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Title: Nuclear HMGA1 nonhistone chromatin proteins directly influence mitochondrial transcription, maintenance, and function

Abstract

We have previously demonstrated that HMGA1 proteins translocate from the nucleus to mitochondria and bind to mitochondrial DNA (mtDNA) at the D-loop control region [G.A. Dement, N.R. Treff, N.S. Magnuson, V. Franceschi, R. Reeves, Dynamic mitochondrial localization of nuclear transcription factor HMGA1, Exp. Cell Res. 307 (2005) 388-401.] [11]. To elucidate possible physiological roles for such binding, we employed methods to analyze mtDNA transcription, mitochondrial maintenance, and other organelle functions in transgenic human MCF-7 cells (HA7C) induced to over-express an HA-tagged HMGA1 protein and control (parental) MCF-7 cells. Quantitative real-time (RT) PCR analyses demonstrated that mtDNA levels were reduced approximately 2-fold in HMGA1 over-expressing HA7C cells and flow cytometric analyses further revealed that mitochondrial mass was significantly reduced in these cells. Cellular ATP levels were also reduced in HA7C cells and survival studies showed an increased sensitivity to killing by 2-deoxy-D-glucose, a glycolysis-specific inhibitor. Flow cytometric analyses revealed additional mitochondrial abnormalities in HA7C cells that are consistent with a cancerous phenotype: namely, increased reactive oxygen species (ROS) and increased mitochondrial membrane potential ({delta}{psi}{sub m}). Additional RT-PCR analyses demonstrated that gene transcripts from both the heavy (ND2, COXI, ATP6) and light (ND6) strands of mtDNA were up-regulated approximately 3-fold in HA7Cmore » cells. Together, these mitochondrial changes are consistent with many previous reports and reveal several possible mechanisms by which HMGA1 over-expression, a common feature of naturally occurring cancers, may affect tumor progression.« less

Authors:
 [1];  [1];  [2]
  1. School of Molecular Biosciences, Washington State University, Rm. 639, Fulmer Hall, Pullman, WA 99164-4660 (United States)
  2. School of Molecular Biosciences, Washington State University, Rm. 639, Fulmer Hall, Pullman, WA 99164-4660 (United States). E-mail: reevesr@mail.wsu.edu
Publication Date:
OSTI Identifier:
20858067
Resource Type:
Journal Article
Resource Relation:
Journal Name: Experimental Cell Research; Journal Volume: 313; Journal Issue: 1; Other Information: DOI: 10.1016/j.yexcr.2006.09.014; PII: S0014-4827(06)00399-5; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ATP; CHROMATIN; GLUCOSE; GLYCOLYSIS; MITOCHONDRIA; OXIDASES; OXYGEN; PHENOTYPE; PHOSPHORYLATION; POLYMERASE CHAIN REACTION; TRANSCRIPTION; TRANSCRIPTION FACTORS

Citation Formats

Dement, Gregory A., Maloney, Scott C., and Reeves, Raymond. Nuclear HMGA1 nonhistone chromatin proteins directly influence mitochondrial transcription, maintenance, and function. United States: N. p., 2007. Web. doi:10.1016/j.yexcr.2006.09.014.
Dement, Gregory A., Maloney, Scott C., & Reeves, Raymond. Nuclear HMGA1 nonhistone chromatin proteins directly influence mitochondrial transcription, maintenance, and function. United States. doi:10.1016/j.yexcr.2006.09.014.
Dement, Gregory A., Maloney, Scott C., and Reeves, Raymond. Mon . "Nuclear HMGA1 nonhistone chromatin proteins directly influence mitochondrial transcription, maintenance, and function". United States. doi:10.1016/j.yexcr.2006.09.014.
@article{osti_20858067,
title = {Nuclear HMGA1 nonhistone chromatin proteins directly influence mitochondrial transcription, maintenance, and function},
author = {Dement, Gregory A. and Maloney, Scott C. and Reeves, Raymond},
abstractNote = {We have previously demonstrated that HMGA1 proteins translocate from the nucleus to mitochondria and bind to mitochondrial DNA (mtDNA) at the D-loop control region [G.A. Dement, N.R. Treff, N.S. Magnuson, V. Franceschi, R. Reeves, Dynamic mitochondrial localization of nuclear transcription factor HMGA1, Exp. Cell Res. 307 (2005) 388-401.] [11]. To elucidate possible physiological roles for such binding, we employed methods to analyze mtDNA transcription, mitochondrial maintenance, and other organelle functions in transgenic human MCF-7 cells (HA7C) induced to over-express an HA-tagged HMGA1 protein and control (parental) MCF-7 cells. Quantitative real-time (RT) PCR analyses demonstrated that mtDNA levels were reduced approximately 2-fold in HMGA1 over-expressing HA7C cells and flow cytometric analyses further revealed that mitochondrial mass was significantly reduced in these cells. Cellular ATP levels were also reduced in HA7C cells and survival studies showed an increased sensitivity to killing by 2-deoxy-D-glucose, a glycolysis-specific inhibitor. Flow cytometric analyses revealed additional mitochondrial abnormalities in HA7C cells that are consistent with a cancerous phenotype: namely, increased reactive oxygen species (ROS) and increased mitochondrial membrane potential ({delta}{psi}{sub m}). Additional RT-PCR analyses demonstrated that gene transcripts from both the heavy (ND2, COXI, ATP6) and light (ND6) strands of mtDNA were up-regulated approximately 3-fold in HA7C cells. Together, these mitochondrial changes are consistent with many previous reports and reveal several possible mechanisms by which HMGA1 over-expression, a common feature of naturally occurring cancers, may affect tumor progression.},
doi = {10.1016/j.yexcr.2006.09.014},
journal = {Experimental Cell Research},
number = 1,
volume = 313,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • It has been well established that high mobility group A1 (HMGA1) proteins act within the nucleus of mammalian cells as architectural transcription factors that regulate the expression of numerous genes. Here, however, we report on the unexpected cytoplasmic/mitochondrial localization of the HMGA1 proteins within multiple cell types. Indirect immunofluorescence, electron microscopic immunolocalization, and Western blot studies revealed that, in addition to the nucleus, HMGA1 proteins could also be found in both the cytoplasm and mitochondria of randomly dividing populations of wild-type murine NIH3T3 cells and transgenic human MCF-7 breast cancer epithelial cells expressing a hemagglutinin tagged-HMGA1a fusion protein. While themore » molecular mechanisms underlying these novel subcellular localization patterns have not yet been determined, initial synchronization studies revealed a dynamic, cell cycle-dependent translocation of HMGA1 proteins from the nucleus into the cytoplasm and mitochondria of NIH3T3 cells. Furthermore, preliminary functionality studies utilizing a modified 'chromatin' immunoprecipitation protocol revealed that HMGA1 retains its DNA binding capabilities within the mitochondria and associates with the regulatory D-loop region in vivo. We discuss potential new biological roles for the classically nuclear HMGA1 proteins with regard to the observed nucleocytoplasmic translocation, mitochondrial internalization, and regulatory D-loop DNA binding.« less
  • Interaction of nonhistone proteins of chromatin (NPC) and proteins of karyolymph (PK) with DNA were studied by two methods: fixation of protein-/sup 14/C-DNA complexes on nitrocellulose filters and chromatography of /sup 32/P-labeled proteins on DNA-containing polyacrylamide-agarose columns. It was shown that irradiated DNA binds somewhat more NPC and PK and much more stably than native DNA. The effect is enhanced with increase in dosage, and it is related to appearance of denatured sites in the DNA. Irradiation of NPC does not have an appreciable effect on their capacity to bind DNA; however, there is impairment of specific interaction between partmore » of the NPC and homologous DNA. The possible role of radiation impairment of interaction between DNA and NPC and PK is discussed, with regard to change in structure and function of chromatin.« less
  • Initial results of an approach to the isolation of functionally active chromatin are described. Slight digestion of mouse myeloma nuclei at 0/sup 0/C with micrococcal nuclease, followed by dialysis against near-physiological saline solution containing 1 mM Mg/sup 2 +/, caused release of up to 17% of the nuclear DNA as soluble nucleoproteins. This soluble (S) fraction was relatively depleted in H1 histones and methylated DNA (5-methylcytosine) but highly enriched in RNA, single-stranded DNA, and nonhistone chromosomal proteins, particularly two species of the high mobility group identified as HMG 1 and HMG 2. The S fraction released most rapidly (6 tomore » 8% of the total DNA) consisted mainly of mono- and small oligonucleosomes. The mononucleosomes appeared normal in terms of sedimentation behavior, DNA length, and content of histones H2A, H2B, H3, and H4, but lacked H1, and instead were associated with approximately stoichiometric amounts of HMG 1 and HMG 2. Studies using isolated, fluorescence-labeled, total mouse HMG proteins indicated that added HMG 1 and MHG 2 do not bind strongly to S-fraction nucleoproteins but that two smaller HMG species (probably HMG 14 and HMG 17) do not bind preferentially to S-fraction mono- and dinucleosomes. These results argue against artifactual redistribution of HMG 1 and HMG 2 during this fractionation but suggest caution in interpreting the distribution of smaller HMG proteins after digestion of chromatin. The potential relaionship of this soluble fraction to transcriptionally active chromatin is discussed.« less
  • The metabolism of various classes of histones and nonhistone proteins in whole nuclei and liver chromatin of albino Wistar rats 1, 3, 12, and 24 months of age was studied. It was shown that in the course of postnatal ontogenesis, the metabolism of nonhistone proteins, extractable by a 0.14 M solution of NaCl, is increased in the animals. The incorporation of labeled precursors into the HMG 14 and HMG 17 proteins decreases with age of the animals; a higher level of specific radioactivity was established for the HMG 1+2 proteins in the 3- and 24-month old animals. The intensity ofmore » the metabolism of nonhistone proteins and histones is higher in the chromatin complex than in the whole nucleus at all stages of postnatal development of the animals. Among the histone proteins, H1 histones possess a higher level of specific radioactivity in animals of all age groups.« less
  • A subclass of nonhistone chromatin proteins from rat liver, previously shown to exhibit high affinity for DNA, has been fractionated by single-stranded DNA-agarose affinity chromatography. The protein fraction that bound to DNA-agarose in 0.19 M NaCl-buffer and was eluted with 2 M NaCl-buffer is enriched for a protein component of approximately 20,000 daltons and exhibits preferential binding to denatured DNA. This nonhistone protein fraction specific for single strands binds to DNA in a non-species-specific manner, and causes helix-coil transition of synthetic poly(d(A-T).d(A-T)) at 25/sup 0/, as indicated by the increase in absorbance of ultraviolet light at 260 nm. The observedmore » hyperchromicity does not result from any nuclease activity in the protein fraction, because addition of Mg/sup +2/ results in partial hypochromic shift, and the protein/DNA complex is retained by nitrocellulose filters.« less