Upstream DNA Research Articles

A Promoter Recognition Mechanism Common to Yeast Mitochondrial and Phage T7 RNA Polymerases

Yeast mitochondrial (YMt) and phage T7 RNA polymerases (RNAPs) are two divergent representatives of a large family of single subunit RNAPs that are also found in the mitochondria and chloroplasts of higher eukaryotes, mammalian nuclei, and many other bacteriophage. YMt and phage T7 promoters differ greatly in sequence and length, and the YMt RNAP uses an accessory factor for initiation, whereas T7 RNAP does not. We obtain evidence here that, despite these apparent differences, both the YMt and T7 RNAPs utilize a similar promoter recognition loop to bind their respective promoters. Mutations in this element in YMt RNAP specifically disrupt mitochondrial promoter utilization, and experiments with site-specifically tethered chemical nucleases indicate that this element binds the mitochondrial promoter almost identically to how the promoter recognition loop from the phage RNAP binds its promoter. Sequence comparisons reveal that the other members of the single subunit RNAP family display loops of variable sequence and size at a position corresponding to the YMt and T7 RNAP promoter recognition loops. We speculate that these elements may be involved in promoter recognition in most or all of these enzymes and that this element's structure allows it to accommodate significant sequence and length variation to provide a mechanism for rapid evolution of new promoter specificities in this RNAP family.

Human Peroxiredoxin 6 (Prdx6) gene expression is regulated by keratinocyte growth factor (KGF) through the same antioxidant response element (ARE) that controls its response to oxidative stress

Peroxiredoxin 6 (Prdx6) can reduce lipid hydroperoxides by a unique mechanism employing a single conserved cysteine and also acts as a phospholipase A2. It is highly expressed in lung, both protecting against oxidative stress and regulating pulmonary surfactant phospholipid metabolism. We reported previously that rodent Prdx6 is up‐regulated in development and by oxidative stress. A 1524 base pair fragment of proximal upstream DNA from the human Prdx6 gene was cloned into the pSEAP‐2 reporter vector (BD Bioscience). Transient transfection of this construct into lung cell lines resulted in measurable production of secreted alkaline phosphatase that was increased 3‐4 fold by keratinocyte growth factor (KGF) treatment. β‐galactosidase expressed from a co‐transfected plasmid was used as a control for transfection efficiency. Using promoter deletions and then site‐directed deletion mutagenesis, a consensus Antioxidant Response Element (ARE), located between 357 and 349 nucleotides before the transcriptional start was identified. This element was important in basal and KGF‐induced expression in both A549 cells and primary rat alveolar type II cells. We have previously shown that this ARE controls the Prdx6 response to H2O2 treatment. We conclude that KGF and antioxidants, both activate transcription through the ARE, and may act through Nrf2, a transcription factor known to bind to the ARE. [HL P01‐75587].

Effect of short term calorie restriction on pro-inflammatory NF-kB and AP-1 in aged rat kidney

To compare the effect of short-term calorie restriction (CR) on aging with that of already known long-term CR, the anti-inflammatory efficacy of 10-day CR was explored in aged rat kidney. Two different age groups, 6 months (young) and 24 months (old) were used. In the old group, one sub-group was control, fed ad libitum (AL) and the other was fed CR for 10 days with 40 % of the food intake of the AL subgroup (n = 5). Reactive species (RS), lipid peroxides and COX-2 activity were measured. The activities of proinflammatory transcription factors NF-kB and AP-1 were measured by electro-mobility shift assay (EMSA). Upstream signaling cascades of NF-kB and AP-1 as well as proinflammatory gene expression were detected by Western blot. 10-day CR suppressed RS, lipid peroxides, and COX-2 activity in aged rat kidney. CR also inhibited upstream signaling cascades and DNA binding activity of NF-kB and AP-1, and thioredoxin/Ref-1 pathway. CR blocked expression of NF-kB-and AP-1-responsive gene COX-2, iNOS, VCAM-1 and ICAM-1. We report for the first time that 10-day CR can attenuate the altered signaling transduction for inflammatory processes which is mediated through RS-induced NF-kB and AP-1 in aged kidney.

Position of the non-template DNA in the Polymerase II elongation complex revealed using single-molecule Fluorescence Resonance Energy Transfer

Single Molecule Fluorescence Resonance Energy Transfer (FRET) was used as a quantitative tool to localize non-template DNA in the RNA Polymerase II (Pol II) elongation complex.We measured the FRET efficiencies between ‘antenna dye molecules’ (ADMs) attached to the non-template DNA and several ‘satellite dye molecules’ (SDMs) attached to known positions within the Pol II elongation complex using the recently developed Nano Positioning System (NPS) (1). NPS combines x-ray crystallographic information, single-molecule FRET data and bayesian parameter estimation, a probability based analysis method, to compute the three dimensional probability density for the position of ADMs. Within the bayesian framework, we were able to account for errors in the determined Forster radii, errors in the measured FRET efficiencies and uncertainties in the SDM positions (due to the attachment of the SDMs via flexible linkers) as well as for geometric constraints.We determined positions of the non-template DNA at +1, -2, -4, -7 (within the transcription bubble) and -12, -15, -18 of the up-stream DNA. Together with the high resolution x-ray structure and our earlier studies (2) determining the position of the nascent RNA this study completes the picture of the Pol II elongation complex.1. A. Muschielok, J. Andrecka, A. Jawhari, F. Bruckner, P. Cramer and J. Michaelis, A nano-positioning system for macromolecular structural analysis. Nature Methods, accepted.2. J. Andrecka, R. Lewis, F. Brueckner, E. Lehmann, P. Cramer and J. Michaelis, Tracking the position of mRNA in RNA polymerase II elongation complexes. PNAS 105, 135-140 (2008).

Identifying and quantifying load-dependence in transcription initiation by T7 RNA Polymerase

To access the genetic code to be transcribed to RNA, RNA polymerases must first melt the DNA, opening a “transcription bubble”. In single molecule optical tweezers studies of initiation by T7 bacteriophage RNA polymerase (T7 RNAP), we have found that the lifetime of the initiation complex is strongly dependent on tension applied to the upstream DNA, indicating, in accord with structural studies, that initiation by T7 RNAP likely involves mechanical deformation of the DNA. The tension dependence diminishes considerably in the absence of free ribonucleotides, suggesting that this mechanical deformation of the DNA template does not coincide with binding, but instead with the opening of the transcription bubble. A new maximum-likelihood method will be presented, in which data do not have to be grouped into force bins, to identify the force-dependent step or steps in initiation by T7 RNAP and to estimate the associated rates and distances to the transition state from these experiments. This method is generalizable to systems with more complicated kinetic pathways, as long as there are two observable states (e.g. bound and unbound) and an irreversible final transition.

Functionally important structural elements of the cyanobacterial clock‐related protein Pex

Pex, a clock-related protein involved in the input pathway of the cyanobacterial circadian clock system, suppresses the expression of clock gene kaiA and lengthens the circadian period. Here, we determined the crystal structure of Anabaena Pex (AnaPex; Anabaena sp. strain PCC 7120) and Synechococcus Pex (SynPex; Synechococcus sp. strain PCC 7942). Pex is a homodimer that forms a winged-helix structure. Using the DNase I protection and electrophoresis mobility shift assays on a Synechococcus kaiA upstream region, we identified a minimal 25-bp sequence that contained an imperfectly inverted repeat sequence as the Pex-binding sequence. Based on crystal structure, we predicted the amino acid residues essential for Pex's DNA-binding activity and examined the effects of various Ala-substitutions in the alpha3 helix and wing region of Pex on in vitro DNA-binding activity and in vivo rhythm functions. Mutant AnaPex proteins carrying a substitution in the wing region displayed no specific DNA-binding activity, whereas those carrying a substitution in the alpha3 helix did display specific binding activity. But the latter were less thermostable than wild-type AnaPex and their in vitro functions were defective. We concluded that Pex binds a kaiA upstream DNA sequence via its wing region and that its alpha3 helix is probably important to its stability.

DISCLOSE : DISsection of CLusters Obtained by SEries of transcriptome data using functional annotations and putative transcription factor binding sites

BackgroundA typical step in the analysis of gene expression data is the determination of clusters of genes that exhibit similar expression patterns. Researchers are confronted with the seemingly arbitrary choice between numerous algorithms to perform cluster analysis.ResultsWe developed an exploratory application that benchmarks the results of clustering methods using functional annotations. In addition, a de novo DNA motif discovery algorithm is integrated in our program which identifies overrepresented DNA binding sites in the upstream DNA sequences of genes from the clusters that are indicative of sites of transcriptional control. The performance of our program was evaluated by comparing the original results of a time course experiment with the findings of our application.ConclusionDISCLOSE assists researchers in the prokaryotic research community in systematically evaluating results of the application of a range of clustering algorithms to transcriptome data. Different performance measures allow to quickly and comprehensively determine the best suited clustering approach for a given dataset.

Sequence-Dependent Upstream DNA–RNA Polymerase Interactions in the Open Complex with λP R and λP RM Promoters and Implications for the Mechanism of Promoter Interference

Upstream interactions of Escherichia coli RNA polymerase (RNAP) in an open promoter complex (RPo) formed at the P R and P RM promoters of bacteriophage λ have been studied by atomic force microscopy. We demonstrate that the previously described 30-nm DNA compaction observed upon RPo formation at P R [Rivetti, C., Guthold, M. & Bustamante, C. (1999). Wrapping of DNA around the E. coli RNA polymerase open promoter complex. EMBO J., 18, 4464–4475.] is a consequence of the specific interaction of the RNAP with two AT-rich sequence determinants positioned from − 36 to − 59 and from − 80 to − 100. Likewise, RPos formed at P RM showed a specific contact between RNAP and the upstream DNA sequence. We further demonstrate that this interaction, which results in DNA wrapping against the polymerase surface, is mediated by the C-terminal domains of α-subunits (carboxy-terminal domain). Substitution of these AT-rich sequences with heterologous DNA reduces DNA wrapping but has only a small effect on the activity of the P R promoter. We find, however, that the frequency of DNA templates with both P R and P RM occupied by an RNAP significantly increases upon loss of DNA wrapping. These results suggest that α carboxy-terminal domain interactions with upstream DNA can also play a role in regulating the expression of closely spaced promoters. Finally, a model for a possible mechanism of promoter interference between P R and P RM is proposed.

Analysis of γH2AX and NHEJ Signaling as Molecular Determinants for GOSensitivity in AML

Most patients with acute myeloid leukemia (AML) display an initial response to high dose chemotherapy but a majority develops resistance and relapse within 1–2 years. The targeted drug gemtuzumab ozogamicin (GO) has shown promising clinical effects. Consisting of a monoclonal CD33 antibody coupled to a toxin, calicheamicin, GO enters the cell after binding to the CD33 antigen, preferentially expressed on myeloid cells. Calicheamicin is then released from the antibody and causes multiple signaling effects resulting in cell death. We have previously shown that GO-induced apoptotic signaling in AML cells involves activation of the Bcl-2 proteins Bax and/or Bak. In this study we aimed to clarify if altered DNA repair or DNA damage signaling are molecular determinants of GO-sensitivity in AML. Moreover, attempts were made to connect the upstream DNA double stranded break (dsb) signaling to GO-induced activation of Bax and Bak. Therefore, we analyzed GO-induced DNA damage signaling in GO responsive (HL60) and resistant (KG1a) AML cells. We can show that GO-resistant cells display a higher basal activity of γH2AX and DNA-PKcs and hence a higher DNA repair capacity. Moreover, by treating AML cells with clinically relevant doses of GO we have observed an increased level of both γH2AX and phosphorylated DNA-PKcs which indicates the prevalence of DNA dsbs. This activation was observed in HL60 as well as in KG1a cells, in vitro. Thus, our data suggest that resistance to GO is not due to lack of DNA dsbs induction, but is rather influenced by the DNA dsbs signaling to the downstream apoptotic components. Key activators downstream of DNA-PK but upstream of Bak/Bax, including c-Abl, caspase-2, Bid, Bad, Bim, Bcl-2 and Bcl-xL, are therefore currently being analyzed. In conclusion, we show that GO induced DNA dsbs in both GO sensitive and GO resistant AML cells in vitro, and that resistance to GO treatment is at least in part due to factors downstream of DNA-PK but upstream of Bak/Bax activation.

Effect of upstream curvature and transcription factors H-NS and LRP on the efficiency of Escherichia coli rRNA promoters P1 and P2 – a phasing analysis

To study the influence of DNA curvature and DNA-binding proteins, which interact with curved DNA on bacterial promoters, we constructed two sets of promoter variants in which a synthetic DNA-bending module was fused at defined distances and angular orientations with respect to the transcription start sites. The distance between the synthetic binding site centre and the transcription start site of the different constructs varied by up to 20 bp, corresponding to almost two complete helical B-DNA turns. The rRNA promoters rrnB P1 and rrnB P2 were selected as target promoters. While in its natural context P1 depends on upstream curved DNA and several transcription factors that bind to this region, promoter P2 is not preceded by curved DNA, nor is it believed to be directly regulated by transcription factors. In vitro transcription measurements of both promoters in the absence of transcription factors varied with the phase of the curved upstream DNA element, underlining the importance of DNA conformation to promoter efficiency. Specific binding of H-NS and LRP to the curved DNA element was demonstrated by gel shift and footprint analysis. Binding affinity was not notably altered for the different distance variants. We demonstrated that the two proteins acted as repressors for both promoters. The extent of H-NS-mediated repression for both promoters did not vary strongly with the phasing of the upstream binding module. In contrast, LRP-dependent repression showed a clear dependence on the angular orientation of the constructs. Phasing-dependent repression is very distinct for P2 but only rudimentary for the P1 promoter.

Structural Modules of RNA Polymerase Required for Transcription from Promoters Containing Downstream Basal Promoter Element GGGA

We recently described a novel basal bacterial promoter element that is located downstream of the -10 consensus promoter element and is recognized by region 1.2 of the sigma subunit of RNA polymerase (RNAP). In the case of Thermus aquaticus RNAP, this element has a consensus sequence GGGA and allows transcription initiation in the absence of the -35 element. In contrast, the Escherichia coli RNAP is unable to initiate transcription from GGGA-containing promoters that lack the -35 element. In the present study, we demonstrate that sigma subunits from both E. coli and T. aquaticus specifically recognize the GGGA element and that the observed species specificity of recognition of GGGA-containing promoters is determined by the RNAP core enzyme. We further demonstrate that transcription initiation by T. aquaticus RNAP on GGGA-containing promoters in the absence of the -35 element requires sigma region 4 and C-terminal domains of the alpha subunits, which interact with upstream promoter DNA. When in the context of promoters containing the -35 element, the GGGA element is recognized by holoenzyme RNAPs from both E. coli and T. aquaticus and increases stability of promoter complexes formed on these promoters. Thus, GGGA is a bona fide basal promoter element that can function in various bacteria and, depending on the properties of the RNAP core enzyme and the presence of additional promoter elements, determine species-specific differences in promoter recognition.

Regulation of DYT1 gene expression by the Ets family of transcription factors

The DYT1 gene encodes for torsinA, a protein with widespread tissue distribution, involved in early onset dystonia (EOD). Numerous studies have focused on torsinA function but no information is available on its transcriptional regulation. We cloned mouse and human 5'-upstream DYT1 DNA fragments, exhibiting high transcriptional activity, as well as tissue specificity. We identified a proximal minimal DYT1 promoter within -141 bp for mouse and -191 bp for human with respect to the ATG codon. Primer extension analysis indicated multiple transcription start sites. In silico analysis of approximately 500 bp 5'-upstream DYT1 fragment demonstrated lack of a classical TATA or CAAT box and the presence of a highly conserved direct repeat of two Ets binding cores within -86 bp to -77 bp and -78 bp to -69 bp of the mouse and human DYT1 gene, respectively. A single or a two base nucleotide alteration within the downstream Ets core resulted in approximately 90% (mouse) or 45-60% (human) drop in activity. Interestingly, a 3-bp distance increase between the two Ets cores dramatically decreased transcriptional activity which was partially restored when the distance was increased up to 10 bp. Ets-like dominant negatives confirmed the Ets factors as DYT1 transcriptional activators.

Identification of the sensory neuron specific regulatory region for the mouse gene encoding the voltage‐gated sodium channel NaV1.8

Voltage-gated sodium channels (VGSC) are critical membrane components that participate in the electrical activity of excitable cells. The type one VGSC family includes the tetrodotoxin insensitive sodium channel, Na(V)1.8, encoded by the Scn10a gene. Na(V)1.8 expression is restricted to small and medium diameter nociceptive sensory neurons of the dorsal root ganglia and cranial sensory ganglia. To understand the stringent transcriptional regulation of the Scn10a gene, the sensory neuron specific promoter was functionally identified. While identifying the mRNA 5'-end, alternative splicing within the 5'-UTR was observed to create heterogeneity in the RNA transcript. Four kilobases of upstream genomic DNA was cloned and the presence of tissue specific promoter activity was tested by microinjection and adenoviral infection of fluorescent protein reporter constructs into primary mouse and rat neurons, and cell lines. The region contained many putative transcription factor-binding sites and strong homology with the predicted rat ortholog. Homology to the predicted human ortholog was limited to the proximal end and several conserved cis elements were noted. Two regulatory modules were identified by microinjection of reporter constructs into dorsal root ganglia and superior cervical ganglia neurons: a neuron specific proximal promoter region between -1.6 and -0.2 kb of the transcription start site cluster, and a distal sensory neuron switch region beyond -1.6 kb that restricted fluorescent protein expression to a subset of primary sensory neurons.

Activation of transcription initiation by Spx: formation of transcription complex and identification of a Cis‐acting element required for transcriptional activation

The Spx protein of Bacillus subtilis interacts with RNA polymerase (RNAP) to activate transcription initiation in response to thiol-oxidative stress. Protein-DNA cross-linking analysis of reactions containing RNAP, Spx and trxA (thioredoxin) or trxB (thioredoxin reductase) promoter DNA was undertaken to uncover the organization of the Spx-activated transcription initiation complex. Spx induced contact between the RNAP sigma(A) subunit and the -10 promoter sequence of trxA and B, and contact of the betabeta' subunits with core promoter DNA. No Spx-DNA contact was detected. Spx mutants, Spx(C10A) and Spx(G52R.), or RNAP alpha C-terminal domain mutants that impair productive Spx-RNAP interaction did not induce heightened sigma and betabeta' contact with the core promoter. Deletion analysis and the activity of hybrid promoter constructs having upstream trxB DNA fused at positions -31, -36 and -41 of the srf (surfactin synthetase) promoter indicated that a cis-acting site between -50 and -36 was required for Spx activity. Mutations at -43 and -44 of trxB abolished Spx-dependent transcription and Spx-induced cross-linking between the sigma subunit and the -10 region. These data are consistent with a model that Spx activation requires contact between the Spx/RNAP complex and upstream promoter DNA, which allows Spx-induced engagement of the sigma and large subunits with the core promoter.

STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF PUTATIVE REGULATORY DNA SEQUENCES OF FCP GENES IN THE CENTRIC DIATOM CYCLOTELLA CRYPTICA

In order to investigate the transcriptional regulation of light harvesting proteins in diatoms we cloned and sequenced the regions encoding the N-termini together with the respective upstream DNA stretches of 11 different fucoxanthin chlorophyll a/c binding polypeptides (FCP) of Cyclotella cryptica. The deduced N-terminal amino acid sequences almost completely matched those deduced earlier from cDNA clones. No introns were found in the regions coding for the N-terminal plastid targeting sequences or shortly downstream of them. A PLACE search for cis-acting DNA elements which are known from higher plants to be involved in the light-dependent or circadian expression of Lhc genes encoding chlorophyll a/b binding light-harvesting polypeptides in green plants resulted in the detection of several motifs. A comparative analysis with DNA stretches, upstream of the Lhc, LhcaR and Fcp genes of the green alga Chlamydomonas reinhardtii, the red alga Cyanidioschyzon merolae and the diatom Thalassiosira pseudonana, however, did not show any conservation of these elements. This indicates that green plants, red algae and diatoms most probably do not share the same conserved elements for light-regulated gene expression. Furthermore a 924 bp DNA fragment habouring a stretch of 292 bp upstream DNA and the entire Fcp6 gene was amplified from genomic DNA of Cyclotella cryptica, cloned into a modified derivative of the transformation vector pPha-T1 and expressed in the pennate diatom Phaeodactylum tricornutum. Reverse transcriptase polymerase chain reaction using RNA of the transformants as template showed that the introduced Fcp6 gene indeed was transcribed, indicating that the regulatory sequence may be generally functional in diatoms.

Conserved hypothetical BB0462 protein enhances the transcription activity of oppAV promoter

Borrelia burgdorferi BB0462 ORF encodes an unknown functional protein with 110 amino acids. A BLAST search in protein databases and the secondary structure being predicted by the program JUFO showed that the conserved hypothetical BB0462 protein was similar to the members of the YbaB protein family in both amino acid composition and protein structure. The co-transformation of BB0462 ORF and oppA upstream regulation DNA into E. coli host cells and β-galactosidase activity assay demonstrated that the BB0462 protein enhanced the transcriptional activity of the oppAV promoter, but does not affect those of oppAI, II, III and IV promoters. Analysis of DNA retardation and competitive repression also confirmed that the BB0462 protein bound to the 409 bp upstream regulation DNA fragment close to the initiation codon of the oppAV gene. All data in our study suggested that the BB0462 protein was involved in the transcriptional regulation of the oppAV gene

Autoregulatory loop and retinoic acid repression regulate pou2/pou5f1 gene expression in the zebrafish embryonic brain

Zebrafish pou5f1, also known as pou2, encodes a POU-family transcription factor that is transiently expressed in the prospective midbrain and anterior hindbrain during gastrulation, governing brain development. In the present study, we found that the main regulatory elements reside in the proximal upstream DNA sequence from -2.2 to -0.12 kb (the -2.2/-0.1 region). The electrophoretic gel mobility shift assay (EMSA) revealed four functional octamer sequences that can associate with zebrafish Pou2/Pou5f1. The expression of mutated reporter constructs, as well as EMSA, suggested that these four octamer sequences operate in a cooperative manner to drive expression in the mid/hindbrain. We also identified a retinoic acid (RA)-responsive element in this proximal region, which was required to repress transcription in the posterior part of the embryo. These data provide a scheme wherein pou2/pou5f1 expression in zebrafish embryos is regulated by both an autoregulatory loop and repression by RA emanating from the posterior mesoderm.

A Myocyte Enhancer Factor 2 (MEF2) Site Located in a Hypersensitive Region of the FGF16 Gene Locus Is Required for Preferential Promoter Activity in Neonatal Cardiac Myocytes

Fibroblast growth factor 16 (FGF16) is preferentially expressed in the heart after birth, suggesting its regulation is associated with tissue-specific chromatin remodeling and DNA-protein interactions. Here we have mapped the transcription initiation site of murine FGF16 to approximately 1.1 kilobases (kb) upstream of the translation start codon (ATG). Hybrid reporter genes directed by about 4.7 kb of upstream FGF16 DNA were expressed specifically in transfected neonatal rat cardiac myocytes, as well as in the heart of transgenic mice. A DNaseI hypersensitive site was mapped to a region about 1.2 kb upstream of the transcription initiation site in heart but not kidney tissue, and a nuclease protection assay gave evidence of a cardiac-specific protein-DNA interaction in this region. Deletion analysis indicated that a hybrid gene with 1205 bp but not 1054 bp of upstream DNA directed FGF16 promoter activity in transfected neonatal rat cardiac myocytes. We identified a putative myocyte enhancer factor 2 (MEF2)-binding site at nucleotides -1159/-1148, confirmed by electrophoretic mobility shift assay and MEF2 antibody binding. Mutation of the MEF2 site resulted in a blunting of FGF16 promoter activity in transfected neonatal rat cardiac myocytes. These data suggest that chromatin remodeling and MEF2 binding in the FGF16 promoter contribute to expression in the postnatal heart.

Regulation of transcription by acetate in Escherichia coli: in vivo and in vitro comparisons

Acetate, even at neutral pH, induces changes in gene expression that allow Escherichia coli to adapt to the diverse chemical stresses of the gastrointestinal tract. These include differential effects on transcription, including both activation and repression. The in vivo studies presented here show that cyclopropane fatty acid synthase transcription induced by neutral acetate proceeds via both the sigma70 and sigma38 forms of RNA polymerase. Upstream DNA elements are required in both cases, but in vitro studies demonstrate that the binding of regulatory factors is not needed. Sigma70 promoters respond towards acetate with very significant differences in vitro, with effects ranging from inhibition to lack of effect to weak stimulation to strong stimulation. By contrast, the effects of acetate on sigma38 transcription at these same promoters in vitro are generally stimulatory. Thus, acetate directly alters transcription complexes in vitro in ways that lead to significant differential transcription, contributing to the diverse effects that are known to allow adaptation in vivo.

Er81is a downstream target of Pax6 in cortical progenitors

BackgroundAlthough the transcription factor Pax6 plays an essential role in neurogenesis, layer formation and arealization in the developing mammalian cortex, the mechanisms by which it accomplishes these regulatory functions are largely unknown. Pax6 and the ETS family transcription factor Er81, which is presumed to play a role in the specification of a sublineage of layer 5 projection neurons, are expressed with a prominent rostrolateral-high to caudomedial-low gradient in cortical progenitors. In the absence of functional Pax6, progenitors do not express Er81 and the rostrolateral cortex lacks Er81-positive layer 5 neurons. In this study, we investigated the transcriptional regulation of Er81 and provide evidence that Er81 is a direct target of Pax6.ResultsWe identified and analyzed the regulatory function of an evolutionarily conserved upstream DNA sequence in the putative mouse Er81 promoter. Three potential Pax6 binding sites were identified in this region. We found that the presence of one of these sites is necessary and sufficient for full activation of the Er81 promoter in Pax6-transfected HeLa cells, while other still unknown factors appear to contribute to Er81 promoter activity in cortical progenitors and neuronal cells. The results suggest that endogenous Pax6, which is expressed at the highest level in progenitors of the rostrolateral cortex, exerts region-specific control of Er81 activity, thus specifying a subpopulation of layer 5 projection neurons.ConclusionWe conclude that the genetic interplay between the transcription factors, Pax6 and Er81, is responsible, in part, for the regional specification of a distinct sublineage of layer 5 projection neurons.