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(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.

Figure 1

Western blot using a specific anti-HMGA1 polyclonal antibody demonstrating that HA7C cells over-express transgenic HA-tagged HMGA1 protein ∼39-fold above the level of endogenous protein found in parental, control MCF-7 cells. Lanes: (1) Recombinant human (rh) HMGA1 protein reference standard; (2) empty; (3) Extract of parental, non-transgenic control MCF-7 cells (12.5 ug of total protein isolated from ∼2 ×10⁶ cells); (4) Extract of induced, transgenic MCF-7 cells (12.5 ug of total protein isolated from ∼2 x10⁶ cells) over-expressing HA-tagged HMGA1 protein. The positions of the endogenous, non-transgenic HMGA1 proteins, plus the HA-tagged transgenic HMGA1 protein in the gels are indicated by the arrows at the right-hand side of the figure.

Figure 2

Fold change data for mtDNA levels and mitochondrial gene transcript NADH Dehydrogenase subunit 2 (ND2), cytochrome c oxidase subunit I (COXI), ATP Synthase 6 (ATP6), and NADH dehydrogenase subunit 6 (ND6) levels as determined by real-time PCR. Results demonstrate an approximately 1.5-2-fold down regulation in the levels of mitochondrial DNA in HA7C cells relative to MCF-7 cells. In contrast, there is an approximately 3-fold increase in the level of the analyzed mitochondrial gene transcripts when adjusted mathematically for the observed decrease in mtDNA in the HA7C cells. Fold change relative to an endogenous control was determined using the comparative Ct method (see Methods and Materials).

Figure 3

Mitochondrial mass within HA7C cells is reduced relative to MCF-7 cells. Histograms shows nonyl acridine orange (NAO) staining intensity on the logarithmic X axis and cell counts on the Y axis. A total of 10,000 events were analyzed by FACS analysis. Panel A: In the NAO(−) samples, autofluorescence was minimal. The shift in the HA7C peak to a lower staining intensity in the NAO(+) column represents an approximately 20% lower level of mitochondria in the majority of these HMGA1 over-expressing cells as compared to MCF-7 cells. Percent difference was calculated using median peak values as reported by Cell Quest Pro FACS analysis software. Panel B: Overlay histogram in which sample detection was reduced to center the peaks and provide a visual representation of the differences represented by the different median values.

Figure 4

Total cellular ATP levels were reduced in HA7C cells as compared to MCF-7 cells. Analysis of ATP levels using a luciferase based bioluminescence assay kit required accurate cell counts and serial dilutions for accurate readings. Thus, data is represented as cell counts (x-axis) determined by serial dilutions of original cell resuspensions versus luminescence (y-axis) which is proportional to cellular ATP levels. Data shows a consistently lower level of ATP in HA7C across multiple serial dilutions and repetitions.

Figure 5

Increased sensitivity of HA7C transgenic cells to treatment with the inhibitor of glycolysis, 2-Deoxyglucose (2-DG), as compared to wild-type MCF-7 cells. Relative fluorescence units represent Hoechst 33258 staining of 2-DG treated samples (1-4mM) relative to untreated samples. A value of 1 represents cell numbers equal to that of the non-treated samples. Decreasing values represent a decrease in the number of cells as compared to the cell type specific non-treated control. The greatest difference between the HA7C and MCF-7 cells lines exists at 3 mM 2-DG.

Figure 6

Transgenic HA7C cells display increased levels of reactive oxygen species (ROS) when compared to wild-type MCF-7 cells. Histograms show H₂DCFDA staining intensity on the logarithmic X axis and cell counts on the Y axis. A total of 10,000 events were analyzed by FACS analysis. Cells that were not stained (H₂DCFDA(−)) showed a lack of significant autofluorescence. The shift in the HA7C peak to a higher staining intensity in the H₂DCFDA(+) column represents an increased level of ROS production in the majority of these HMGA1 over-expressing cells. Treatment with H₂O₂ or menadione sodium bisulfite was used as a positive control and showed expected increased in ROS detection. Median peak values were those reported using Cell Quest Pro FACS analysis software.

Figure 7

Mitochondrial membrane potential is increased in HA7C cells as compared to wild-type MCF-7 cells. Histograms show DiOC6 staining intensity on the logarithmic X axis and cell counts on the Y axis. A total of 10,000 events were analyzed by FACS analysis. CCCP treatments showed reduced membrane potential as expected and verifying the efficacy of the staining method. Median peak values were reported by Cell Quest Pro FACS analysis software.

Figure 8

Postulated dual function for HMGA1 binding at a single D-loop site. As adapted from [23], the schematic displays several important regulatory features of the D-loop region of mtDNA including the heavy and light strand promoter start site (HSP and LSP, respectively) and the conserved sequence blocks (CSB's). HMGA1, as determined in living cells by ChIP analysis, associates with the D-loop DNA close to CSB I [11] which is an essential region for proper termination of LSP transcription, a process required for the production of RNA ‘primer’transcripts involved in the initiation of mtDNA replication [23, 24]. Due to the ability of HMGA1 to bend DNA upon binding [1], this close proximity of HMGA1 association to CSB I potentially inhibits termination of LSP transcription and thus, as a consequence, prevents replication initiation. This possibility is supported by the observations that LSP regulated transcription of the ND6 gene (coded for by mtDNA light strand) is up-regulated ∼3-fold in HA7C cells. Similarly, binding of HMGA1 to the same D-loop site, which is upstream of the HSP start site, could potentially also enhance transcription of the mtDNA heavy strand and explain the increase observed in ND2, COXI and ATP6 gene transcripts in over-expressing cells.