Initiation Of Gene Transcription Research Articles

Novel Functions for TAF7, a Regulator of TAF1-independent Transcription

The transcription factor TFIID components TAF7 and TAF1 regulate eukaryotic transcription initiation. TAF7 regulates transcription initiation of TAF1-dependent genes by binding to the acetyltransferase (AT) domain of TAF1 and inhibiting the enzymatic activity that is essential for transcription. TAF7 is released from the TAF1-TFIID complex upon completion of preinitiation complex assembly, allowing transcription to initiate. However, not all transcription is TAF1-dependent, and the role of TAF7 in regulating TAF1-independent transcription has not been defined. The IFNγ-induced transcriptional co-activator CIITA activates MHC class I and II genes, which are vital for immune responses, in a TAF1-independent manner. Activation by CIITA depends on its intrinsic AT activity. We now show that TAF7 binds to CIITA and inhibits its AT activity, thereby repressing activated transcription. Consistent with this TAF7 function, siRNA-mediated depletion of TAF7 resulted in increased CIITA-dependent transcription. A more global role for TAF7 as a regulator of transcription was revealed by expression profiling analysis: expression of 30-40% of genes affected by TAF7 depletion was independent of either TAF1 or CIITA. Surprisingly, although TAF1-dependent transcripts were largely down-regulated by TAF7 depletion, TAF1-independent transcripts were predominantly up-regulated. We conclude that TAF7, until now considered only a TFIID component and regulator of TAF1-dependent transcription, also regulates TAF1-independent transcription.

Eukaryotic and prokaryotic promoter prediction using hybrid approach

Promoters are modular DNA structures containing complex regulatory elements required for gene transcription initiation. Hence, the identification of promoters using machine learning approach is very important for improving genome annotation and understanding transcriptional regulation. In recent years, many methods have been proposed for the prediction of eukaryotic and prokaryotic promoters. However, the performances of these methods are still far from being satisfactory. In this article, we develop a hybrid approach (called IPMD) that combines position correlation score function and increment of diversity with modified Mahalanobis Discriminant to predict eukaryotic and prokaryotic promoters. By applying the proposed method to Drosophila melanogaster, Homo sapiens, Caenorhabditis elegans, Escherichia coli, and Bacillus subtilis promoter sequences, we achieve the sensitivities and specificities of 90.6 and 97.4% for D. melanogaster, 88.1 and 94.1% for H. sapiens, 83.3 and 95.2% for C. elegans, 84.9 and 91.4% for E. coli, as well as 80.4 and 91.3% for B. subtilis. The high accuracies indicate that the IPMD is an efficient method for the identification of eukaryotic and prokaryotic promoters. This approach can also be extended to predict other species promoters.

Regulation of Dendritic Development by BDNF Requires Activation of CRTC1 by Glutamate

Dendritic growth is essential for the establishment of a functional nervous system. Among extrinsic signals that control dendritic development, substantial evidence indicates that BDNF regulates dendritic morphology. However, little is known about the underlying mechanisms by which BDNF controls dendritic growth. In this study, we show that the MAPK signaling pathway and the transcription factor cAMP response element-binding protein (CREB) mediate the effects of BDNF on dendritic length and complexity. However, phosphorylation of CREB alone is not sufficient for the stimulation of dendritic growth by BDNF. Thus, using a mutant form of CREB unable to bind CREB-regulated transcription coactivator (CRTC1), we demonstrate that this effect also requires a functional interaction between CREB and CRTC1. Moreover, inhibition of CRTC1 expression by shRNA-mediated knockdown abolished BDNF-induced dendritic growth of cortical neurons. Interestingly, we found that nuclear translocation of CRTC1 results from activation of NMDA receptors by glutamate, a process that is essential for the effects of BDNF on dendritic development. Together, these data identify a previously unrecognized mechanism by which CREB and the coactivator CRTC1 mediate the effects of BDNF on dendritic growth.

Genome-wide characterization of methylguanosine-capped and polyadenylated small RNAs in the rice blast fungus Magnaporthe oryzae

Small RNAs are well described in higher eukaryotes such as mammals and plants; however, knowledge in simple eukaryotes such as filamentous fungi is limited. In this study, we discovered and characterized methylguanosine-capped and polyadenylated small RNAs (CPA-sRNAs) by using differential RNA selection, full-length cDNA cloning and 454 transcriptome sequencing of the rice blast fungus Magnaporthe oryzae. This fungus causes blast, a devastating disease on rice, the principle food staple for over half the world’s population. CPA-sRNAs mapped primarily to the transcription initiation and termination sites of protein-coding genes and were positively correlated with gene expression, particularly for highly expressed genes including those encoding ribosomal proteins. Numerous CPA-sRNAs also mapped to rRNAs, tRNAs, snRNAs, transposable elements and intergenic regions. Many other 454 sequence reads could not be mapped to the genome; however, inspection revealed evidence for non-template additions and chimeric sequences. CPA-sRNAs were independently confirmed using a high affinity variant of eIF-4E to capture 5′-methylguanosine-capped RNA followed by 3′-RACE sequencing. These results expand the repertoire of small RNAs in filamentous fungi.

Mechanism and regulation of acetylated histone binding by the tandem PHD finger of DPF3b

Histone lysine acetylation and methylation are important during gene transcription in a chromatin context1,2. Our knowledge about the types of protein modules that can interact with acetyl-lysine has so far been limited to bromodomains1. Recently, a tandem PHD (plant homeodomain) finger3 (PHD12) of human DPF3b, which functions in association with the BAF chromatin remodelling complex to initiate transcription in the heart and muscle development, was reported to bind histones H3 and H4 in an acetylation sensitive manner4, making it a first alternative to bromodomains for acetyl-lysine binding5. Here, we report the structural mechanism of acetylated histone binding by the double PHD fingers of DPF3b. Our three-dimensional solution structures and biochemical analysis of DPF3b illuminate the molecular basis of the integrated tandem PHD finger, which acts as one functional unit in the sequence-specific recognition of lysine 14-acetylated histone H3 (H3K14ac). Whereas the interaction with H3 is promoted by acetylation at lysine 14, it is inhibited by methylation at lysine 4, and these opposing influences are important during transcriptional activation of DPF3b target genes Pitx2 and Jmjd1c. Binding of this tandem protein module to chromatin can thus be regulated by different histone modifications during the initiation of gene transcription.

Modular Utilization of Distal cis-Regulatory Elements Controls Ifng Gene Expression in T Cells Activated by Distinct Stimuli

Distal cis-regulatory elements play essential roles in the T lineage-specific expression of cytokine genes. We have mapped interactions of three trans-acting factors-NF-kappaB, STAT4, and T-bet-with cis elements in the Ifng locus. We find that RelA is critical for optimal Ifng expression and is differentially recruited to multiple elements contingent upon T cell receptor (TCR) or interleukin-12 (IL-12) plus IL-18 signaling. RelA recruitment to at least four elements is dependent on T-bet-dependent remodeling of the Ifng locus and corecruitment of STAT4. STAT4 and NF-kappaB therefore cooperate at multiple cis elements to enable NF-kappaB-dependent enhancement of Ifng expression. RelA recruitment to distal elements was similar in T helper 1 (Th1) and effector CD8(+) T (Tc1) cells, although T-bet was dispensable in CD8 effectors. These results support a model of Ifng regulation in which distal cis-regulatory elements differentially recruit key transcription factors in a modular fashion to initiate gene transcription induced by distinct activation signals.

A precise equation of equilibrium of four steps of TBP binding with the TATA box for prognosis of phenotypic manifestation of mutations

Among the central events of transcription initiation of TATA-containing genes in eukaryotes are the recognition and binding of the TATA box by the TATA-binding protein (TBP) to start the preinitiation complex formation on nucleosomal DNA. Using the equation of equilibrium for step-by-step TBP/TATA binding, we have analyzed 69 experimental datasets for the characteristics of biologically important features altered by TATA-box mutations. Among these features, the TBP/TATA-complex parameters, the transcription level, the activity of gene products, yeast colony growth at a dose of growth inhibitor (phenotype), and the heterogeneity of the response of a population to unspecific environmental stress have been described. Significant correlations were found between in silico prediction for TBP/TATA affinity and experimental data for in vivo and in vitro test systems based on 15 cell types of 19 species, RNA polymerases II and III, and natural, recombinant or mutant TBP. Such an invariant impact of the step-by-step TBP/TATA binding on the biological activity of complex systems, from a molecule to a population, might be due to the fact that TBP/TATA-complex formation precedes specific steps of transcription machinery assembly, which provide the multivariant jigsaw puzzle according to the expression pattern of each eukaryotic gene.

Achieving Singularity in Mammalian Odorant Receptor Gene Choice

Odor discrimination requires differential expression of odor detectors. In fact, olfactory input to the brain is organized in units (glomeruli) innervated only by olfactory sensory neurons that express the same odorant receptor (OR). Therefore, discriminatory capacity is maximized if each sensory neuron expresses only one allele of a single OR gene, a postulate sometimes canonized as the "one neuron-one receptor rule." OR gene choice appears to result from a hierarchy of processes: differential availability of the alleles of each OR gene, zonal exclusion (or selection), OR gene switching during the initiation of OR gene transcription, and OR-dependent feedback to solidify the choice of one OR gene. The mechanisms underlying these processes are poorly understood, though a few elements are known or suspected. For example, the mechanism of activation of OR gene transcription appears to work in part through a few homeobox transcription factors (Emx2, and perhaps Lhx2) and the Ebf family of transcription factors. Further insights will probably come from several directions, but a promising hypothesis is that epigenetic mechanisms contribute to all levels of the hierarchical control of OR gene expression, especially the repressive events that seem to be necessary to achieve the singularity of OR gene choice.

Differential Requirements of Hsp90 and DNA for the Formation of Estrogen Receptor Homodimers and Heterodimers

The two estrogen receptor (ER) subforms, ERalpha and ERbeta, are capable of forming DNA-binding homodimers and heterodimers. Although binding to DNA is thought to stabilize ER dimers, how ERalpha/alpha, ERbeta/beta, and ERalpha/beta dimerization is regulated by DNA and the chaperone protein Hsp90 is poorly understood. Using our highly optimized bioluminescence resonance energy transfer assays in conjunction with assays for transcriptional activation of ERs, we determined that DNA binding appears to play a minor role in the stabilization of ER dimers, especially in the case of ERbeta/beta homodimers. These findings suggest that ER dimers form before they associate with chromatin and that DNA binding plays a minor role in stabilizing ER dimers. Additionally, although Hsp90 is essential for the proper dimerization of ERalpha/alpha and ERalpha/beta, it is not required for the proper dimerization of ERbeta/beta. Despite this, Hsp90 is critical for the estrogen-dependent transcriptional activity of the ERbeta/beta homodimer. Thus, Hsp90 is implicated as an important regulator of distinct aspects of ERalpha and ERbeta action.

Genome-wide analysis of regions similar to promoters of histone genes

BackgroundThe purpose of this study is to: i) develop a computational model of promoters of human histone-encoding genes (shortly histone genes), an important class of genes that participate in various critical cellular processes, ii) use the model so developed to identify regions across the human genome that have similar structure as promoters of histone genes; such regions could represent potential genomic regulatory regions, e.g. promoters, of genes that may be coregulated with histone genes, and iii/ identify in this way genes that have high likelihood of being coregulated with the histone genes.ResultsWe successfully developed a histone promoter model using a comprehensive collection of histone genes. Based on leave-one-out cross-validation test, the model produced good prediction accuracy (94.1% sensitivity, 92.6% specificity, and 92.8% positive predictive value). We used this model to predict across the genome a number of genes that shared similar promoter structures with the histone gene promoters. We thus hypothesize that these predicted genes could be coregulated with histone genes. This hypothesis matches well with the available gene expression, gene ontology, and pathways data. Jointly with promoters of the above-mentioned genes, we found a large number of intergenic regions with similar structure as histone promoters.ConclusionsThis study represents one of the most comprehensive computational analyses conducted thus far on a genome-wide scale of promoters of human histone genes. Our analysis suggests a number of other human genes that share a high similarity of promoter structure with the histone genes and thus are highly likely to be coregulated, and consequently coexpressed, with the histone genes. We also found that there are a large number of intergenic regions across the genome with their structures similar to promoters of histone genes. These regions may be promoters of yet unidentified genes, or may represent remote control regions that participate in regulation of histone and histone-coregulated gene transcription initiation. While these hypotheses still remain to be verified, we believe that these form a useful resource for researchers to further explore regulation of human histone genes and human genome. It is worthwhile to note that the regulatory regions of the human genome remain largely un-annotated even today and this study is an attempt to supplement our understanding of histone regulatory regions.

Genome‐wide identification of vitamin D receptor (VDR) binding sites in DNA from prostate epithelial cells (PEC) by chromatin immunoprecipitation‐sequencing (ChIP‐seq)

The 1,25 dihydroxyvitamin D (1,25D) activates the VDR to bind DNA and initiate gene transcription. While microarray studies show 1,25D regulates expression of many transcripts, few of these genes have been examined for VDR binding. Using ChIP‐seq, we mapped VDR binding sites in DNA from the human PEC line, RWPE1. Cells were treated with vehicle or 1,25D (10 nM, 3 h) and subjected to ChIP using either VDR antibody or IgG. Using IgG as the reference, 1236 and 1175 VDR binding peaks were found in 1,25D and vehicle‐treated cells, respectively (0.1 FDR, 200 bp window, Cisgenome). Genes with known VDREs were confirmed e.g. CYP24 and IGFBP3. Correlation between ChIP‐seq peaks and a previous microarray study found that only 39% were associated with 1,25D‐regulated transcripts, including novel peaks close to the transcription factors (TF) JunB, FOS and TBP. Also only 5% of VDR peaks were found within 5 kb of the transcription start site of a gene and the known DR3 VDR binding motif was found in just 15% of the peaks. However, several other DNA‐binding motifs were enriched under the peaks suggesting indirect DNA association of VDR through other TF. Our data suggest the relationship between VDR binding and transcription is more complex that previously reported.Grant Funding Source: Showalter Trust to JCF

Heavy Involvement of Stringent Transcription Control Depending on the Adenine or Guanine Species of the Transcription Initiation Site in Glucose and Pyruvate Metabolism inBacillus subtilis

In Bacillus subtilis cells, the GTP level decreases and the ATP level increases upon a stringent response. This reciprocal change in the concentrations of the substrates of RNA polymerase affects the rate of transcription initiation of certain stringent genes depending on the purine species at their transcription initiation sites. DNA microarray analysis suggested that not only the rrn and ilv-leu genes encoding rRNAs and the enzymes for synthesis of branched-chain amino acids, respectively, but also many genes, including genes involved in glucose and pyruvate metabolism, might be subject to this kind of stringent transcription control. Actually, the ptsGHI and pdhABCD operons encoding the glucose-specific phosphoenolpyruvate:sugar phosphotransferase system and the pyruvate dehydrogenase complex were found to be negatively regulated, like rrn, whereas the pycA gene encoding pyruvate carboxylase and the alsSD operon for synthesis of acetoin from pyruvate were positively regulated, like ilv-leu. Replacement of the guanine at position 1 and/or position 2 of ptsGHI and at position 1 of pdhABCD (transcription initiation base at position 1) by adenine changed the negative stringent control of these operons in the positive direction. The initiation bases for transcription of pdhABCD and pycA were newly determined. Then the promoter sequences of these stringent operons were aligned, and the results suggested that the presence of a guanine(s) and the presence of an adenine(s) at position 1 and/or position 2 might be indispensable for negative and positive stringent control, respectively. Such stringent transcription control that affects the transcription initiation rate through reciprocal changes in the GTP and ATP levels likely occurs for numerous genes of B. subtilis.

DNA dynamics play a role as a basal transcription factor in the positioning and regulation of gene transcription initiation

We assess the role of DNA breathing dynamics as a determinant of promoter strength and transcription start site (TSS) location. We compare DNA Langevin dynamic profiles of representative gene promoters, calculated with the extended non-linear PBD model of DNA with experimental data on transcription factor binding and transcriptional activity. Our results demonstrate that DNA dynamic activity at the TSS can be suppressed by mutations that do not affect basal transcription factor binding–DNA contacts. We use this effect to establish the separate contributions of transcription factor binding and DNA dynamics to transcriptional activity. Our results argue against a purely ‘transcription factor-centric’ view of transcription initiation, suggesting that both DNA dynamics and transcription factor binding are necessary conditions for transcription initiation.

RNA polymerase II–TFIIB structure and mechanism of transcription initiation

To initiate gene transcription, RNA polymerase II (Pol II) requires the transcription factor IIB (B). Here we present the crystal structure of the complete Pol II-B complex at 4.3 A resolution, and complementary functional data. The results indicate the mechanism of transcription initiation, including the transition to RNA elongation. Promoter DNA is positioned over the Pol II active centre cleft with the 'B-core' domain that binds the wall at the end of the cleft. DNA is then opened with the help of the 'B-linker' that binds the Pol II rudder and clamp coiled-coil at the edge of the cleft. The DNA template strand slips into the cleft and is scanned for the transcription start site with the help of the 'B-reader' that approaches the active site. Synthesis of the RNA chain and rewinding of upstream DNA displace the B-reader and B-linker, respectively, to trigger B release and elongation complex formation.

Gene promoter analysis in molecular diagnostics: do or don’t?

Approximately 1% of single basepair substitutions causing human genetic disease occur within gene promoter regions where they disrupt the normal processes of gene activation and transcriptional initiation, and usually decrease or increase the level of mRNA and, thus, protein [1]. Although promoter mutations are known to have functionally important consequences for gene expression, promoter analysis is not a regular part of molecular diagnostics. One reason is that the effect of promoter mutations can be very subtle. For instance, the majority of missense mutations cause a fairly easy to identify qualitative defect. By contrast, promoter mutations may cause small quantitative defects that may be hard to detect. Even if the promoter of an autosomal gene is completely downregulated as result of mutation, half of the normal amount of protein is present, which is often enough to prevent severe disease. Another reason for not performing promoter analysis is that it is complex and the assays that are needed to investigate the functional relationship between the mutation and disease are laborious and difficult to perform. As a result, thorough studies of promoter mutations are scarce and often confined to research laboratories. By contrast, in epidemiological studies in which large amounts of single-nucleotide polymorphisms (SNPs) are studied in relation to disease, several promoter SNPs have recently been shown to be associated with specific diseases. Unfortunately, appropri ate functional analysis of these SNPs is often lacking and it can be questioned if they are themselves causing disease. Gene promoter mutations

PKR acts early in infection to suppress Semliki Forest virus production and strongly enhances the type I interferon response.

The double-stranded RNA-activated protein kinase (PKR) is a key regulator of protein translation, interferon (IFN) expression and cell survival. Upon infection of vertebrate cells in continuous culture, the alphavirus Semliki Forest virus (SFV) initiates apoptosis and IFN synthesis. To determine the effect of PKR on SFV infection, we studied the course of infection in wild-type (wt) mice, mice with a genetic deletion of PKR (PKR−/−) and mouse embryo fibroblasts (MEFs) derived from these mice. In MEFs, PKR delayed virus protein synthesis, production of infectious virus and caspase-3-activated cell death and reduced the yield of infectious virus by 90 %. Small interfering RNA suppression of PKR levels in NIH-3T3 cells also reduced virus production and apoptosis. In MEFs, PKR was not required for initiation of IFN-β gene transcription, but contributed strongly to the magnitude of this response. Levels of IFN-β transcripts in PKR−/− MEFs at 8 h were 80 % lower than those in wt MEFs and levels of functional IFN at 24 h were 95 % lower. Following infection of wt and PKR−/− mice, SFV4 and SFV A7(74) were avirulent. PKR increased levels of serum IFN and the rate of clearance of infectious virus from the brain. In summary, in response to SFV, PKR exerts an early antiviral effect that delays virus protein production and release of infectious virus and, whilst PKR is not required for induction of apoptosis or activation of the type I IFN response, it strongly augments the type I IFN response and contributes to clearance of infectious virus from the mouse brain.

The ABC transporter gene family of Daphnia pulex

BackgroundThe large gene superfamily of ABC (ATP-binding cassette) transporters encodes membrane proteins involved in trafficking processes across biological membranes and further essential cell biological functions. ABC transporters are evolutionary ancient and involved in the biochemical defence against toxicants. We report here a genome-wide survey of ABC proteins of Daphnia pulex, providing for the first time information on ABC proteins in crustacea, a primarily aquatic arthropod subphylum of high ecological and economical importance.ResultsWe identified 64 ABC proteins in the Daphnia genome, which possesses members of all current ABC subfamilies A to H. To unravel phylogenetic relationships, ABC proteins of Daphnia were compared to those from yeast, worm, fruit fly and human. A high conservation of Daphnia of ABC transporters was observed for proteins involved in fundamental cellular processes, including the mitochondrial half transporters of the ABCB subfamily, which function in iron metabolism and transport of Fe/S protein precursors, and the members of subfamilies ABCD, ABCE and ABCF, which have roles in very long chain fatty acid transport, initiation of gene transcription and protein translation, respectively. A number of Daphnia proteins showed one-to-one orthologous relationships to Drosophila ABC proteins including the sulfonyl urea receptor (SUR), the ecdysone transporter ET23, and the eye pigment precursor transporter scarlet. As the fruit fly, Daphnia lacked homologues to the TAP protein, which plays a role in antigene processing, and the cystic fibrosis transmembrane conductance regulator (CFTR), which functions as a chloride channel. Daphnia showed two proteins homologous to MDR (multidrug resistance) P-glycoproteins (ABCB subfamily) and six proteins homologous to MRPs (multidrug resistance-associated proteins) (ABCC subfamily). However, lineage specific gene duplications in the ABCB and ABCC subfamilies complicated the inference of function. A particularly high number of gene duplications were observed in the ABCG and ABCH subfamilies, which have 23 and 15 members, respectively.ConclusionThe in silico characterisation of ABC transporters in the Daphnia pulex genome revealed that the complement of ABC transporters is as complex in crustaceans as that other metazoans. Not surprisingly, among currently available genomes, Daphnia ABC transporters most closely resemble those of the fruit fly, another arthropod.

Nanoparticle-Mediated Drug Delivery and Pulmonary Hypertension

Nanotechnology offers great potential benefits for drug delivery and therapy of respiratory and systemic diseases. Nanoparticles (NPs) have been of significant interest for some time because they can be designed to simultaneously carry a drug payload, specifically target features of diseased tissues, and carry an imaging molecule to track drug accumulation and clearance in tissues. Moreover, they can be engineered to tailor drug delivery and improve pharmacokinetics. A variety of NPs have been investigated in experimental animal models as tools to improve the delivery and therapeutic efficacy of drugs or genes delivered to the lung or other organ systems.1 The nanotechnology platform for drug delivery contains a number of very different types of nanostructures with widely varying properties. Examples of these NPs include dendrimers, fullerenes, carbon nanotubes, and polymeric NPs. In this issue of Hypertension , Kimura et al2 report that nuclear factor κB (NF-κB) decoy oligodeoxynucleotides (ODNs) encapsulated in poly-(ethylene glycol)- block -lactide/glycolide copolymer NPs and delivered to the lungs of rats by intratracheal instillation reduced pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT). They showed that NP-encapsulated NF-κB decoy, visualized by fluorescein isothiocyanate labeling, reached the distal regions of the lungs and was present in alveolar macrophages and small pulmonary arteries for ≤14 days after a single instillation. The small pulmonary arteries were also found to be a site of NF-κB activation and NF-κB–dependent inflammatory cytokine production (monocyte chemoattractant protein 1, interleukin 1, and tumor necrosis factor α) in patients with PAH and in rats with MCT-induced PAH. The decoy ODNs, unlike antisense ODNs, which bind specific regions in mRNA, bind directly to the transcription factor and inhibit transcription factor binding to target DNA and initiation of gene transcription (Figure). It was speculated by the authors that cellular uptake of the NPs might slowly release encapsulated …

Toward a Detailed Description of the Thermally Induced Dynamics of the Core Promoter

Establishing the general and promoter-specific mechanistic features of gene transcription initiation requires improved understanding of the sequence-dependent structural/dynamic features of promoter DNA. Experimental data suggest that a spontaneous dsDNA strand separation at the transcriptional start site is likely to be a requirement for transcription initiation in several promoters. Here, we use Langevin molecular dynamic simulations based on the Peyrard-Bishop-Dauxois nonlinear model of DNA (PBD LMD) to analyze the strand separation (bubble) dynamics of 80-bp-long promoter DNA sequences. We derive three dynamic criteria, bubble probability, bubble lifetime, and average strand separation, to characterize bubble formation at the transcriptional start sites of eight mammalian gene promoters. We observe that the most stable dsDNA openings do not necessarily coincide with the most probable openings and the highest average strand displacement, underscoring the advantages of proper molecular dynamic simulations. The dynamic profiles of the tested mammalian promoters differ significantly in overall profile and bubble probability, but the transcriptional start site is often distinguished by large (longer than 10 bp) and long-lived transient openings in the double helix. In support of these results are our experimental transcription data demonstrating that an artificial bubble-containing DNA template is transcribed bidirectionally by human RNA polymerase alone in the absence of any other transcription factors.

Identification and characterization of stem cell-specific transcription of PSF1 in spermatogenesis

PSF1 is an evolutionarily conserved DNA replication factor, which forms the GINS complex with PSF2, PSF3, and SLD5. The mouse PSF1 homolog has been identified from a stem cell-specific cDNA library. To investigate its transcriptional regulatory mechanisms during differentiation, we studied PSF1 mRNA expression in testis and characterized its promoter. No canonical TATA or CAAT boxes could be found in the PSF1 5′-flanking region, whereas several consensus AML1, GATA, and Sry putative binding sequences are predicted within 5 kb of the putative transcription start site. In addition, binding sites for oncoproteins such as Myb and Ets were also found in the promoter. In testis, various PSF1 gene transcription initiation sites are present and short transcripts encoding two novel isoforms, PSF1b and 1c, were found. However, spermatogonium stem cells specifically express transcripts for PSF1a. These data suggest that PSF1 is tightly regulated at the transcriptional level in stem cells.