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Neuron-specific specificity protein 4 bigenomically regulates the transcription of all mitochondria- and nucleus-encoded cytochrome c oxidase subunit genes in neurons - PubMed

. 2013 Nov;127(4):496-508.

doi: 10.1111/jnc.12433. Epub 2013 Sep 30.

Affiliations

Neuron-specific specificity protein 4 bigenomically regulates the transcription of all mitochondria- and nucleus-encoded cytochrome c oxidase subunit genes in neurons

Kaid Johar et al. J Neurochem. 2013 Nov.

Abstract

Neurons are highly dependent on oxidative metabolism for their energy supply, and cytochrome c oxidase (COX) is a key energy-generating enzyme in the mitochondria. A unique feature of COX is that it is one of only four proteins in mammalian cells that are bigenomically regulated. Of its thirteen subunits, three are encoded in the mitochondrial genome and ten are nuclear-encoded on nine different chromosomes. The mechanism of regulating this multisubunit, bigenomic enzyme poses a distinct challenge. In recent years, we found that nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) mediate such bigenomic coordination. The latest candidate is the specificity factor (Sp) family of proteins. In N2a cells, we found that Sp1 regulates all 13 COX subunits. However, we discovered recently that in primary neurons, it is Sp4 and not Sp1 that regulates some of the key glutamatergic receptor subunit genes. The question naturally arises as to the role of Sp4 in regulating COX in primary neurons. The present study utilized multiple approaches, including chromatin immunoprecipitation, promoter mutational analysis, knockdown and over-expression of Sp4, as well as functional assays to document that Sp4 indeed functionally regulate all 13 subunits of COX as well as mitochondrial transcription factors A and B. The present study discovered that among the specificity family of transcription factors, it is the less known neuron-specific Sp4 that regulates the expression of all 13 subunits of mitochondrial cytochrome c oxidase (COX) enzyme in primary neurons. Sp4 also regulates the three mitochondrial transcription factors (TFAM, TFB1M, and TFB2M) and a COX assembly protein SURF-1 in primary neurons.

Keywords: Sp4; TTX; cytochrome c oxidase; depolarization; mitochondria; transcription factor.

© 2013 International Society for Neurochemistry.

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Figures

Fig. 1
Fig. 1

Mouse brain (visual cortex) and cultured primary neurons (from mouse visual cortex) express more Sp4 than N2a cells. A, Western blots of proteins obtained from mouse brain, cultured mouse primary neurons (PN), and N2a cells labeled with Sp1 or Sp4 antibodies. β actin served as the loading control. N = 3 for each group. B, Quantification of bands indicated that both brain and primary neurons have much more Sp4 than N2a cells. On the other hand, N2a cells have significantly more Sp1 than either brain or primary neurons. C, Western blots of Sp4 proteins from cultured primary neurons subjected to KCl or TTX. β actin was the loading control. N = 3 for each group. D, Quantification of bands indicated that Sp4 protein was up-regulated by KCl depolarizing stimulation and down-regulated by TTX impulse blockade as compared to controls. ***= P < 0.001.

Fig. 2
Fig. 2

Sp4 binds to the promoters of all 10 nucleus-encoded COX subunits as well as TFAM, TFB1M, and TFB2M in vivo. ChIP assays were done on mouse visual cortical tissue immunoprecipitated with anti-Sp4 antibodies (Sp4 lanes), anti-nerve growth factor receptor p75 antibodies (negative control, NGFR lanes), or no antibody (negative control, Blank). Input lanes indicate 0.5% of input chromatin without immunoprecipitation. NT3 promoter served as the positive control and β-actin (Actb) served as the negative control for Sp factors. Promoters of all 10 nucleus-encoded COX subunit, TFAM, TFB1M, and TFB2M were immunoprecipitated with anti-Sp4 antibodies and not with anti-NGRF or without exogenous antibodies.

Fig. 3
Fig. 3

Silencing of Sp4 in N2a cells (A–C) or in primary neurons (D) suppressed expression of all thirteen COX subunits. A, Down-regulation of Sp4, COX1, and COX4i1 protein levels in Sp4-silenced cells as compared to those transfected with scrambled vector controls. Lane 1, scrambled vectors; lane 2, Sp4 shRNA. β-actin (Actb) served as a loading control. N = 6 for each group. B, Quantification of protein levels shown in A. Solid bars, controls; open bars, Sp4 shRNA. C, Sp4 shRNA-transfected N2a cells had a significant down-regulation of transcript levels for Sp4, the mitochondrial-encoded COX subunits (COX1, COX2, and COX3), mitochondrial transcription factors (TFAM, TFB1M, and TFB2M), SURF1, and nucleus-encoded COX subunits (COX4i1 - COX8a) as compared to those transfected with scrambled vectors. N = 5 for each data point; ***= P < 0.001 as compared to scrambled vectors. D, Sp4 shRNA-transfected primary neurons also had a significant down-regulation of Sp4, mitochondrial encoded COX subunits (COX1, COX2, and COX3), mitochondrial transcription factors (TFAM, TFB1M, and TFB2M), SURF1, and nucleus-encoded subunits (COX4i1 - COX8a) compared with scrambled vector controls. Silencing of Sp4 induced a slight but significant increase in the expressions of Sp1. Silencing of Sp1 also induced a slight but significant increase in the transcripts of Sp1. However, Sp1 shRNA did not lead to any change in the expressions of all 13 COX subunit genes, nor those of TFAM, TFB1M, TFB2M, and SURF1 in primary neurons. N = 3 for each data point; *= P < 0.05; **= P < 0.01; ***= P < 0.001. X= Non-significant as compared to scrambled vector controls.

Fig. 4
Fig. 4

Over-expression of Sp4 in N2a cells (A – C) or in primary neurons (D) increased expression of all thirteen COX subunits. A, Up-regulation of Sp4, COX1, and COX4i1 protein levels in cells transfected with Sp4 expression vectors as compared to empty vector controls. Lane 1, empty vectors; Lane 2, Sp4 over-expression. N = 6 for each group. B, Quantification of protein levels shown in A. Solid bars, controls; open bars, Sp4 over-expression. C, Sp4 over-expression led to a significant up-regulation of Sp4, mitochondrial encoded COX subunits (COX1, COX2, and COX-3), mitochondrial transcription factors (TFAM, TFB1M, and TFB2M), SURF1, and nucleus-encoded COX subunits (COX4i1 - COX8a) as compared to empty vector controls. N = 6 for each group. All * P values were compared to empty vectors. ** P < 0.01 and *** P < 0.001 when compared to empty vectors. D, Sp4 over-expression in primary neurons also led to a significant up-regulation of Sp4, mitochondrial encoded COX subunits (COX1, COX2, and COX-3), mitochondrial transcription factors (TFAM, TFB1M, and TFB2M), SURF1, and all 10 nucleus-encoded subunits (COX4i1 - COX8a) compared to empty vector controls. N = 3 for each group. All * P values were compared to empty vectors. ** P < 0.01 and *** P < 0.001.

Fig. 5
Fig. 5

Sp4 and its target genes are all regulated by neuronal activity in N2a cells. A. KCl-induced depolarization significantly increased mRNA levels of Sp4, 3 mitochondrial-encoded COX subunits, 3 mitochondrial transcription factors, SURF1, and 10 nucleus-encoded COX subunits. In the presence of Sp4 shRNA, however, KCl was not able to up-regulate the levels of all transcripts. B. TTX-induced impulse blockade led to reduced levels of Sp4, 3 mitochondrial-encoded COX subunits, 3 mitochondrial transcription factors, SURF1, and 10 nucleus-encoded COX subunits. Over-expression of Sp4, however, rescued all 13 COX subunits and 3 mitochondrial transcription factors as well as SURF1 from TTX-induced suppression N = 6 for each data point; *= P < 0.05; **= P < 0.01; ***= P < 0.001 when compared to either scrambled (A) or empty vector (B) controls. ### = P < 0.001 when compared to control + KCl (A) or control + TTX (B). C. Optical densitometric analysis of reaction product of cytochrome c oxidase histochemistry in N2a cells. KCl induced a significant increase in the relative activity of COX as compared to scrambled vector controls. However, no up-regulation was observed in cells transfected with Sp4 shRNA. N for optical densitometric (OD) analysis = 122 cells for control, 125 for control + KCl, and 128 for Sp4 shRNA + KCl. *** = P < 0.001 when compared to controls. ### = P < 0.001 when compared to control + KCl. No significance was found between Sp4 shRNA + KCl and controls. D. Three days of TTX treatment led to significant reduction of COX relative activity. However, in N2a cells transfected with Sp4 expression vectors, the activity was increased rather than down-regulated. N for OD analysis = 115 cells for empty vector controls, 162 for empty vector + TTX, and 207 for Sp4 over-expression + TTX. **= P < 0.01; ***= P < 0.001 when compared to controls. ### = P < 0.001 when compared to control + KCl.

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