed with the SV40 large T antigen gene invariably develop lymphoid tumors confined to the thymus. In the current work, transgenic mice bearing a 2.6-kb fragment of the human distal promoter fused to the SV40 large T antigen gene express large T antigen in thymocytes and in peripheral lymphoid cells, and develop tumors of both the thymus and the peripheral lymphoid organs. The ability of the human distal promoter to function appropriately in transgenic mice is consistent with the strong similarity observed between the mouse and human distal promoter sequences. With the exception of a single short interval that serves as a target for binding of nuclear factors, significant sequence similarity is not seen when the distal and proximal promoter sequences are compared. Hence, developmentally regulated, lineage-specific transcription of the lck gene is mediated by distinct promoter sequences that appear to be capable of functioning independently.