Browsing by Subject "Promoter"

Now showing 1 - 4 of 4
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    Analysis of the cryptic promoter in the 5'-UTR of P27
    (2012-03-19) Francis, Zachary T.; Zhang, Jian-Ting; Safa, Ahmad R.; Hocevar, Barbara A.
    Cyclin Dependent Kinase regulation is often manipulated by cancer cells to promote unlimited proliferation. P27 is an important regulator of Cyclin E/CDK 2, which has been found in low amounts in many types of malignant cancers. Lovastatin has been shown to cause cell cycle arrest in the G1 phase of the cell cycle by increasing the P27 protein. There has been some question, however, if lovastatin regulates P27 at the transcriptional or translational level. Although it has been claimed that P27 expression regulation is due to an IRES located in its 5’UTR, other studies suggested that P27 expression is regulated at the level of transcription. To further investigate the regulation mechanism of P27 expression, the 5’-UTR of P27 and its deletion mutants were examined using a luciferase reporter gene in HeLa cells following exposure to lovastatin. It was found that lovastatin stimulated a significant 1.4 fold increase in its promoter activity of the full length 5’UTR (575). Deletion of 35 nucleotides from the 5’ end of the UTR eliminated the lovastatin-induced increase in promoter activity. Further mapping analyses of the first 35 bases showed that two regions, M1 (575-559) and M3 (543-527), were less sensitive to lovastatin than the other mutated constructs. Since M1 and M3 still showed some activity, a construct was created with deletions in both the M1 and M3 regions. This showed no increase in luciferase activity when exposed to lovastatin. Looking at RNA levels, there was a 1.5 fold increase in RNA when the full length 5’UTR was inserted into HeLa cells and exposed to 81 µM of lovastatin. In contrast, there was no increase in RNA when M1/M3 (575-559; 543-527) was inserted into HeLa cells and exposed to 81 µM of lovastatin. In addition, there was a 1.6 fold increase in endogenous P27 RNA levels after HeLa cells were exposed to 81 µM of lovastatin. In all of these experiments, there seems to be two promoters that work cooperatively: M1 (575-559) and M3 (543-527).
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    Global approaches for profiling transcription initiation
    (Elsevier, 2021) Policastro, Robert A.; Zentner, Gabriel E.; Medicine, School of Medicine
    Transcription start site (TSS) selection influences transcript stability and translation as well as protein sequence. Alternative TSS usage is pervasive in organismal development, is a major contributor to transcript isoform diversity in humans, and is frequently observed in human diseases including cancer. In this review, we discuss the breadth of techniques that have been used to globally profile TSSs and the resulting insights into gene regulation, as well as future prospects in this area of inquiry.
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    Structural and functional characterization of H2 haplotype MAPT promoter: unique neurospecific domains and a hypoxia-inducible element would enhance rationally targeted tauopathy research for Alzheimer's disease
    (Elsevier, 2012) Maloney, Bryan; Lahiri, Debomoy K.; Psychiatry, School of Medicine
    Alzheimer's disease (AD) is the leading cause of dementia in the elderly. Extraneuronal plaque comprising mostly the amyloid β peptide and intraneuronal tangles of hyperphosphorylated microtubule-associated τ protein (τ, gene MAPT) are typical of AD. Misfolded τ is also implicated in Parkinson's disease and frontotemporal dementia. We aim to understand the regulation of the human MAPT promoter by mapping its functional domains. We subcloned a 4868 base pair (bp) fragment from human BAC RPCI-11 100C5. Sequence analysis revealed an H2 haplotype MAPT promoter, 5'-UTR, and intronal fragment. Database analysis of the fragment showed 50%-75% homology with mouse and >90% with rhesus monkey. Comparison with human H1 sequences revealed differences that crossed predicted transcription factor sites. DNA-protein interaction studies by electrophoretic mobility shift assay suggested hypoxia response and an active specificity protein 1 (SP1) site in the 5'-untranslated region. Transfection of a series of MAPT promoter deletions revealed unique functional domains. The distal-most had different activities in neuronal vs. non-neuronal cells. We have cloned, sequenced, and functionally characterized a 4868bp fragment of the human MAPT 5'-flanking region, including the core promoter region (-302/+4), neurospecific domains (-4364/-1992 and +293/+504, relative to +1 TSS), and a hypoxia-inducible element (+60/+84). Our work extended functional analysis of the MAPT sequence further upstream, and explores cell-type specificity of MAPT promoter activity. Finally, we provided direct comparison of likely transcription factor binding sites, which are useful to understand differences between H1/H2 pathogenic associations.
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    The common murine retroviral integration site activating Hhex marks a distal regulatory enhancer co-opted in human early T-cell precursor leukemia
    (American Society for Biochemistry and Molecular Biology, 2025) Hardwick, Joyce; Rodriguez-Hernaez, Javier; Gambi, Giovanni; Venters, Bryan J.; Guo, Yan; Li, Liqi; Love, Paul E.; Copeland, Neal G.; Jenkins, Nancy A.; Papaioannou, Dimitrios; Aifantis, Iannis; Tsirigos, Aristotelis; Ivan, Mircea; Davé, Utpal P.; Medicine, School of Medicine
    The Hhex gene encodes a transcription factor that is important for both embryonic and post-natal development, especially of hematopoietic tissues. Hhex is one of the most common sites of retroviral integration in mouse models. We found the most common integrations in AKXD (recombinant inbred strains) T-ALLs occur 57-61kb 3′ of Hhex and activate Hhex gene expression. The genomic region of murine leukemia virus (MLV) integrations has features of a developmental stage-specific cis regulatory element (CRE), as evidenced by ATAC-seq in murine progenitor cells and high H3K27 acetylation at the syntenic CRE in human hematopoietic cell lines. With ChIP-exonuclease, we describe occupancy of LIM domain binding protein 1 (LDB1), the constitutive partner of the LIM Only-2 (LMO2), GATA1, and TAL1 transcription factors at GATA sites and at a composite GATA-E box within the CRE. With virtual 4C analysis, we observed looping between this +65kb CRE and the proximal intron one enhancer of HHEX in primary human ETP-ALLs and in normal progenitor cells. Our results show that retroviral integrations at intergenic sites can mark and take advantage of CREs. Specifically, in the case of HHEX activation, this newly described +65kb CRE is co-opted in the pathogenesis of ETP-ALL by the LMO2/LDB1 complex.