Browsing by Subject "Gene Regulation"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    In Vivo Analysis of Human LHX3 Gene Regulation
    (2011-02) Mullen, Rachel D.; Rhodes, Simon J.; Herring, B. Paul; Skalnik, David Gordon; Thurmond, Debbie C.; Walvoord, Emily C.
    LHX3 is a transcription factor important in pituitary and nervous system development. Patients with mutations in coding regions of the gene have combined pituitary hormone deficiency (CPHD) that causes growth, fertility, and metabolic problems. Promoter and intronic elements of LHX3 important for basal gene expression in vitro have been identified, but the key regulatory elements necessary for in vivo expression were unknown. With these studies, I sought to elucidate how LHX3 gene expression is regulated in vivo. Based on sequence conservation between species in non-coding regions, I identified a 7.9 kilobase (kb) region 3' of the human LHX3 gene as a potential regulatory element. In a beta galactosidase transgenic mouse model, this region directed spatial and temporal expression to the developing pituitary gland and spinal cord in a pattern consistent with endogenous LHX3 expression. Using a systematic series of deletions, I found that the conserved region contains multiple nervous system enhancers and a minimal 180 base pair (bp) enhancer that direct expression to both the pituitary and spinal cord in transgenic mice. Within this minimal enhancer, TAAT/ATTA sequences that are characteristic of homeodomain protein binding sites are required to direct expression. I performed DNA binding experiments and chromatin immunoprecipitation assays to reveal that the ISL1 and PITX1 proteins specifically recognize these elements in vitro and in vivo. Based on in vivo mutational analyses, two tandem ISL1 binding sites are required for enhancer activity in the pituitary and spine and a PITX1 binding site is required for spatial patterning of gene expression in the pituitary. Additional experiments demonstrated that these three elements cannot alone direct gene expression, suggesting a combination of factors is required for enhancer activity. This study reveals that the key regulatory elements guiding developmental regulation of the human LHX3 gene lie in this conserved downstream region. Further, this work implicates ISL1 as a new transcriptional regulator of LHX3 and describes a possible mechanism for the regulation of LHX3 by a known upstream factor, PITX1. Identification of important regulatory regions will also enable genetic screening in candidate CPHD patients and will thereby facilitate patient treatment and genetic counseling.
  • Thumbnail Image
    Item
    Internal Tandem Duplication in FLT3 Attenuates Proliferation and Regulates Resistance to the FLT3 Inhibitor AC220 by Modulating p21Cdkn1a and Pbx1 in Hematopoietic Cells
    (Public Library of Science (PLoS), 2016) Abe, Mariko; Pelus, Louis M.; Singh, Pratibha; Hirade, Tomohiro; Onishi, Chie; Purevsuren, Jamiyan; Taketani, Takeshi; Yamaguchi, Seiji; Fukuda, Seiji; Department of Microbiology and Immunology, IU School of Medicine
    Internal tandem duplication (ITD) mutations in the Fms-related tyrosine kinase 3 (FLT3) gene (FLT3-ITD) are associated with poor prognosis in patients with acute myeloid leukemia (AML). Due to the development of drug resistance, few FLT3-ITD inhibitors are effective against FLT3-ITD+ AML. In this study, we show that FLT3-ITD activates a novel pathway involving p21Cdkn1a (p21) and pre-B cell leukemia transcription factor 1 (Pbx1) that attenuates FLT3-ITD cell proliferation and is involved in the development of drug resistance. FLT3-ITD up-regulated p21 expression in both mouse bone marrow c-kit+-Sca-1+-Lin- (KSL) cells and Ba/F3 cells. The loss of p21 expression enhanced growth factor-independent proliferation and sensitivity to cytarabine as a consequence of concomitantly enriching the S+G2/M phase population and significantly increasing the expression of Pbx1, but not Evi-1, in FLT3-ITD+ cells. This enhanced cell proliferation following the loss of p21 was partially abrogated when Pbx1 expression was silenced in FLT3-ITD+ primary bone marrow colony-forming cells and Ba/F3 cells. When FLT3-ITD was antagonized with AC220, a selective inhibitor of FLT3-ITD, p21 expression was decreased coincident with Pbx1 mRNA up-regulation and a rapid decline in the number of viable FLT3-ITD+ Ba/F3 cells; however, the cells eventually became refractory to AC220. Overexpressing p21 in FLT3-ITD+ Ba/F3 cells delayed the emergence of cells that were refractory to AC220, whereas p21 silencing accelerated their development. These data indicate that FLT3-ITD is capable of inhibiting FLT3-ITD+ cell proliferation through the p21/Pbx1 axis and that treatments that antagonize FLT3-ITD contribute to the subsequent development of cells that are refractory to a FLT3-ITD inhibitor by disrupting p21 expression.
  • Thumbnail Image
    Item
    Mechanisms of Gene Regulation by Soy Peptide Lunasin in Innate Immune Cells
    (Office of the Vice Chancellor for Research, 2015-04-17) Casiano-Rivera, Félix M.; Tung, Chun-Yu; Chang, Hua-Chen
    Lunasin is a seed peptide containing 43 amino acids, originally isolated from soybeans. Recently, a novel function of lunasin was discovered, as it acts as an immune modulating agent regulating gene expression of various innate immune cells. Lunasin strongly activated the expression of genes encoding for type I interferon and inflammatory cytokines. Nonetheless, the molecular mechanisms of lunasin’s gene regulation are relatively unknown. Our current hypothesis states that lunasin is able to induce activation of various transcription factors, resulting in gene expression in immune cells. Human dendritic cells (DCs) or monocytes were purified from peripheral blood mononuclear cells (PBMCs) in order to determine the activation of transcription factors. Phosphorylation of STAT1 and NF-ĸB (p65) were evident in cells treated with lunasin using flow cytometry and Western blotting. The results will ultimately lead to the signaling pathways involved in gene expression regulated by lunasin in innate immune cells. By defining the signaling pathway of lunasin, we can have a better understanding of its application in immune modulation.
  • Thumbnail Image
    Item
    MicroRNA-339-5p down-regulates protein expression of β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) in human primary brain cultures and is reduced in brain tissue specimens of Alzheimer disease subjects
    (ASBMB, 2014-02-21) Long, Justin M.; Ray, Balmiki; Lahiri, Debomoy; Department of Medical & Molecular Genetics, IU School of Medicine
    Alzheimer disease (AD) results, in part, from the excess accumulation of the amyloid-β (Aβ) peptide as neuritic plaques in the brain. The short Aβ peptide is derived from the large transmembrane Aβ precursor protein (APP). The rate-limiting step in the production of Aβ from APP is mediated by the β-site APP-cleaving enzyme 1 (BACE1). Dysregulation of BACE1 levels leading to excess Aβ deposition is implicated in sporadic AD. Thus, elucidating the full complement of regulatory pathways that control BACE1 expression is key to identifying novel drug targets central to the Aβ-generating process. MicroRNAs (miRNAs) are expected to participate in this molecular network. Here, we identified a known miRNA, miR-339-5p, as a key contributor to this regulatory network. Two distinct miR-339-5p target sites were predicted in the BACE1 3'-UTR by in silico analyses. Co-transfection of miR-339-5p with a BACE1 3'-UTR reporter construct resulted in significant reduction in reporter expression. Mutation of both target sites eliminated this effect. Delivery of the miR-339-5p mimic also significantly inhibited expression of BACE1 protein in human glioblastoma cells and human primary brain cultures. Delivery of target protectors designed against the miR-339-5p BACE1 3'-UTR target sites in primary human brain cultures significantly elevated BACE1 expression. Finally, miR-339-5p levels were found to be significantly reduced in brain specimens isolated from AD patients as compared with age-matched controls. Therefore, miR-339-5p regulates BACE1 expression in human brain cells and is most likely dysregulated in at least a subset of AD patients making this miRNA a novel drug target.