Octamer Binding Site Research Articles

Transcriptional Activity of Full-Length and Truncated Metallothionein Promoters ofCrassostrea hongkongensisin Response to Metal Stress

ABSTRACT A 717-bp 5′-flanking regulatory sequence of the metallothionein gene in Crassostrea hongkongensis (ChMT) was isolated using anchored polymerase chain reaction. Several regulatory elements embedded in the flanking sequence, including the central promoter region, five putative metal-responsive elements (MRE), and an octamer-binding site (OBS), were analyzed using online prediction software. Then, the effects of element-based promoter truncations in the upstream regulatory region of ChMT on the metal stress response were examined. The expression levels of the promoters of various lengths were determined individually using a luciferase reporter gene in HEK293T cells. Transfection assays revealed that MRE1- and MRE5-containing DNA fragments inhibited transcription, whereas MRE4-containing DNA sequences activated promoter expression under both metal-stressed and unstressed conditions. A 39-bp DNA fragment containing the OBS was shown to respond positively to metal stress induced by cadmium and copper, ...

Interactions of the ubiquitous octamer-binding transcription factor-1 with both the signal transducer and activator of transcription 5 and the glucocorticoid receptor mediate prolactin and glucocorticoid-induced β-casein gene expression in mammary epithelial cells

Regulation of milk protein gene expression by lactogenic hormones (prolactin and glucocorticoids) provides an attractive model for studying the mechanisms by which protein and steroid hormones synergistically regulate gene expression. β-Casein is one of the major milk proteins and its expression in mammary epithelial cells is stimulated by lactogenic hormones. The signal transducer and activator of transcription 5 and glucocorticoid receptor are essential downstream mediators of prolactin and glucocorticoid signaling, respectively. Previous studies have shown that mutating the octamer-binding site of the β-casein gene proximal promoter dramatically reduces the hormonal induction of the promoter activity. However, little is known about the underlying molecular mechanisms. In this report, we show that lactogenic hormones rapidly induce the binding of octamer-binding transcription factor-1 to the β-casein promoter and this induction is not mediated by either increasing the expression of octamer-binding transcription factor-1 or inducing its translocation to the nucleus. Rather, lactogenic hormones induce physical interactions between the octamer-binding transcription factor-1, signal transducer and activator of transcription 5, and glucocorticoid receptor to form a ternary complex, and these interactions enhance or stabilize the binding of these transcription factors to the promoter. Abolishing these interactions significantly reduces the hormonal induction of β-casein gene transcription. Thus, our study indicates that octamer-binding transcription factor-1 may serve as a master regulator that facilitates the DNA binding of both signal transducer and activator of transcription 5 and glucocorticoid receptor in hormone-induced β-casein expression, and defines a novel mechanism of regulation of tissue-specific gene expression by the ubiquitous octamer-binding transcription factor-1.

Micrococcal Nuclease Does Not Substantially Bias Nucleosome Mapping

We have mapped sequence-directed nucleosome positioning on genomic DNA molecules using high-throughput sequencing. Chromatins, prepared by reconstitution with either chicken or frog histones, were separately digested to mononucleosomes using either micrococcal nuclease (MNase) or caspase-activated DNase (CAD). Both enzymes preferentially cleave internucleosomal (linker) DNA, although they do so by markedly different mechanisms. MNase has hitherto been very widely used to map nucleosomes, although concerns have been raised over its potential to introduce bias. Having identified the locations and quantified the strength of both the chicken or frog histone octamer binding sites on each DNA, the results obtained with the two enzymes were compared using a variety of criteria. Both enzymes displayed sequence specificity in their preferred cleavage sites, although the nature of this selectivity was distinct for the two enzymes. In addition, nucleosomes produced by CAD nuclease are 8–10 bp longer than those produced with MNase, with the CAD cleavage sites tending to be 4–5 bp further out from the nucleosomal dyad than the corresponding MNase cleavage sites. Despite these notable differences in cleavage behaviour, the two nucleases identified essentially equivalent patterns of nucleosome positioning sites on each of the DNAs tested, an observation that was independent of the histone type. These results indicate that biases in nucleosome positioning data collected using MNase are, under our conditions, not significant.

Direct Interactions of Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 ORF50/Rta Protein with the Cellular Protein Octamer-1 and DNA Are Critical for Specifying Transactivation of a Delayed-Early Promoter and Stimulating Viral Reactivation

The Kaposi's sarcoma-associated herpesvirus (KSHV) delayed-early K-bZIP promoter contains an ORF50/Rta binding site whose sequence is conserved with the ORF57 promoter. Mutation of the site in the full-length K-bZIP promoter reduced Rta-mediated transactivation by greater than 80%. The K-bZIP element contains an octamer (Oct) binding site that overlaps the Rta site and is well conserved with Oct elements found in the immediate-early promoters of herpes simplex virus type 1(HSV-1). The cellular protein Oct-1, but not Oct-2, binds to the K-bZIP element in a sequence-specific fashion in vitro and in vivo and stimulates Rta binding to the promoter DNA. The coexpression of Oct-1 enhances Rta-mediated transactivation of the wild type but not the mutant K-bZIP promoter, and Oct-1 and Rta proteins bind to each other directly in vitro. Mutations of Rta within an amino acid sequence conserved with HSV-1 virion protein 16 eliminate Rta's interactions with Oct-1 and K-bZIP promoter DNA but not RBP-Jk. The binding of Rta to both Oct-1 and DNA contributes to the transactivation of the K-bZIP promoter and viral reactivation, and Rta mutants deficient for both interactions are completely debilitated. Our data suggest that the Rta/Oct-1 interaction is essential for optimal KSHV reactivation. Transfections of mouse embryo fibroblasts and an endothelial cell line suggest cell-specific differences in the requirement for Oct-1 or RBP-Jk in Rta-mediated transactivation of the K-bZIP promoter. We propose a model in which Rta transactivation of the promoter is specified by the combination of DNA binding and interactions with several cellular DNA binding proteins including Oct-1.

Production of Specific mRNA Transcripts, Usage of an Alternate Promoter, and Octamer-Binding Transcription Factors Influence the Surface Expression Levels of the HIV Coreceptor CCR5 on Primary T Cells

Surface levels of CCR5 on memory CD4(+) T cells influence HIV-1/AIDS susceptibility. Alternative promoter usage results in the generation of CCR5 mRNA isoforms that differ based on whether they contain or lack the untranslated exon 1. The impact of exon 1-containing transcripts on CCR5 surface expression is unknown. In this study, we show that the increased cell surface expression of CCR5 on primary T cells is associated with selective enrichment of exon 1-containing transcripts. The promoter that drives exon 1-containing transcripts is highly active in primary human T cells but not in transformed T cell lines. The transcription factors Oct-1 and -2 inhibit and enhance, respectively, the expression of exon 1-containing transcripts and CCR5 surface levels. However, polymorphisms at homologous octamer-binding sites in the CCR5 promoter of nonhuman primates abrogate the binding of these transcription factors. These results identify exon 1-containing transcripts, and the cis-trans factors that regulate the expression levels of these mRNA isoforms as key parameters that affect CCR5 surface expression levels, and by extension, susceptibility to HIV/AIDS among humans, and possibly, the observed interspecies differences in susceptibility to lentiviral infection.

Sox2 and Pou2f1 interact to control lens and olfactory placode development

Sox2, which encodes an SRY-like HMG box transcription factor, is critical for vertebrate development. Sox2 mediates its transcriptional effects through the formation of complexes with specific co-factors, many of which are unknown. In this report, we identify Oct-1, encoded by the Pou2f1 gene, as a co-factor for Sox2 in the context of mouse lens and nasal placode induction. Oct-1, Sox2, and Pax6 are co-expressed during lens and nasal placode induction and during subsequent developmental stages. Genetic combination of Sox2 and Pou2f1 mutant alleles results in impaired induction of the lens placode, an ocular phenotype that includes anophthalmia, and a complete failure of nasal placode induction. These ocular and nasal phenotypes closely resemble those observed in Pax6 null embryos. Moreover, we identify DNA-binding sites that support the cooperative formation of a complex between Sox2 and Oct-1 and mediate Sox2/Oct-1-dependent transactivation of the Pax6 lens ectoderm enhancer in vitro. We demonstrate that the same Sox- and Octamer-binding sites are essential for Pax6 enhancer activity in the lens placode and its derivatives in transgenic mouse embryos. Collectively, these results indicate that Pou2f1, Sox2 and Pax6 are interdependent components of a molecular pathway utilized in both lens and nasal placode induction.

Differential lineage-specific regulation of murine CD45 transcription by Oct-1 and PU.1

Although it has been established that CD45 expression is regulated at the transcriptional level, neither the regulatory elements that are responsible for its unique expression pattern nor the relevance of its three distinct transcriptional start sites (P1a, P1b, and P2) has been fully characterized. We studied the contribution of the three start sites to CD45 mRNA production in haematopoietic cell lines and primary haematopoietic cells. In myeloid and lymphoid cells and cell lines most CD45 transcripts originate from P1b with the exception of the thymoma-derived T cell line EL4, in which ∼90% of CD45 transcripts originate from P1a. The degree of contribution of P1a is highest in lymphoid cells and increases in T cells following mitogen stimulation. In vitro evaluation of sequence upstream of the start sites shows that the P2 start site is sufficient for CD45 expression in lymphoid but not in myeloid cells, confirms the presence of a PU.1-binding site essential for myeloid expression of CD45, and reveals an Octamer-binding site that interacts with both Oct-1 and Oct-2 and activates CD45 transcription in lymphoid and myeloid cells. These findings are the first evidence that Octamer-binding factors are involved in the control of CD45 expression.

Identification of an octamer-binding site controlling the activity of the small breast epithelial mucin gene promoter

e human small breast epithelial mucin (SBEM) gene has been identified as being preferentially expressed in mammary epithelial cells and over-expressed in breast tumors. In this report, we have characterized the promoter of SBEM gene in order to identify sequences responsible for this strong mammary expression. A series of SBEM promoter/luciferase constructs were transiently transfected into both breast (MCF-7, BT-20) and non-breast (HeLa and HepG2) cell lines. In addition to the minimal promoter and to a repressor region, we have identified an 87-bp sequence (-357/-270) driving a strong breast-specific expression. Site-directed mutagenesis of a putative octamer-binding transcription factor binding site located within this latter region led to a strong decrease of the transcriptional activity of the SBEM promoter. Furthermore, transient over-expression of Oct1 and Oct2 not only increased SBEM promoter reporter activity, but also enhanced endogenous SBEM mRNA level. Overall, the data suggest that octamer-binding transcription factors participate in the strong expression of SBEM gene in breast tissues. Clarifying the SBEM gene regulation will help to dissect mechanisms underlying transcription of normal breast and breast cancer-associated genes.

The promoter competition assay (PCA): a new approach to identify motifs involved in the transcriptional activity of reporter genes

Identifying particular motifs responsible for promoter activity is a crucial step toward the development of new gene-based preventive and therapeutic strategies. However, to date, experimental methods to study promoter activity remain limited. We present in this report a promoter competition assay designed to identify, within a given promoter region, motifs critical for its activity. This assay consists in co-transfecting the promoter to be analyzed and double-stranded oligonucleotides which will compete for the binding of transcription factors. Using the recently characterized SBEM promoter as model, we first delineated the feasibility of the method and optimized the experimental conditions. We then identified, within an 87-bp region responsible for a strong expression of the reporter gene, an octamer-binding site essential for its transcriptional regulation. The importance of this motif has been confirmed by site-directed mutagenesis. The promoter competition assay appears to be a fast and efficient approach to identify, within a given promoter sequence, sites critical for its activity.

Relative Affinities of DNA Sequences for the Histone Octamer Depend Strongly upon Both the Temperature and Octamer Concentration

Using a novel competition assay to determine the relative strength of different histone octamer-binding sites, we have compared three natural and two synthetic sites. We show that the relative affinities of these sites for the histone octamer depend upon both the temperature and octamer concentration. In particular, under certain conditions, a natural octamer-binding site from a yeast promoter outcompetes a synthetic sequence of comparable affinity to the strongest previously described positioning sequence. Under other conditions, this synthetic sequence is the preferred octamer ligand. We infer that sequence selection by the histone octamer depends strongly upon both the sequence-dependent anisotropy of DNA bending and on DNA deformability and that these parameters may contribute differently to nucleosome formation. These findings indicate that previous studies designed to identify strong octamer-binding sites may fail to select some natural strong binding sites.

An Oct-1 binding site mediates activation of the gata2 promoter by BMP signaling

The gata2 gene encodes a transcription factor implicated in regulating early patterning of ectoderm and mesoderm, and later in numerous cell-specific gene expression programs. Activation of the gata2 gene during embryogenesis is dependent on the bone morphogenetic protein (BMP) signaling pathway, but the mechanism for how signaling controls gene activity has not been defined. We developed an assay in Xenopus embryos to analyze regulatory sequences of the zebrafish gata2 promoter that are necessary to mediate the response to BMP signaling during embryogenesis. We show that activation is Smad dependent, since it is blocked by expression of the inhibitory Smad6. Deletion analysis identified an octamer binding site that is necessary for BMP-mediated induction, and that interacts with the POU homeodomain protein Oct-1. However, this element is not sufficient to transfer a BMP response to a heterologous promoter, requiring an additional more proximal cooperating element. Based on recent studies with other BMP-dependent promoters (Drosophila vestigial and Xenopus Xvent-2), our studies of the gata2 gene suggest that POU-domain proteins comprise a common component of the BMP signaling pathway, cooperating with Smad proteins and other transcriptional activators.

Patterns of gene divergence and VL promoter activity in immunoglobulin light chain clusters of the channel catfish.

The structure and genomic organization of V, J, and C segments in multiple gene clusters of the G class of catfish light (L) chain were determined to study evolutionary patterns of cluster divergence and regulatory function. The results showed that the organizational pattern is conserved; two VL segments reside in opposite transcriptional orientation to a pseudogene JL segment (psiJ), a functional JL segment, and a CL segment. Structures within the central V-psiJ-J-C regions indicate that cluster duplication occurred after the V-V-psiJ-J-C organization became fixed. VL divergence in gene clusters subsequently occurred by mechanisms that principally targeted complementarity-determining regions. The sequence of the VL-flanking regions, which contained regulatory octamer, TATA, and Pax-5 (BSAP) binding site motifs, was conserved during G cluster duplication and within VL-flanking regions in divergent lineages of bony fish. Reporter assays of catfish B cells transfected with a 742-bp VL-flanking fragment showed promoter activity in the absence of enhancer elements. The promoter's activity doubled when coupled with the catfish IgH enhancer. A 136-bp fragment containing the motifs conserved in bony fish phylogeny and located between the leader initiation codon and the initiation sites of sterile transcripts served as a minimal promoter and provided the highest B-cell activity. These constructs, however, did not act as promoters in catfish non-lymphoid cells or mammalian BJA-B B cells or fibroblasts. These results indicate that the structure and function of VL promoter regions in the regulation and tissue specificity of L-chain gene expression evolved early in phylogeny.

The human involucrin gene is transcriptionally repressed through a tissue-specific silencer element recognized by Oct-2

Involucrin is an important marker of epithelial differentiation which expression is upregulated just after basal cells are pushed into the suprabasal layer in stratified epithelia. Several transcription factors and regulatory elements had been described as responsible for turning on the gene. However, it is evident that in basal cell layer, additional mechanisms are involved in keeping the gene silent before the differentiation process starts. In this work, we located a potential transcriptional silencer in a 52 bp sequence whose integrity is necessary for silencing the proximal enhancer promoter element (PEP) in multiplying keratinocytes. Octamer-binding sites were noticed in this fragment and the specific binding of Oct-2 transcription factor was detected. Oct-2 appears to be implicated in an epithelial-specific repression activity recorded only in keratinocytes and C33-A cell line. Overexpression of Oct-2 repressed the involucrin promoter activity in epithelial cells and in the presence of the silencer element.

Episodic activation of the rat GnRH promoter: role of the homeoprotein oct-1.

Recent reports demonstrate that the rat GnRH promoter is activated in an episodic fashion in immortalized GnRH neurons, but little information is available on molecular processes that contribute to this phenomenon. In this study, we dissected the regions of the rat GnRH promoter that mediate these effects by testing a series of 5' deletion luciferase reporter constructs on the pattern of photonic emissions from single, living GT1-7 GnRH neuronal cells. Deletion analysis revealed that the region -2012/-1597 that contains the neuron-specific enhancer (NSE) was required for the elaboration of pulses of GnRH promoter activity. The importance of this region was supported by observations that episodic reporter activity could be transferred to a neutral nonpulsatile promoter (Rous sarcoma virus, RSV(180)). Immunoneutralization of Oct-1 as well as mutation of an octamer binding site located at -1787/-1783 (AT-a site) blocked the pulsatile GnRH promoter activity in GT1-7 neuronal cells. Taken together, our findings indicate that episodic GnRH gene expression is a promoter-dependent phenomenon, which is mediated by Oct-1 interaction with regulatory elements in the NSE region.

Identification of Sox-2 regulatory region which is under the control of Oct-3/4-Sox-2 complex.

Sox-2 is a transcriptional cofactor expressed in embryonic stem (ES) cells as well as in neuronal cells. It has been demonstrated that Sox-2 plays an important role in supporting gene expression in ES cells, especially by forming a complex with embryonic Octamer factor, Oct-3/4. Here, we have analyzed the regulatory regions of the Sox-2 gene and identified two enhancers which stimulate transcription in ES cells as well as in embryonal carcinoma cells. These regulatory regions, which we termed Sox regulatory regions (SRR) 1 and 2, exert their function specifically when cells are in an undifferentiated state. Interestingly, like the regulatory elements of FGF-4 and UTF1 genes, combinatorial action of Octamer and Sox-2 binding sites support the SRR2 activity. However, biochemical analyses reveal that, due to the unique sequence and/or its organization, the SRR2 bears distinct characteristics from those of FGF-4 and UTF1 regulatory elements. That is, unlike the FGF-4 gene enhancer, the SRR2 precludes the binding of the Oct-1-Sox-2 complex. The difference between the SRR2 and UTF1 regulatory element is in the ability of SRR2 to recruit the Oct-6-Sox-2 complex as well as the Oct-3/4-Sox-2 complex. Co-transfection analyses confirm that both complexes are able to stimulate transcription through the SRR2 element.

Androgen receptor interactions with Oct-1 and Brn-1 are physically and functionally distinct

POU domain proteins interact positively or negatively with steroid hormone receptors, depending on the precise array of these and other factors assembled on target gene promoters. Octamer transcription factor 1 (Oct-1), a ubiquitous POU factor, is implicated in androgen induction of the mouse sex-limited protein ( Slp) gene based on protein–DNA interaction studies. However, direct evidence for a role of Oct-1 in the hormone response has been difficult to obtain. Brain 1 (Brn-1), another POU factor, is more tissue-specific, expressing in brain and also in kidney, which is a major site of Slp synthesis. We compared the interaction of the androgen receptor (AR) with Oct-1 and Brn-1 to reveal the more likely candidate for regulation of Slp. In transfection, addition of either Oct-1 or Brn-1 reduced AR activation, regardless of the presence of an octamer-like sequence in the enhancer, suggesting interference was indirect. However, when the octamer-like element was changed to a consensus octamer site, Brn-1, but not Oct-1, strongly enhanced androgen activation. This correlated with Brn-l's preference for the consensus octamer sequence in DNA binding assays. Direct interaction of AR with glutathione-S-transferase-(GST)–fused Oct-1 was DNA-dependent, while Brn-l–AR association was not. Chimeric Brn-1 and Oct-1 POU domains demonstrated that the DNA-dependent AR interaction relied on the origin of the POU homeodomain. However, in the context of full-length Brn-1 and Oct-1 chimeric proteins, the POU homedomain was not sufficient to confer the distinct behaviors of these factors in vivo, but instead revealed the importance of an N-terminal transactivation domain in Brn-1. These results demonstrate that functional interaction of Oct-1 and Brn-1 with AR is determined by the precise sequence of the octamer binding site, and by differential interaction of the POU factors with AR and other components of the transcriptional machinery.

Octamer proteins inhibit IL-4 gene transcription in normal human CD4 T cells.

The balance of Th1 (eg, interleukin-2 (IL-2)) and Th2 (eg, IL-4) cytokines produced by CD4 T cells markedly influences the outcome of the adaptive immune response. Although octamer transcription factor proteins increase IL-2 transcription in T cells, their role in IL-4 gene transcription remains controversial. We have previously shown and now confirm that the proximal octamer binding site of the human IL-4 promoter, which separates the two most proximal NFAT binding sites, is bound prior to, but not after, activation in vivo. Since these two NFAT sites are essential for optimal IL-4 promoter activity, this suggested that prior engagement by octamer proteins might prevent adjacent NFAT binding and inhibit IL-4 gene transcription. In support of this hypothesis, here we show that NFAT proteins are unable to bind to a combined octamer/NFAT site unless the octamer proteins are competed away. Moreover, activity of an IL-4 reporter gene mutated in the proximal octamer binding site is increased compared to the wild-type promoter in human peripheral blood CD4 T cells. In addition, over-expression of either Oct-1 or Oct-2 decreased wild-type IL-4 promoter activity, while increasing IL-2 promoter activity. No decrease in promoter activity was seen when Oct-1 or Oct-2 was over-expressed with the octamer-mutant IL-4 reporter gene. Thus, octamer proteins are candidates to promote a Th1 rather than Th2 pattern of cytokine gene expression by activated CD4 T cells.

POU Homeodomain Protein OCT1 Is Implicated in the Expression of the Caudal-related Homeobox Gene Cdx-2

The caudal homeobox gene Cdx-2 is a transcriptional activator for approximately a dozen genes specifically expressed in pancreatic islets and intestinal cells. It is also involved in preventing the development of colorectal tumors. Studies using "knockout" approaches demonstrated that Cdx-2 is haplo-insufficient in certain tissues including the intestines but not the pancreatic islets. The mechanisms, especially transcription factors, which regulate Cdx-2 expression, are virtually unknown. We found previously that Cdx-2 expression could be autoregulated in a cell type-specific manner. In this study, we located an octamer (OCT) binding site within the mouse Cdx-2 gene promoter. This site, designated as Cdx-2(P)OCT, is involved in the expression of the Cdx-2 promoter. Both pancreatic and intestinal cell lines were found to express a number of POU (OCT binding) homeodomain proteins examined by electrophoretic mobility shift assay. However, it appears that Cdx-2(P)OCT interacts only with OCT1 in the nuclear extracts of the intestinal cell lines examined, although it interacts with OCT1 and at least two other POU proteins that are to be identified in the pancreatic InR1-G9 cell nuclear extract. Co-transfecting OCT1 cDNA but not five other POU gene cDNAs activates the Cdx-2 promoter in the pancreatic InR1-G9 and the intestinal Caco-2 cell lines. In contrast, Cdx-2(P)OCT cannot act as an enhancer element if it is fused to a thymidine kinase promoter. Furthermore, Cdx-2(P)OCT-thymidine kinase fusion promoters cannot be activated by OCT1 co-transfection. Cell type-specific expression, cell type-specific binding affinity of POU proteins to the cis-element Cdx-2(P)OCT, and the DNA content-dependent activation of Cdx-2 promoter via Cdx-2(P)OCT by OCT1 suggest that POU proteins play important and complicated roles in modulating Cdx-2 expression in cell type-specific manners.

The Upstream Region of the Rpe65 Gene Confers Retinal Pigment Epithelium-specific Expression in Vivo and in Vitro and Contains Critical Octamer and E-box Binding Sites

RPE65 is essential for all-trans- to 11-cis-retinoid isomerization, the hallmark reaction of the retinal pigment epithelium (RPE). Here, we identify regulatory elements in the Rpe65 gene and demonstrate their functional relevance to Rpe65 gene expression. We show that the 5' flanking region of the mouse Rpe65 gene, like the human gene, lacks a canonical TATA box and consensus GC and CAAT boxes. The mouse and human genes do share several cis-acting elements, including an octamer, a nuclear factor one (NFI) site, and two E-box sites, suggesting a conserved mode of regulation. A mouse Rpe65 promoter/beta-galactosidase transgene containing bases -655 to +52 (TR4) of the mouse 5' flanking region was sufficient to direct high RPE-specific expression in transgenic mice, whereas shorter fragments (-297 to +52 or -188 to +52) generated only background activity. Furthermore, transient transfection of analogous TR4/luciferase constructs also directed high reporter activity in the human RPE cell line D407 but weak activity in the non-RPE cell lines HeLa, HepG2, and HS27. Functional binding of potential transcription factors to the octamer sequence, AP-4, and NFI sites was demonstrated by directed mutagenesis, electrophoretic mobility shift assay, and cross-linking. Mutations of these sites abolished binding and corresponding transcriptional activity and indicated that octamer and E-box transcription factors synergistically regulate the RPE65 promoter function. Thus, we have identified the regulatory region in the Rpe65 gene that accounts for tissue-specific expression in the RPE and found that octamer and E-box transcription factors play a critical role in the transcriptional regulation of the Rpe65 gene.

Octamer-primed sequencing technology: development of primer identification software.

Octamer sequencing technology (OST) is a primer-directed sequencing strategy in which an individual octamer primer is selected from a pre-synthesized octamer primer library and used to sequence a DNA fragment. However, selecting candidate primers from such a library is time consuming and can be a bottleneck in the sequencing process. To accelerate the sequencing process and to obtain high quality sequencing data, a computer program, electronic OST or eOST, was developed to automatically identify candidate primers from an octamer primer library. eOST integrates the base calling software PHRED to provide a quality assessment for target sequences and identifies potential primer binding sites located within a high quality target region. To increase the sequencing success rate, eOST includes a simple dynamic folding algorithm to automatically calculate the free energy and predict the secondary structure within the template in the vicinity of the octamer-binding site. Several parameters were found to be important, including base quality threshold, the window size of the template sequence segment, and the threshold [Delta] G value. OST, coupled with the eOST software, can be used to sequence short DNA fragments or in the finishing assembly stage of large-scale sequencing of genomic DNA.