Isolation, sequencing, and functional analysis of the TATA-less murine ATPase II promoter and structural analysis of the ATPase II gene.
ABSTRACT: The P-type Mg2+-ATPase, termed ATPase II (Atp8a1), is a putative aminophospholipid transporting enzyme, which helps to maintain phospholipid asymmetry in cell membranes. In this project we have elucidated the organization of the mouse ATPase II gene and identified its promoter. Located within chromosome 5, this gene spans about 224 kb and consists of 38 exons, three of which are alternatively spliced (exons 7, 8 and 16), giving rise to two transcript variants. Translation of these transcripts results in two ATPase II isoforms (1 and 2) composed of 1164 and 1149 amino acids, respectively. Using RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) we identified multiple transcription start sites (TSS) in messages obtained from heart, lung, liver, and spleen. The mouse ATPase II promoter is TATA-less and lacks a consensus initiator sequence. Luciferase reporter analysis of full and core promoters revealed strong activity and little cell type specificity, possibly because more flanking, regulatory sequences are required to cause such tissue specificity. In the neuronal HN2, N18, SN48 cells and the NIH3T3 fibroblast cells, but not in the B16F10 melanoma cells, the core promoter (-318/+193 with respect to the most common TSS) displayed significantly higher activity than the full promoter (-1026/+193). Serial 5' deletion of the core promoter revealed significant cell type-specific activity of the fragments, suggesting differential expression and use of transcription factors in the five cell lines tested. Additionally distribution of the TSS was organ specific. Such observations suggest tissue-specific differences in transcription initiation complex assembly and regulation of ATPase II gene expression. Information presented here form the groundwork for further studies on the expression of this gene in apoptotic cells.
Project description:The molecule responsible for the enzyme activity plasma membrane (PM) aminophospholipid translocase (APLT), which catalyzes phosphatidylserine (PS) translocation from the outer to the inner leaflet of the plasma membrane, is unknown in mammals. A Caenorhabditis elegans study has shown that ablation of transbilayer amphipath transporter-1 (TAT-1), which is an ortholog of a mammalian P-type ATPase, Atp8a1, causes PS externalization in the germ cells. We demonstrate here that the hippocampal cells of the dentate gyrus, and Cornu Ammonis (CA1, CA3) in mice lacking Atp8a1 exhibit a dramatic increase in PS externalization. Although their hippocampi showed no abnormal morphology or heightened apoptosis, these mice displayed increased activity and a marked deficiency in hippocampus-dependent learning, but no hyper-anxiety. Such observations indicate that Atp8a1 plays a crucial role in PM-APLT activity in the neuronal cells. In corroboration, ectopic expression of Atp8a1 but not its close homolog, Atp8a2, caused an increase in the population (V(max) ) of PM-APLT without any change in its signature parameter K(m) in the neuronal N18 cells. Conversely, expression of a P-type phosphorylation-site mutant of Atp8a1 (Atp8a1*) caused a decrease in V(max) of PM-APLT without significantly altering its K(m) . The Atp8a1*-expressing N18 cells also exhibited PS externalization without apoptosis. Together, our data strongly indicate that Atp8a1 plays a central role in the PM-APLT activity of some mammalian cells, such as the neuronal N18 and hippocampal cells.
Project description:P4-ATPases are a subfamily of P-type ATPases that flip phospholipids across membranes to generate lipid asymmetry, a property vital to many cellular processes. Mutations in several P4-ATPases have been linked to severe neurodegenerative and metabolic disorders. Most P4-ATPases associate with one of three accessory subunit isoforms known as CDC50A (TMEM30A), CDC50B (TMEM30B), and CDC50C (TMEM30C). To identify P4-ATPases that associate with CDC50A, in vivo, and determine their tissue distribution, we isolated P4-ATPases-CDC50A complexes from retina, brain, liver, testes, and kidney on a CDC50A immunoaffinity column and identified and quantified P4-ATPases from their tryptic peptides by mass spectrometry. Of the 12 P4-ATPase that associate with CDC50 subunits, 10 P4-ATPases were detected. Four P4-ATPases (ATP8A1, ATP11A, ATP11B, ATP11C) were present in all five tissues. ATP10D was found in low amounts in liver, brain, testes, and kidney, and ATP8A2 was present in significant amounts in retina, brain, and testes. ATP8B1 was detected only in liver, ATP8B3 and ATP10A only in testes, and ATP8B2 primarily in brain. We also show that ATP11A, ATP11B and ATP11C, like ATP8A1 and ATP8A2, selectively flip phosphatidylserine and phosphatidylethanolamine across membranes. These studies provide new insight into the tissue distribution, relative abundance, subunit interactions and substrate specificity of P4-ATPase-CDC50A complexes.
Project description:Genomic clones coding for one of the two identified Artemia franciscana Na/K-ATPase alpha subunits, the alpha 1 subunit, have been isolated. Several overlapping clones were obtained, although their restriction maps showed a large heterogeneity. Sequencing of their exons showed that they differ in up to 3.46% of their nucleotides in translated regions and 8.18% in untranslated regions. Southern blot analysis of DNA purified from different lots of A. franciscana cysts and from isolated individuals suggests that the variation is due to the existence of multiple Na/K-ATPase alpha 1 subunit alleles in A. franciscana. The Na/K-ATPase alpha 1 subunit gene is divided into 15 exons. Ten of the 14 introns are located in identical positions in this gene as in the human Na/K-ATPase alpha 3 subunit gene. Analysis of the 5' flanking region of the gene has allowed identification of the transcription-initiation sites. The adjacent upstream region has been shown to have functional promoter activity in cultured mammalian cells, suggesting the evolutionary conservation of some of the promoter regulatory sequences.
Project description:The regulation of transcription initiation is critical for developmental and cellular processes. RNA polymerase II (Pol II) is recruited by the basal transcription machinery to the core promoter where Pol II initiates transcription. The core promoter encompasses the region from -40 to +40 bp relative to the +1 transcription start site (TSS). Core promoters may contain one or more core promoter motifs that confer specific properties to the core promoter, such as the TATA box, initiator (Inr) and motifs that are located downstream of the TSS, namely, motif 10 element (MTE), the downstream core promoter element (DPE) and the Bridge, a bipartite core promoter element. We had previously shown that Caudal, an enhancer-binding homeodomain transcription factor and a key regulator of the Hox gene network, is a DPE-specific activator. Interestingly, pair-rule proteins have been implicated in enhancer-promoter communication at the engrailed locus. Fushi tarazu (Ftz) is an enhancer-binding homeodomain transcription factor encoded by the ftz pair-rule gene. Ftz works in concert with its co-factor, Ftz-F1, to activate transcription. Here, we examined whether Ftz and Ftz-F1 activate transcription with a preference for a specific core promoter motif. Our analysis revealed that similarly to Caudal, Ftz and Ftz-F1 activate the promoter containing a TATA box mutation to significantly higher levels than the promoter containing a DPE mutation, thus demonstrating a preference for the DPE motif. We further discovered that Ftz target genes are enriched for a combination of functional downstream core promoter elements that are conserved among Drosophila species. Thus, the unique combination (Inr, Bridge and DPE) of functional downstream core promoter elements within Ftz target genes highlights the complexity of transcriptional regulation via the core promoter in the transcription of different developmental gene regulatory networks.
Project description:A core promoter is a stretch of DNA surrounding the transcription start site (TSS) that integrates regulatory inputs and recruits general transcription factors to initiate transcription. The nature and causative relationship of the DNA sequence and chromatin signals that govern the selection of most TSSs by RNA polymerase II remain unresolved. Maternal to zygotic transition represents the most marked change of the transcriptome repertoire in the vertebrate life cycle. Early embryonic development in zebrafish is characterized by a series of transcriptionally silent cell cycles regulated by inherited maternal gene products: zygotic genome activation commences at the tenth cell cycle, marking the mid-blastula transition. This transition provides a unique opportunity to study the rules of TSS selection and the hierarchy of events linking transcription initiation with key chromatin modifications. We analysed TSS usage during zebrafish early embryonic development at high resolution using cap analysis of gene expression, and determined the positions of H3K4me3-marked promoter-associated nucleosomes. Here we show that the transition from the maternal to zygotic transcriptome is characterized by a switch between two fundamentally different modes of defining transcription initiation, which drive the dynamic change of TSS usage and promoter shape. A maternal-specific TSS selection, which requires an A/T-rich (W-box) motif, is replaced with a zygotic TSS selection grammar characterized by broader patterns of dinucleotide enrichments, precisely aligned with the first downstream (+1) nucleosome. The developmental dynamics of the H3K4me3-marked nucleosomes reveal their DNA-sequence-associated positioning at promoters before zygotic transcription and subsequent transcription-independent adjustment to the final position downstream of the zygotic TSS. The two TSS-defining grammars coexist, often physically overlapping, in core promoters of constitutively expressed genes to enable their expression in the two regulatory environments. The dissection of overlapping core promoter determinants represents a framework for future studies of promoter structure and function across different regulatory contexts.
Project description:<h4>Background</h4>More than 80% of mammalian protein-coding genes are driven by TATA-less promoters which often show multiple transcriptional start sites (TSSs). However, little is known about the core promoter DNA sequences or mechanisms of transcriptional initiation for this class of promoters.<h4>Methodology/principal findings</h4>Here we identify a new core promoter element XCPE2 (X core promoter element 2) (consensus sequence: A/C/G-C-C/T-C-G/A-T-T-G/A-C-C/A(+1)-C/T) that can direct specific transcription from the second TSS of hepatitis B virus X gene mRNA. XCPE2 sequences can also be found in human promoter regions and typically appear to drive one of the start sites within multiple TSS-containing TATA-less promoters. To gain insight into mechanisms of transcriptional initiation from this class of promoters, we examined requirements of several general transcription factors by in vitro transcription experiments using immunodepleted nuclear extracts and purified factors. Our results show that XCPE2-driven transcription uses at least TFIIB, either TFIID or free TBP, RNA polymerase II (RNA pol II) and the MED26-containing mediator complex but not Gcn5. Therefore, XCPE2-driven transcription can be carried out by a mechanism which differs from previously described TAF-dependent mechanisms for initiator (Inr)- or downstream promoter element (DPE)-containing promoters, the TBP- and SAGA (Spt-Ada-Gcn5-acetyltransferase)-dependent mechanism for yeast TATA-containing promoters, or the TFTC (TBP-free-TAF-containing complex)-dependent mechanism for certain Inr-containing TATA-less promoters. EMSA assays using XCPE2 promoter and purified factors further suggest that XCPE2 promoter recognition requires a set of factors different from those for TATA box, Inr, or DPE promoter recognition.<h4>Conclusions/significance</h4>We identified a new core promoter element XCPE2 that are found in multiple TSS-containing TATA-less promoters. Mechanisms of promoter recognition and transcriptional initiation for XCPE2-driven promoters appear different from previously shown mechanisms for classical promoters that show single "focused" TSSs. Our studies provide insight into novel mechanisms of RNA Pol II transcription from multiple TSS-containing TATA-less promoters.
Project description:Nucleosome depletion at transcription start sites (TSS) has been documented genome-wide in multiple eukaryotic organisms. However, the mechanisms that mediate this nucleosome depletion and its functional impact on transcription remain largely unknown. We have studied these issues at human MHC class II (MHCII) genes. Activation-induced nucleosome free regions (NFR) encompassing the TSS were observed at all MHCII genes. Nucleosome depletion was exceptionally strong, attaining over 250-fold, at the promoter of the prototypical HLA-DRA gene. The NFR was induced primarily by the transcription factor complex that assembles on the conserved promoter-proximal enhancer situated upstream of the TSS. Functional analyses performed in the context of native chromatin demonstrated that displacing the NFR without altering the sequence of the core promoter induced a shift in the position of the TSS. The NFR thus appears to play a critical role in transcription initiation because it directs correct TSS positioning in vivo. Our results provide support for a novel mechanism in transcription initiation whereby the position of the TSS is controlled by nucleosome eviction rather than by promoter sequence.
Project description:The transcription start site (TSS) determines the length and composition of the 5' UTR and therefore can have a profound effect on translation. Yet, little is known about the mechanism underlying start site selection, particularly from promoters lacking conventional core elements such as TATA-box and Initiator. Here we report a novel mechanism of start site selection in the TATA- and Initiator-less promoter of miR-22, through a strictly localized downstream element termed DTIE and an upstream distal element. Changing the distance between them reduced promoter strength, altered TSS selection and diminished Pol II recruitment. Biochemical assays suggest that DTIE does not serve as a docking site for TFIID, the major core promoter-binding factor. TFIID is recruited to the promoter through DTIE but is dispensable for TSS selection. We determined DTIE consensus and found it to be remarkably prevalent, present at the same TSS downstream location in ≈20.8% of human promoters, the vast majority of which are TATA-less. Analysis of DTIE in the tumor suppressor p53 confirmed a similar function. Our findings reveal a novel mechanism of transcription initiation from TATA-less promoters.
Project description:Telomerase is often upregulated during initiation and/or progression of human tumors, suggesting that repression of telomerase might inhibit cancer growth or progression. Here, we report that BRG1, the ATPase subunit of the SWI/SNF chromatin remodeling complex, is a general suppressor of hTERT transcription in human cancer cells. While overexpression of BRG1 inhibits hTERT transcription, depletion of BRG1 stimulates transcription of hTERT, leading to higher telomerase activity and longer telomeres. Chromatin-immunoprecipitation assays revealed that BRG1 binds to the transcription start site (TSS) of the hTERT promoter and forms a ternary complex with histone deacetylase 2 (HDAC2). BRG1 remodels chromatin structure to facilitate the action of HDAC2, leading to deacetylation of H3K9ac and H4ac at the TSS and suppression of hTERT transcription. On the other hand, ?-catenin binds to the TSS and stimulates hTERT transcription. Thus, BRG1/HDAC2 and ?-catenin constitute a manipulative apparatus at the TSS to play opposite but complementary roles in regulating hTERT expression. These results uncover a yin-yang mechanism in modulating hTERT transcription and provide explanation for limited transcription of hTERT in human cancer cells. BRG1/HDAC2 may have a potential as an anti-cancer therapeutic and/or for reactivating cellular proliferative capacity in the context of in vitro tissue engineering.
Project description:The MHC class I gene, PD1, has neither functional TATAA nor Initiator (Inr) elements in its core promoter and initiates transcription at multiple, dispersed sites over an extended region in vitro. Here, we define a novel core promoter feature that supports regulated transcription through selective transcription start site (TSS) usage. We demonstrate that TSS selection is actively regulated and context dependent. Basal and activated transcriptions initiate from largely nonoverlapping TSS regions. Transcripts derived from multiple TSS encode a single protein, due to the absence of any ATG triplets within approximately 430 bp upstream of the major transcription start site. Thus, the PD1 core promoter is embedded within an "ATG desert". Remarkably, extending this analysis genome-wide, we find that ATG deserts define a novel promoter subclass. They occur nonrandomly, are significantly associated with non-TATAA promoters that use multiple TSS, independent of the presence of CpG islands (CGI). We speculate that ATG deserts may provide a core promoter platform upon which complex upstream regulatory signals can be integrated, targeting multiple TSS whose products encode a single protein.