TATA Sequence Research Articles

A Global Transcriptional Regulator in Thermococcus kodakaraensis Controls the Expression Levels of Both Glycolytic and Gluconeogenic Enzyme-encoding Genes

We identified a novel regulator, Thermococcales glycolytic regulator (Tgr), functioning as both an activator and a repressor of transcription in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Tgr (TK1769) displays similarity (28% identical) to Pyrococcus furiosus TrmB (PF1743), a transcriptional repressor regulating the trehalose/maltose ATP-binding cassette transporter genes, but is more closely related (67%) to a TrmB paralog in P. furiosus (PF0124). Growth of a tgr disruption strain (Deltatgr) displayed a significant decrease in growth rate under gluconeogenic conditions compared with the wild-type strain, whereas comparable growth rates were observed under glycolytic conditions. A whole genome microarray analysis revealed that transcript levels of almost all genes related to glycolysis and maltodextrin metabolism were at relatively high levels in the Deltatgr mutant even under gluconeogenic conditions. The Deltatgr mutant also displayed defects in the transcriptional activation of gluconeogenic genes under these conditions, indicating that Tgr functions as both an activator and a repressor. Genes regulated by Tgr contain a previously identified sequence motif, the Thermococcales glycolytic motif (TGM). The TGM was positioned upstream of the Transcription factor B-responsive element (BRE)/TATA sequence in gluconeogenic promoters and downstream of it in glycolytic promoters. Electrophoretic mobility shift assay indicated that recombinant Tgr protein specifically binds to promoter regions containing a TGM. Tgr was released from the DNA when maltotriose was added, suggesting that this sugar is most likely the physiological effector. Our results strongly suggest that Tgr is a global transcriptional regulator that simultaneously controls, in response to sugar availability, both glycolytic and gluconeogenic metabolism in T. kodakaraensis via its direct binding to the TGM.

Gene structure and promoter functional analysis of the marmoset type II GnRH receptor

The marmoset type II GnRH receptor (GnRH-R) gene has the same structure and genomic organisation as the human and other type II GnRH-R genes. The gene consists of three exons and two introns and overlaps in the antisense orientation on its 5' end with peroxisomal membrane protein 11beta and on its 3' end with the RNA-binding motif protein 8A. However, these genes occur only at one locus in the marmoset genome, while in the human at two loci. Employing 5' rapid amplification of cDNA ends demonstrated that the marmoset type II GnRH-R gene has two transcriptional start sites at -341 and -567 nucleotides relative to the translational start codon and both start sites lack TATA and CAAT consensus sequences. A luciferase reporter construct with a 2.3 kb 5' flanking region of the type II GnRH-R gene was active in a wide variety of cell lines tested, consistent with the wide tissue expression of the receptor. Progressive 5' and 3' deletions were employed to identify sequences required for basal expression of the type II GnRH-R gene. This analysis identified negative regulatory elements in the regions -2342/-1995, -1679/-1084 and -458/-1 and positive regulatory elements in the regions -1995/-1679, -1084/-458 and -458/-1 relative to the translational start site. The strongest of the positive regions located between -766/-665 has enhancer activity when cloned in front of a heterologous minimal promoter and is critical for basal expression of the type II GnRH-R.

Real-time monitoring of functional interactions between upstream and core promoter sequences in living cells of sea urchin embryos

There are some functional compatibilities between upstream and core promoter sequences for transcriptional activation in yeast, Drosophila and mammalian cells. Here we examined whether similar compatibilities exist in sea urchin embryos, and if so, whether they are dynamically regulated during early development. Two reporter plasmids, each containing a test promoter conjugated to either CFP or YFP, were concurrently introduced into embryos, and their expression patterns were studied by fluorescence microscopy. The upstream sequence of the Hemicentrotus pulcherrimus (Hp) OtxE promoter drives the expression of its own core promoter and that of Strongylocentrotus purpuratus (Sp) Spec2a in different embryonic regions, especially at the late gastrula stage. Interestingly, when the four putative transcription factor binding sites of this upstream sequence were individually mutated, the resulting sequences directed different spatiotemporal expression from the same set of two core promoters, indicating that combinations of upstream factors may determine core promoter usage in sea urchin embryos. In addition, the insertion or deletion of consensus or nonconsensus TATA sequences changed the expression profile significantly, irrespective of whether the upstream sequence was intact or mutated. Thus, the TATA sequence may serve as a primary determinant for core promoter selection in these cells.

Distribution of the UGT1A1*28 polymorphism in Caucasian and Asian populations in the US: a genomic analysis of 138 healthy individuals

The hepatic isoform 1A1 of uridine diphosphate glucuronosyltransferase is responsible for glucuronidation and detoxification of SN-38, the active metabolite of irinotecan. The presence of an additional TA repeat in the TATA sequence of the UGT1A1 promoter leads to a significant decrease in SN-38 glucuronidation. Patients with the UGT1A1 (TA)7 allele are more likely to experience severe neutropenia and diarrhea following irinotecan chemotherapy. We assessed the distribution of the UGT1A1 (TA)n polymorphism in healthy male and female US residents of European and Asian descent. We used a fluorescent polymerase chain reaction-based assay to detect UGT1A1 (TA)n polymorphisms in 138 healthy volunteers (56 Caucasians, 37 Chinese, 37 Filipino and eight Japanese) between the ages of 18 and 65 years. The chi-test was used to assess between-group differences in the distribution of UGT1A1 (TA)n genotypes. The UGT1A1 (TA)6/6 genotype was significantly more common in Asians than in Caucasians (76 vs. 46%), whereas the (TA)6/7 (39 vs. 20%) and (TA)7/7 (13 vs. 5%) genotypes were more common in Caucasians than in Asians. Genotype distributions did not differ significantly between men and women in either group. The UGT1A1 (TA)5/5 genotype was detected in one Caucasian woman. In conclusion, consistent with previous reports, the UGT1A1 (TA)7/7 genotype was significantly more common in Caucasians than in Asians. UGT1A1 (TA)n/n genotype distribution did not vary with sex in individuals of European or Asian descent.

Transcriptional regulation of human CYP11A1 in gonads and adrenals

The CYP11A1 gene encodes the cholesterol side-chain cleavage enzyme, also termed cytochrome P450scc, which catalyzes the conversion of cholesterol to pregnenolone in the first step of steroid biosynthesis in mitochondria. The adrenal- and gonad-selective, hormonally and developmentally regulated expression of CYP11A1 is principally driven by its 2.3 kb promoter. Multiple trans-acting factors like SF-1, Sp1, AP-2, TReP-132, LBP-1b, LBP-9, AP-1, NF-1, and Ets control CYP11A1 transcription either through DNA-protein interaction with their specific cis-acting elements or through protein-protein interaction between each other, wherein SF-1 plays a central role in adrenals and testes. In addition to binding with its proximal and upstream motifs, SF-1 also physically interacts with TFIIB, CBP/p300, TReP-132, and c-Jun/AP-1 to specifically transmit the regulatory signals of cAMP. Other factors like Sp1 family members, AP-2, and LBP-1b/LBP-9 may be other factors that play a role in CYP11A1 transcription, particularly in placental cells. The TATA sequence could also contribute to tissue-specificity and hormonal regulation of CYP11A1 transcription. This article reviews recent studies focusing on adrenals and gonads.

Angiotensin II Type 1 Receptor Gene Regulation

Angiotensin II (Ang II), the major bioactive peptide of the renin–angiotensin system, was originally described as a potent vasoconstrictor. It is now recognized as a multifunctional hormone influencing many cellular processes, including cell growth, apoptosis, migration, inflammation, and fibrosis.1,2 Under pathological conditions, the vasoconstrictor, mitogenic, proinflammatory, and profibrotic actions of Ang II contribute to altered vascular tone, structural remodeling, and endothelial dysfunction.3–6 Thus, Ang II plays a key role in the pathogenesis of cardiovascular diseases. The biological responses of Ang II are mediated by its interaction with 2 distinct high-affinity G protein–coupled receptors now designated Ang II type 1 receptor (AT1R) and Ang II type 2 receptor.7 Most of the known physiological and pathophysiological effects of Ang II are mediated via the AT1R.1–7 This receptor subtype is expressed in cardiovascular-relevant cell types including vascular smooth muscle cells (VSMCs), endothelial cells, cardiac myocytes, cardiac fibroblasts, and renal mesangial cells.7 The multiple actions of Ang II, mediated through the AT1R, are a result of complex intracellular signaling pathways including stimulation of the phospholipase C/inositol 1,4,5-trisphosphate/diacylglycerol cascade, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases, tyrosine kinases, and RhoA/Rho kinase.1,2,4,6,7 In addition, AT1Rs mediate many of their pathophysiological effects by stimulating reactive oxygen species generation via an reduced nicotinamide-adenine dinucleotide/reduced nicotinamide-adenine dinucleotide phosphate oxidase-dependent mechanism.2,8 Reactive oxygen species, in turn, influences downstream signaling molecules, including transcription factors, tyrosine kinases/phosphatases, Ca2+ channels, and MAPKs.2,8 The expression level of the AT1R defines the biological efficacy of Ang II; hence, overexpression of the AT1R is one potential mechanism by which Ang II can contribute to cardiovascular disease. Importantly, aberrant expression of the AT1R has been shown to have pathophysiological relevance in cell culture, animal studies, …

Presence of the anti-leukemic nucleotide analog, 2-chloro-2′-deoxyadenosine-5′-monophosphate, in a promoter sequence alters DNA binding of TATA-binding protein (TBP)

2-Chlorodeoxyadenosine (CldAdo, Cladribine), a nucleoside analog used in the treatment of hairy cell leukemia, is phosphorylated and incorporated into DNA, but is not an absolute chain terminator. We hypothesized that the presence of a chlorine molecule projecting into the DNA minor groove would affect DNA:protein-binding interactions. Here, we investigated recognition of and binding to double-stranded CldAMP-substituted TATA promoter sequences by human TATA-binding protein (TBP) using mobility shift assays. Depending on the site, CldAMP in place of dAMP within a TATA sequence decreased in vitro TBP binding by ∼30% to 55% compared to control sites. When bound to a CldAMP-substituted TATA box, however, the TBP complex was more resistant to polyanions, suggesting enhanced stability. Limited exposure of the TBP:DNA complex to proteases indicated that TBP conformation was altered on CldAMP-substituted DNA compared to control. Further, binding of transcription factor IIB to TBP was diminished on analog-containing TATA sequences. These results suggest normal TBP-binding interactions—specifically recognition, stability, and conformation—are disrupted by CldAMP insertion into eukaryotic promoter sequences.

Modulation of Rad26- and Rpb9-mediated DNA Repair by Different Promoter Elements

Rad26, the yeast homologue of human Cockayne syndrome group B protein, and Rpb9, a nonessential subunit of RNA polymerase II, have been shown to mediate two subpathways of transcription-coupled DNA repair in yeast. Here we show that Rad26- and Rpb9-mediated repair in the yeast GAL1 gene is differently modulated by different promoter elements. The initiation site and efficiency of Rad26-mediated repair in the transcribed strand are determined by the upstream activating sequence (UAS) but not by the TATA or local sequences. The role of UAS in determining the Rad26-mediated repair is not through loading of RNA polymerase II or the transcriptional regulatory complex SAGA. However, both the UAS and the TATA sequences are essential for confining Rad26-mediated repair to the transcribed strand. Mutation of the TATA sequence, which greatly reduces transcription, or deletion of the TATA or mutation of the UAS, which completely abolishes transcription, causes Rad26-mediated repair to occur in both strands. Rpb9-mediated repair only occurs in the transcribed strand and is efficient only in the presence of both TATA and UAS sequences. Also, the efficiency of Rpb9-mediated repair is dependent on the SAGA complex. Our results suggest that Rad26-mediated repair can be either transcription-coupled, provided that a substantial level of transcription is present, or transcription-independent, if the transcription is too low or absent. In contrast, Rpb9-mediated repair is strictly transcription-coupled and is efficient only when the transcription level is high.

Non-optimal TATA Elements Exhibit Diverse Mechanistic Consequences

To reveal mechanistic differences in transcription initiation between variant TATA elements, in vivo and in vitro assays of the functional activity of 14 different sequences were compared. Variant elements exhibited particular degrees of activation in vivo but universally were unable to support the -fold activation observed for an element consisting of TATAAA. Each element was classified by its functional activity for in vitro interaction with TATA-binding protein (TBP), TFIIA, and TFIIB. Certain off-consensus TATA elements form poor binding sites for TBP and this compromised interaction interferes with higher order complex formation with TFIIA and/or TFIIB. Other elements are only modestly decreased for TBP binding but dramatically affected for higher order complex formation. Another distinct category is comprised of two elements (CATAAA and TATAAG), which are not affected in the initial formation of the TBP, TFIIA-TBP, or TFIIB-TBP complexes. However, CATAAA and TATAAG are unable to form a stable TFIIA-TBP-DNA complex in vitro. Moreover, fusion of TFIIA to TBP specifically restores activity from these two elements in vivo. Taken together, these results indicate that the interplay between the sequence of the TATA element and the components of the general transcription machinery can lead to variations in the formation of functional complexes and/or the stability of these complexes. These differences offer distinct opportunities for an organism to exploit diverse steps in the regulation of gene expression depending on the precise TATA element sequence at a given gene.

Characterization of the mitochondrial aconitase promoter and the identification of transcription factor binding

Mitochondrial (m) aconitase plays an important role in the unique pathway of citrate accumulation in prostate epithelial cells through its limited activity. In the current study, we characterized the human m-aconitase gene promoter. A 1,411-bp 5'-flanking fragment of the human m-aconitase gene was cloned, followed by 5' serial deletion analysis of promoter activity. Transcriptional start sties and transcription factors bound to the promoter were identified by 5' RACE, DNA pull-down assay and transcription factor array analysis. Two transcriptional start sites were identified. The promoter fragment pulled down 15 transcription factors, some without consensus sequences in the promoter. Deletion of one Sp1 site eliminated all promoter activity. The m-aconitase promoter is contained in a 153-bp 5' fragment lacking a TATA or CAAT sequence. Sp1 binding to a specific Sp1 site is required for promoter activity while other transcription factors are recruited through protein-protein interactions.

Distribution of UGT1A1 (TA) polymorphisms in Caucasian and Asian subjects

2063 Background: The hepatic isoform 1A1 of uridine diphosphate glucuronosyltransferase (UGT) is responsible for glucuronidation and detoxification of SN-38, the active metabolite of irinotecan. The presence of an additional TA repeat in the TATA sequence of the UGT1A1 gene is a common polymorphism, leading to a significant decrease in SN-38 glucuronidation. Patients with the UGT1A1 (TA)7 allele (either [TA]6/7 or [TA]7/7 ) are more likely to experience severe neutropenia and diarrhea following irinotecan chemotherapy. We assessed the distribution of the UGT1A1 (TA) polymorphism in Caucasian and Asian subjects. Methods: We used a fluorescent PCR-based assay to detect UGT1A1 (TA) polymorphisms in 129 healthy subjects (52 Caucasian, 34 Chinese, 36 Filipino, and 7 Japanese). The chi-square test was used to assess between-group differences in the distribution of UGT1A1 (TA) genotypes. Results: UGT1A1 (TA) genotype distribution differed significantly between Caucasian and Asian subjects (P = 0.003). The UGT1A1 (TA)6/7 and (TA)7/7 genotypes were more common in Caucasians than Asians. Genotype distributions did not differ significantly between men and women in either group ( Table ). Conclusions: The frequency of the deleterious UGT1A1 (TA)7 polymorphism was greater in Caucasians than in Asians; genotype frequencies were consistent with previous reports. In both groups, UGT1A1 (TA) genotype distributions were similar in men and women. [Table: see text] No significant financial relationships to disclose.

HESR1/CHF2 suppresses VEGFR2 transcription independent of binding to E-boxes

The bHLH transcription factor HESR1 (CHF2) acts downstream of notch to regulate cardiovascular development and angiogenesis, at least in part through down-regulation of the VEGF receptor, VEGFR2. Surprisingly, we find that HESR1 interacts with the promoter in endothelial cells (EC) not through direct binding to the E-boxes, but through intermediary interactions with GC-box-binding proteins. The bHLH and orange domains of HESR1 are sufficient for repression in EC, likely through recruitment of co-repressors, however, the C-terminal YRPW motif is not required. The VEGFR2 promoter contains a functional initiator element but no TATA box, however, addition of a TATA sequence renders the promoter resistant to inhibition by HESR1. In agreement with this finding, the NrCAM, TK, and CMV promoters, which have TATA boxes, cannot be repressed. Thus, HESR1 represses VEGFR2 through interactions with SP-1-like factors and requires an Inr element in the absence of a TATA box. Our findings illuminate an important mechanism for notch/HESR1 regulation of VEGF-induced angiogenesis.

Transcription of individual tRNAGly1 genes from within a multigene family is regulated by transcription factor TFIIIB

Members of a multigene family from the silkworm Bombyx mori have been classified based on their transcriptions in homologous nuclear extracts, into three groups of highly, moderately and poorly transcribed genes. Because all these gene copies have identical coding sequences and consequently identical promoter elements (the A and B boxes), the flanking sequences modulate their expression levels. Here we demonstrate the interaction of transcription factor TFIIIB with these genes and its role in regulating differential transcriptions. The binding of TFIIIB to the poorly transcribed gene -6,7 was less stable compared with binding of TFIIIB to the highly expressed copy, -1. The presence of a 5' upstream TATA sequence closer to the coding region in -6,7 suggested that the initial binding of TFIIIC to the A and B boxes sterically hindered anchoring of TFIIIB via direct interactions, leading to lower stability of TFIIIC-B-DNA complexes. Also, the multiple TATATAA sequences present in the flanking regions of this poorly transcribed gene successfully competed for TFIIIB reducing transcription. The transcription level could be enhanced to some extent by supplementation of TFIIIB but not by TATA box binding protein. The poor transcription of -6,7 was thus attributed both to the formation of a less stable transcription complex and the sequestration of TFIIIB. Availability of the transcription factor TFIIIB in excess could serve as a general mechanism to initiate transcription from all the individual members of the gene family as per the developmental needs within the tissue.

HnRNP K Binds a Core Polypyrimidine Element in the Eukaryotic Translation Initiation Factor 4E (eIF4E) Promoter, and Its Regulation of eIF4E Contributes to Neoplastic Transformation

Translation initiation factor eukaryotic translation initiation factor 4E (eIF4E) plays a key role in regulation of cellular proliferation. Its effects on the m7GpppN mRNA cap are critical because overexpression of eIF4E transforms cells, and eIF4E function is rate-limiting for G1 passage. Although we identified eIF4E as a c-Myc target, little else is known about its transcriptional regulation. Previously, we described an element at position -25 (TTACCCCCCCTT) that was critical for eIF4E promoter function. Here we report that this sequence (named 4EBE, for eIF4E basal element) functions as a basal promoter element that binds hnRNP K. The 4EBE is sufficient to replace TATA sequences in a heterologous reporter construct. Interactions between 4EBE and upstream activator sites are position, distance, and sequence dependent. Using DNA affinity chromatography, we identified hnRNP K as a 4EBE-binding protein. Chromatin immunoprecipitation, siRNA interference, and hnRNP K overexpression demonstrate that hnRNP K can regulate eIF4E mRNA. Moreover, hnRNP K increased translation initiation, increased cell division, and promoted neoplastic transformation in an eIF4E-dependent manner. hnRNP K binds the TATA-binding protein, explaining how the 4EBE might replace TATA in the eIF4E promoter. hnRNP K is an unusually diverse regulator of multiple steps in growth regulation because it also directly regulates c-myc transcription, mRNA export, splicing, and translation initiation.

The Yeast FACT Complex Has a Role in Transcriptional Initiation

A crucial step in eukaryotic transcriptional initiation is recognition of the promoter TATA by the TATA-binding protein (TBP), which then allows TFIIA and TFIIB to be recruited. However, nucleosomes block the interaction between TBP and DNA. We show that the yeast FACT complex (yFACT) promotes TBP binding to a TATA box in chromatin both in vivo and in vitro. The SPT16 gene encodes a subunit of yFACT, and we show that certain spt16 mutations are synthetically lethal with TBP mutants. Some of these genetic defects can be suppressed by TFIIA overexpression, strongly suggesting a role for yFACT in TBP-TFIIA complex formation in vivo. Mutations in the TOA2 subunit of TFIIA that disrupt TBP-TFIIA complex formation in vitro are also synthetically lethal with spt16. In some cases this spt16 toa2 lethality is suppressed by overexpression of TBP or the Nhp6 architectural transcription factor that is also a component of yFACT. The Spt3 protein in the SAGA complex has been shown to regulate TBP binding at certain promoters, and we show that some spt16 phenotypes can be suppressed by spt3 mutations. Chromatin immunoprecipitations show TBP binding to promoters is reduced in single spt16 and spt3 mutants but increases in the spt16 spt3 double mutant, reflecting the mutual suppression seen in the genetic assays. Finally, in vitro studies show that yFACT promotes TBP binding to a TATA sequence within a reconstituted nucleosome in a TFIIA-dependent manner. Thus, yFACT functions in establishing transcription initiation complexes in addition to the previously described role in elongation.

1053. Novel Exogenously Regulated Stem Cell–Mediated Gene Therapy Platform for Spinal Fusion

Cell-mediated gene therapy involving mesenchymal stem cells (MSCs) and bone morphogenetic protein (BMP) genes can provide a local supply of precursor cells and osteoinductive molecules that may promote bone formation. Spinal fusion has become a popular surgical technique for different pathological spinal conditions. We hypothesized that MSCs that overexpress the human (h)BMP-2 gene under the tetracycline (Tet)-controlled expression system (Tet-Off) can induce regulated bone formation leading to spinal fusion. We used a Tet-Off system based on genetically engineered MSCs containing an inducible hBMP-2 expression vector (ptTATop-BMP2) that had a bidirectional promoter (TATA sequence) utilizing elements of the tetracycline regulatory system. The system was regulated by the administration of doxycycline (Dox), a tetracycline analogue. In C3H/HeN mice 2 |[times]| 10^6 genetically engineered MSCs were injected into the paravertebral muscle of the lumbar spine (L2|[ndash]|6). Ten animals served as a negative control group (Dox was added to their drinking water) and 20 animals constituted the experimental group (Dox was supplied for different time periods leading to different duration of hBMP-2 expression). A longitudinal quantitative analysis of vertebrae fusion was performed using micro|[ndash]|computerized tomography (|[mu]|CT) to observe bone formation. In addition, engineered MSCs were injected in a similar manner to transgenic mice expressing the luciferase gene under human osteocalcin promoter. In these mice measuring the luciferase signal using a bioluminescence imaging system can noninvasively and quantitatively monitor osteogenic activity. From Week 1 postinjection the |[mu]|CT analysis demonstrated extensive bone formation leading to vertebrae fusion (Fig. 1A). No bone formation was observed in the control animals, in which hBMP-2 was inhibited. A minimum of 7 days of hBMP-2 expression was required to induce bone formation (Fig. 1B). Detection of luciferase gene expression in the transgenic mice 2 and 4 weeks postinjection indicated an increase in the osteogenic activity in vivo, which could be attributed to the paracrine effect of the engineered MSCs (Fig. 2). Our data demonstrate a regulated system for the induction of spinal fusion. We conclude that exogenously controlled gene therapy for spinal fusion can be achieved using the Tet-regulated hBMP-2 system.

Efficient Binding of NC2·TATA-binding Protein to DNA in the Absence of TATA

Negative cofactor 2 (NC2) forms a stable complex with TATA-binding protein (TBP) on promoters. This prevents the assembly of transcription factor (TF) IIA and TFIIB and leads to repression of RNA polymerase II transcription. Here we have revisited the interactions of NC2.TBP with DNA. We show that NC2.TBP complexes exhibit a significantly reduced preference for TATA box sequences compared with TBP and TBP.TFIIA complexes. In chromatin immunoprecipitations, NC2 is found on a variety of human TATA-containing and TATA-less promoters. Substantial amounts of NC2 are present in a complex with TBP in bulk chromatin. A complex of NC2.TBP displays a K(D) for DNA of approximately 2 x 10(-9) m for a 35-bp major late promoter oligonucleotide. While preferentially recognizing promoter-bound TBP, NC2 also accelerates TBP binding to promoters and stabilizes TBP.DNA complexes. Our data suggest that NC2 controls TBP binding and maintenance on DNA that is largely independent of a canonical TATA sequence.

Transcriptional regulation of the human DNA methyltransferase 3A and 3B genes by Sp3 and Sp1 zinc finger proteins.

The DNMT3A (DNA methyltransferase 3A) and DNMT3B genes encode putative de novo methyltransferases and show complex transcriptional regulation in the presence of three and two different promoters respectively. All promoters of DNMT3A and DNMT3B lack typical TATA sequences adjacent to their transcription start sites and contain several Sp1-binding sites. The importance of these Sp1-binding sites was demonstrated by using a GC-rich DNA-binding protein inhibitor, mithramycin A, i.e. on the basis of decrease in the promoter activities and mRNA expression levels of DNMT3A and DNMT3B. Overexpression of Sp1 and Sp3 up-regulated the promoter activities of these two genes. The physical binding of Sp1 and Sp3 to DNMT3A and DNMT3B promoters was confirmed by a gel shift assay. Interestingly, Sp3 overexpression in HEK-293T cells (human embryonic kidney 293T cells) resulted in 3.3- and 4.0-fold increase in DNMT3A and DNMT3B mRNA expression levels respectively by quantitative reverse transcriptase-PCR, whereas Sp1 overexpression did not. Furthermore, an antisense oligonucleotide to Sp3 significantly decreased the mRNA levels of DNMT3A and DNMT3B. These results indicate the functional importance of Sp proteins, particularly Sp3, in the regulation of DNMT3A and DNMT3B gene expression.

Structural organization of the mouse neurochondrin gene

Neurochondrin is a brain and bone specific leucine-rich protein. We previously cloned the two types of mRNAs (neurochondrin-1; 729 amino acids and neurochondrin-2; 712 amino acids) from mouse and human species. As a first step, to better understand the mechanism of the bone and brain specific and developmentally regulated expression of the neruochondrin gene, the genomic organization of murine neurochondrin was determined. It consists of 7 exons and spans about 10 kb; all splice junctions conform to the GT/AG rule. It codes for two alternatively spliced messenger RNAs, neurochondrin-1 containing all 7 exons and neurochondrin-2 lacking exon 1b but containing the other exons. Cap site analysis showed that the major transcription initiation occurs at 765 bp upstream of the ATG start codon of neurochondrin-1. The promoter region has no TATA and CAAT box-like sequence but contains potential AP-1 and SP-1 binding sites. The neurochondrin gene is localized to mouse chromosome 4D1 and rat chromosome 5q36.11.

Role for Nhp6, Gcn5, and the Swi/Snf complex in stimulating formation of the TATA-binding protein-TFIIA-DNA complex.

The TATA-binding protein (TBP), TFIIA, and TFIIB interact with promoter DNA to form a complex required for transcriptional initiation, and many transcriptional regulators function by either stimulating or inhibiting formation of this complex. We have recently identified TBP mutants that are viable in wild-type cells but lethal in the absence of the Nhp6 architectural transcription factor. Here we show that many of these TBP mutants were also lethal in strains with disruptions of either GCN5, encoding the histone acetyltransferase in the SAGA complex, or SWI2, encoding the catalytic subunit of the Swi/Snf chromatin remodeling complex. These synthetic lethalities could be suppressed by overexpression of TOA1 and TOA2, the genes encoding TFIIA. We also used TFIIA mutants that eliminated in vitro interactions with TBP. These viable TFIIA mutants were lethal in strains lacking Gcn5, Swi2, or Nhp6. These lethalities could be suppressed by overexpression of TBP or Nhp6, suggesting that these coactivators stimulate formation of the TBP-TFIIA-DNA complex. In vitro studies have previously shown that TBP binds very poorly to a TATA sequence within a nucleosome but that Swi/Snf stimulates binding of TBP and TFIIA. In vitro binding experiments presented here show that histone acetylation facilitates TBP binding to a nucleosomal binding site and that Nhp6 stimulates formation of a TBP-TFIIA-DNA complex. Consistent with the idea that Nhp6, Gcn5, and Swi/Snf have overlapping functions in vivo, nhp6a nhp6b gcn5 mutants had a severe growth defect, and mutations in both nhp6a nhp6b swi2 and gcn5 swi2 strains were lethal.