Hypothesis Genomic Lens on Neuroglobin Transcription
Santina Cutrupi1,2 Giulio Ferrero1,3 Stefania Reineri4 Francesca Cordero1,3 Michele De Bortoli1,2*
1
Center for Molecular Systems Biology, University of Turin, Orbassano, Turin, Italy 2 Department of Clinical and Biological Sciences, University of Turin, Orbassano, Turin, Italy 3 Department of Computer Science, University of Turin, Turin, Italy 4 Bioindustry Park Silvano Fumero, Colleretto Giacosa, Turin, Italy
Abstract Neuroglobin is a brain globin with neuroprotective effects against ischemia and related pathological processes, acting as O2 sensor and antiapoptotic pathway transducer. Here, we survey data on neuroanatomical coexpression of transcription factors, epigenetic signature and predictive transcription factor
binding sites at the neuroglobin gene locus to find hints of pathways to neuroglobin transcriptional regulation. These data provide a glimpse of how neuroglobin expression may translate into neuronal diversity and function, as well as disease. C 2014 IUBMB Life, 66(1):46–51, 2014. V
Keywords: neuroglobin; transcriptional regulation; genomics
Neuroglobin (NGB) is the first vertebrate nerve globin identified in the nervous tissues of mice and humans (1). It is induced by hypoxia and oxidative stress and leads to neuroprotection, but the molecular mechanisms of induction and protection are poorly understood. NGB overexpression is protective in animal models of several diseases such as stroke, Alzheimer’s disease, tumor progression and glaucoma (2–4). There are two main hypotheses on the potential mechanisms of neuroprotection by NGB: reactive oxigen species (ROS)scavenging in mitochondria metabolism and interference with apoptosis signaling. NGB binds several ligands, including O2, CO and NO; the ligand-linked conformational changes provide a surface for formation of protein complexes that may account for the involvement of NGB in a protective signaling mechanism. NGB interacts with Rho GTPase family members and in association with cytochrome c, it prevents its release in the cytosol resulting in neuroprotection against death induced by injuring stimuli (5). Therefore, understanding how NGB is
C 2014 International Union of Biochemistry and Molecular Biology V
Volume 66, Number 1, January 2014, Pages 46–51 *Address correspondence to: Michele De Bortoli, Center for Molecular Systems Biology, University of Turin, Ospedale San Luigi 10043, Orbassano, Turin, Italy. Tel.: 1390125561427; E-mail: [email protected] Received 12 October 2013; Accepted 24 November 2013 DOI 10.1002/iub.1235 Published online 6 January 2014 in Wiley Online Library (wileyonlinelibrary.com)
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regulated and which factors orchestrate its transcriptional activation is pivotal to underpinning its cell function and developing pharmacological approaches to its control. Systems biology approaches, integrating gene expression profiling and anatomical maps with genomic data of transcription factor binding sites (TFBSs) and chromatin epigenetic modifications, provide correlative networks extremely powerful to support hypothesis driven studies on specific proteins in the brain (6). To apply this concept to NGB regulation, in the first part of this article we go through available data on NGB expression in specific brain regions and its coexpression with TFs. Next, we analyze epigenetic marks and predicted consensus TF binding motifs at the NGB genomic locus. We used the Allen Human Brain Atlas (AHBA) (http:// www.brain-map.org), a comprehensive map of transcripts with anatomically complete coverage of the entire adult brain (7,8). NGB expression is observed in focal regions of the brain (9), and the pattern is similar in rat and human brain, as reported in Fig. 1A. NGB is highly expressed in the hypothalamus, in particular in the anterior hypothalamic area and in the lateral hypothalamic area (mammillary region), in the paraventricular nucleus and in the arcuate nucleus, in the dorsomedial hypothalamic nucleus and in the preoptic area. In addition, NGB is expressed in the laterodorsal and pontine tegmental nucleus and in the anterior basomedial and posterodorsal medial amygdaloid nucleus. It appears that, in basal conditions, NGB is expressed in metabolically active, oxygenconsuming cell types, such as neurons of the hypothalamus and retinal rod cells. The level of NGB is 100-fold higher in the
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Fig 1
(A): NGB expression in human brain by AHBA. From left to right, magnetic resonance (MR) images in axial section, coronal section and 3D reconstruction are reported. The NGB expression data are shown by red and green dots indicating upregulation and downregulation, respectively. MNI coordinates at cross-hairs are indicated. The heat map reported in the bottom part of (A) shows NGB expression in dissected substructures. (B): Heat map showing coexpression of TFs and NGB in specific neuroanatomic regions. The rows legend is indicated on the left part of the panel. The TFs are selected on the basis of NGB expression (Z-Score > 1). Columns represent TFs ordered left to right by decreasing Pearson coefficient computed respect NGB expression. (C): Heat map representing Pearson coefficient of the most correlated/anti-correlated TFs (absolute coefficient > 0.4) with NGB expression considering the data provided by both NGB probes. Expression levels are reported as Z-scores.
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retina than in the brain, where it may help to control the high metabolic demands required for vision and protection against oxidative stress derived from external exposure. In retinal cells, the prevalent subcellular localization of NGB is in mitochondria, in agreement with the reported role of NGB in mitochondrial metabolism and integrity (10–13). To identify TFs potentially involved in NGB regulation, we explored coexpression profiles in neuroanatomically defined regions. We analyzed the two available experimental datasets provided by the AHBA and we integrated them, obtaining a Pearson coefficient between their expression values of 0.8995, suggesting highly reproducible results. In the first step, we selected a number of specific NGB expressing human brain areas and then we searched for TFs coexpression. The correlation data between NGB and TFs expression are graphically represented as heat maps (Fig. 1B). Considering the subregional location, we found high correlation of NGB expression with estrogen receptor alpha (ESR1) in ventromedial hypothalamic nucleus, supramammillary nucleus, emboliform nucleus, globus pallidus and lateral nucleus. In supramammillary nucleus, paraventricular nucleus of hypothalamus and supraoptic neurons, SIM1 is coexpressed with NGB, consistent with the expression pattern in the mouse (14). SIM1 is involved in food intake and energy expenditure (15) in accordance with a possible role of NGB in maintaining energy homeostasis in neurons. In the neurogenic subventricular zone in adult rat brain SOX-3 and SOX-4, which are involved in the maintenance of progenitor neuronal cells, are coexpressed with NGB, coherent with a role of NGB in neurogenesis, migration and differentiation (16,17). Correlative evidence presented in Fig. 1 finds only limited support from the experimental data available on NGB transcriptional regulation, which is restricted to the promoter region (2549 to 16). This region is well conserved among human, mouse and rat and is characterized by the presence of binding sites for several TFs such as members of the Nuclear Factor NF-jB (NFjB) family (p65, p50, cRel), Egr1, Sp1 and CREB, which regulate basal NGB expression via specific interactions with the mouse promoter. NFkB, Sp1 and HypoxiaInducible Factor 1 (HIF-1) are required for NGB overexpression in hypoxic conditions. However, there is no consensus sequence for HIF-1 in NGB promoter, and it has been suggested that HIF-1 forms a complex with NFkB (18–21). To investigate functional genomic elements that can contribute to NGB transcriptional regulation, we explored the ENCODE Project database (http://genome.ucsc.edu/ENCODE/). We focused on a 6-kbp region containing the gene locus of NGB (Chromosome 14: 77,731,826–77,737,655). Genomic and epigenetic features of NGB gene locus are available in the ENCODE database, generated from experiments in astrocytes, glial and neuroblastoma cell lines. These include SK-N-SH and SH-SY5Y, SK-N-MC, SK-N-SH_RA (retinoic acid [RA]-differentiated neuroblastoma), NH-A, HA-sp, HAc astrocytes (Fig. 2). To better understand the regulation of NGB, we analyzed DNasehypersensitive sites in the NGB locus, corresponding to regions
48
accessible to transcriptional regulatory complexes, commonly defined as open chromatin state. In ENCODE Open Chromatin by DNase-Duke University and Open Chromatin by DNaseWashington University, a very evident peak is visible in the first NGB intron, which is present in diverse cell types, as shown in Fig. 2. To understand the function of these open chromatin regions, we explored histone modifications known to be associated with transcriptionally active or regulatory regions, such as H3K4me3/2, H3K36me3, H3K27ac or to silenced, heterochromatic regions, such as H3K27me3 and H3K9me3. There are only few studies on histone modifications in neuronal cell lines and astrocytes (ENCODE Histone Modifications ChIP-SeqUniversity of Washington). A weak H3K27me3 signal is present only in SK-N-SH cells treated with RA, suggesting that this DNase-hypersensitive site is in poised status and these regulatory regions are used in response to specific stimuli. In RNASeq assay, a high signal is present in the first intron region in undifferentiated cells, which decreases upon RA-induced differentiation. These transcripts are most likely noncoding eRNAs (RNA transcribed from enhancers). Altogether, these data indicate that the NGB locus is silenced in RA-differentiated cells. In accordance with the fact that NGB expression is very low in all cell lines studied, no active H3K4me3 histone mark is observed on the NGB promoter. In correspondence with the area of the first intron, positive to DNAse analysis, the binding of several TFs was observed by Chromatin Immunoprecipitation followed by high-troughtput sequencing (ChIP-Seq) in these cell lines. In addition, this region displays a positive signal of H3K27 acetylation and p300 coactivator. All together, these data suggest that NGB first intron contains an enhancer in a preprogrammed poised status. It should be stressed, however, that enhancer-bound transcriptional complexes can regulate genes in a distanceindependent fashion, even jumping over intervening genes (22). Taking into consideration this evidence, we can assume that this enhancer may either regulate NGB promoter activity or form long-range interactions with other regions, in basal normal culture conditions of the cell types in investigation. Interestingly, next to this enhancer there are two Single-Nucleotide Polymorphisms (SNPs) (rs10133981 and rs7149300) associated with Alzheimer’s disease, suggesting that this genomic region plays a central role in the NGB regulation in pathological conditions. A single base change within a TFs binding sequence can impair TF association and therefore inhibit biological functions, as described in the case of allelespecific activity (23). Predictive analysis of the consensus sequences that can be recognized by various TFs in NGB promoter was already reported and some TFs have been experimentally validated by chromatin immunoprecipitation, for example, NFkB and Sp1 (20,24). Figure 3 shows the results we obtained using matrixscan algorithms (Jaspar CORE 2009, NHRScan) reported in top and bottom panels, respectively. We discovered different TFBSs in the regions indicated as A (promoter) and B (putative enhancer). In the NGB promoter region, we found putative
Genomic Lens On Neuroglobin Transcription
Fig 2
Integration at NGB locus of ENCODE high-throughput experiments performed in astrocytes and neuronal cell lines. Respect to NGB locus at top, black horizontal bars A and B refer to gene promoter and candidate enhancer regions, respectively. From top to bottom, each row reports the genomic coverage obtained by RNA-Seq, DNAse-Seq and ChIP-Seq against different histone modifications and TFs. Letters on the right column indicate the cell line analyzed, row by row. Sample information is reported at bottom. The image was generated using WashU Epigenome Browser (http://epigenomegateway.wustl.edu).
response elements for Peroxisome Proliferator-Activated Receptor c (PPARc) and retinoid X receptor (RXR), Zinc Finger Protein, X-linked (ZFX), Nuclear Transcription Factor Y alpha (NFYA), Transcriptional enhancer domain 1 (TEAD1), which was also detected in the AHBA analysis (Fig. 1C) as the TF showing the highest correlation with NGB. PPARc and RXR are involved,
Cutrupi et al.
together with NGB, in Alzheimer’s disease experimental models, and therefore they may act as NGB modulators (3). Recent evidence indicates that NGB expression is induced by estradiol in neuroblastoma cells and primary astrocytes (25,26), building up a possible mechanistic link between the known neuroprotective effect of estrogen and NGB action.
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Fig 3
Predictive analysis of TFBSs at NGB promoter (A) and the candidate intronic enhancer (B). Top panels report the most enriched predictions obtained using Jaspar website (matrix score > 10). Bottom panels report nuclear receptor BSs predicted by NHRScan with their enrichment expressed as Viterbi probability. ER6 and ER8: everted repeat with 6-length and 8-length bp spacing between repeats, respectively; IR0: inverted repeat with no spacing between repeats.
Indeed, a significant correlation between NGB and ERa expression in the hypothalamic regions exists, indicating a possible direct regulation of NGB transcription by ERa. Both NHRScan, which finds putative BSs for nuclear receptors based on the presence of direct, inverted or everted repeats, and other algorithms confirm the presence of partially conserved nuclear receptor response elements in both the promoter and putative enhancer regions. However, binding of ERs in these regions awaits direct demonstration. It should be stressed that both ERa and ERb may regulate NGB transcription not only directly, but also indirectly, by interacting with other TFs bound to their DNA elements, for example, Sp1, as demonstrated previously (27). In this article, we provide a hypothesis on NGB regulation, by integrating published experimental data with bioinformatic analysis. In physiological conditions, NGB is highly expressed in the hypothalamus, which is a key integrative center in the brain, consisting of many different cell types required for a variety of functions including homeostasis, reproduction, stress response, social and cognitive behavior. During neurogenesis, the fine tuning of NGB may contribute to support high cellular
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growth. In the retina, NGB localizes in optic nerve glial cells for ensuring visual information transmission to the brain and protects against oxidative stress induced by direct exposure to the extracellular milieu. Under basal condition, NGB may act as modulator of cell energy balance, whereas it will regulate mitochondrial activity under stress conditions. The molecular mechanisms leading to neuronal differentiation and specialization in the different hypothalamic areas are poorly understood. The convergent expression of NGB and specific TFs in defined neuroanatomic regions may help identifying molecular mechanisms linked to specific biological functions. In pathological conditions, NGB protects cells against oxidative stress. It is well demonstrated that NGB has a neuroprotective role in hypoxia condition, such as stroke and Alzheimer’s disease. NGB is expressed at higher levels in neurons than astrocytes and glial cells in hypoxia condition-adapted mammals (28), suggesting that cell type-specific NGB may increase upon low O2 concentration in restricted locations. We focused our analysis on basal coexpression of NGB with TFs in specific brain regions. We observed that TFs potentially involved in NGB regulation, such as ESR1, SIM1,
Genomic Lens On Neuroglobin Transcription
SOX-3, SOX-4 and PPARc, are coherent with NGB role in cell metabolism and neuroprotection. The integration of genomic, epigenetic and experimental data represents a step forward to understand the mechanisms of NGB transcriptional regulation, which rely on complex networks of regulatory proteins acting in concert to provide cell-specific functions.
Acknowledgements The authors thank Silvia De Marchis and Stefano Gotti, NICO Neuroscience Institute Cavalieri Ottolenghi for discussions. This work was supported by grants from Italian MURST (PRIN Grant20109MXHMR_005) to M.D.B.
References [1] Burmester, T., Weich, B., Reinhardt, S., and Hankeln, T. (2000) A vertebrate globin expressed in the brain. Nature 407, 520–523. [2] Yu, Z., Liu, N., Liu, J., Yang, K., and Wang, X. (2012) Neuroglobin, a novel target for endogenous neuroprotection against stroke and neurodegenerative disorders. Int. J. Mol. Sci. 13, 6995–7014. [3] Cramer, P. E., Cirrito, J. R., Wesson, D. W., Lee, C. Y., Karlo, J. C., et al. (2012) ApoE-directed therapeutics rapidly clear b-amyloid and reverse deficits in AD mouse models. Science 335, 1503–1506. [4] Emara, M., Turner, A. R., and Allalunis-Turner, J. (2010) Hypoxic regulation of cytoglobin and neuroglobin expression in human normal and tumor tissues. Cancer Cell Int. 10, 33–49. [5] Fiocchetti, M., De Marinis, E., Ascenzi, P., and Marino, M. (2013). Neuroglobin and neuronal cell survival. Biochim. Biophys. Acta 1834, 1744–1749. [6] Geschwind, D. H. and Konopka, G. (2009) Neuroscience in the era of functional genomics and systems biology. Nature 461, 908–915. [7] Hundahl, C. A., Allen, G. C., Nyengaard, J. R., Dewilde, S., Carter, B. D., et al. (2008) Neuroglobin in the rat brain: localization. Neuroendocrinology 88, 173–182. [8] Hawrylycz, M. J., Lein, E. S., Guillozet-Bongaarts, A. L., Shen, E. H., Ng, L., et al. (2012) An anatomically comprehensive atlas of the adult human brain transcriptome. Nature 489, 391–399. [9] Mammen, P. P., Shelton, J. M., Goetsch, S. C., Williams, S. C., Richardson, J. A., et al. (2002) Neuroglobin, a novel member of the globin family, is expressed in focal regions of the brain. J. Histochem. Cytochem. 50, 1591– 1598. [10] Lechauve, C., Augustin, S., Roussel, D., Sahel, J. A., and Corral-Debrinski, M. (2013) Neuroglobin involvement in visual pathways through the optic nerve. Biochim. Biophys. Acta 1834, 1772–1778. [11] Hundahl, C. A., Allen, G. C., Hannibal, J., Kjaer, K., Rehfeld, J. F., et al. (2010) Anatomical characterization of cytoglobin and neuroglobin mRNA and protein expression in the mouse brain. Brain Res.1331, 58–73.
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[12] Fago, A., Mathews, A. J., and Brittain, T. (2008) A role for neuroglobin: resetting the trigger level for apoptosis in neuronal and retinal cells. IUBMB Life 60, 398–401. [13] Schmidt, M., Giessl, A., Laufs, T., Hankeln, T., Wolfrum, U., et al. (2003) How does the eye breathe? Evidence for neuroglobin-mediated oxygen supply in the mammalian retina. J. Biol. Chem. 278, 1932–1935. [14] Xu, C. and Fan, C. M. (2007) Allocation of paraventricular and supraoptic neurons requires Sim1 function: a role for a Sim1 downstream gene fPlexinC1. Mol. Endocrinol. 2, 1234–1245. [15] Xi, D., Gandhi, N., Lai, M., and Kublaoui, B. M. (2012) Ablation of Sim1 neurons causes obesity through hyperphagia and reduced energy expenditure. PLoS One. 7, e36453. [16] Haines, B., Mao, X., Xie, L., Spusta, S., Zeng, X., et al. (2013) Neuroglobin expression in neurogenesis. Neurosci. Lett. 549, 3–6. [17] Chew, L. J., and Gallo, V. (2009) The Yin and Yang of Sox proteins: activation and repression in development and disease. J. Neurosci. Res. 87, 3277– 3287. [18] Zhang, W., Tian, Z., Sha, S., Yuk, L., Cheng, L., et al. (2011) Functional and sequence analysis of human neuroglobin gene promoter region. Biochim. Biophys. Acta 1809, 236–244. [19] Haines, B., Demaria, M., Mao, X., Xie, L., Campisi, J., et al. (2012) Hypoxia-inducible factor-1 and neuroglobin expression. Neurosci. Lett. 514, 137–140. €rnlund-Wolf, A., et al. (2012) Transcrip[20] Liu, N., Yu, Z., Xiang, S., Zhao, S., Tja tional regulation mechanisms of hypoxia-induced neuroglobin gene expression. Biochem. J. 443, 153–164. [21] Liu, N., Yu, Z., Li, Y., Yuan, J., Zhang, J., et al. (2013) Transcriptional regulation of mouse neuroglobin gene by cyclic AMP responsive element binding protein (CREB) in N2a cells. Neurosci. Lett. 534, 333–337. [22] Calo, E. and Wysocka, J. (2013) Modification of enhancer chromatin: what, how, and why? Mol. Cell. 49, 825–837. [23] Gerstein, M. B., Kundaje, A., Hariharan, M., Landt, S. G., Yan, K. K., et al. (2012) Architecture of the human regulatory network derived from ENCODE data. Nature 489, 91–100. € ge, J., Pande, A., Englander, E. W., and Makałowski, W. (2012) [24] Dro Comparative genomics of neuroglobin reveals its early origins. PLoS One 7, e47972. [25] De Marinis, E., Ascenzi, P., Pellegrini, M., Galluzzo, P., Bulzomi, P., et al. (2010) 17b-Estradiol-a new modulator of neuroglobin levels in neurons: role in neuroprotection against H2O2-induced toxicity. Neurosignals 18, 223–235. [26] De Marinis, E., Acaz-Fonseca, E., Arevalo, M. A., Ascenzi, P., Fiocchetti, M., et al. (2013) 17b-oestradiol anti-inflammatory effects in primary astrocytes require oestrogen receptor b-mediated neuroglobin up-regulation. J. Neuroendocrinol. 25, 260–270. [27] Safe, S. (2001) Transcriptional activation of genes by 17 beta-estradiol through estrogen receptor-Sp1 interactions. Vitam. Horm. 62, 231–252. [28] Avivi, A., Gerlach, F., Joel, A., Reuss, S., Burmester, T., et al. (2010) Neuroglobin, cytoglobin, and myoglobin contribute to hypoxia adaptation of the subterranean mole rat Spalax. Proc. Natl. Acad. Sci. USA 107, 21570–21575.
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Santina Cutrupi1,2 Giulio Ferrero1,3 Stefania Reineri4 Francesca Cordero1,3 Michele De Bortoli1,2*
1
Center for Molecular Systems Biology, University of Turin, Orbassano, Turin, Italy 2 Department of Clinical and Biological Sciences, University of Turin, Orbassano, Turin, Italy 3 Department of Computer Science, University of Turin, Turin, Italy 4 Bioindustry Park Silvano Fumero, Colleretto Giacosa, Turin, Italy
Abstract Neuroglobin is a brain globin with neuroprotective effects against ischemia and related pathological processes, acting as O2 sensor and antiapoptotic pathway transducer. Here, we survey data on neuroanatomical coexpression of transcription factors, epigenetic signature and predictive transcription factor
binding sites at the neuroglobin gene locus to find hints of pathways to neuroglobin transcriptional regulation. These data provide a glimpse of how neuroglobin expression may translate into neuronal diversity and function, as well as disease. C 2014 IUBMB Life, 66(1):46–51, 2014. V
Keywords: neuroglobin; transcriptional regulation; genomics
Neuroglobin (NGB) is the first vertebrate nerve globin identified in the nervous tissues of mice and humans (1). It is induced by hypoxia and oxidative stress and leads to neuroprotection, but the molecular mechanisms of induction and protection are poorly understood. NGB overexpression is protective in animal models of several diseases such as stroke, Alzheimer’s disease, tumor progression and glaucoma (2–4). There are two main hypotheses on the potential mechanisms of neuroprotection by NGB: reactive oxigen species (ROS)scavenging in mitochondria metabolism and interference with apoptosis signaling. NGB binds several ligands, including O2, CO and NO; the ligand-linked conformational changes provide a surface for formation of protein complexes that may account for the involvement of NGB in a protective signaling mechanism. NGB interacts with Rho GTPase family members and in association with cytochrome c, it prevents its release in the cytosol resulting in neuroprotection against death induced by injuring stimuli (5). Therefore, understanding how NGB is
C 2014 International Union of Biochemistry and Molecular Biology V
Volume 66, Number 1, January 2014, Pages 46–51 *Address correspondence to: Michele De Bortoli, Center for Molecular Systems Biology, University of Turin, Ospedale San Luigi 10043, Orbassano, Turin, Italy. Tel.: 1390125561427; E-mail: [email protected] Received 12 October 2013; Accepted 24 November 2013 DOI 10.1002/iub.1235 Published online 6 January 2014 in Wiley Online Library (wileyonlinelibrary.com)
46
regulated and which factors orchestrate its transcriptional activation is pivotal to underpinning its cell function and developing pharmacological approaches to its control. Systems biology approaches, integrating gene expression profiling and anatomical maps with genomic data of transcription factor binding sites (TFBSs) and chromatin epigenetic modifications, provide correlative networks extremely powerful to support hypothesis driven studies on specific proteins in the brain (6). To apply this concept to NGB regulation, in the first part of this article we go through available data on NGB expression in specific brain regions and its coexpression with TFs. Next, we analyze epigenetic marks and predicted consensus TF binding motifs at the NGB genomic locus. We used the Allen Human Brain Atlas (AHBA) (http:// www.brain-map.org), a comprehensive map of transcripts with anatomically complete coverage of the entire adult brain (7,8). NGB expression is observed in focal regions of the brain (9), and the pattern is similar in rat and human brain, as reported in Fig. 1A. NGB is highly expressed in the hypothalamus, in particular in the anterior hypothalamic area and in the lateral hypothalamic area (mammillary region), in the paraventricular nucleus and in the arcuate nucleus, in the dorsomedial hypothalamic nucleus and in the preoptic area. In addition, NGB is expressed in the laterodorsal and pontine tegmental nucleus and in the anterior basomedial and posterodorsal medial amygdaloid nucleus. It appears that, in basal conditions, NGB is expressed in metabolically active, oxygenconsuming cell types, such as neurons of the hypothalamus and retinal rod cells. The level of NGB is 100-fold higher in the
IUBMB Life
Fig 1
(A): NGB expression in human brain by AHBA. From left to right, magnetic resonance (MR) images in axial section, coronal section and 3D reconstruction are reported. The NGB expression data are shown by red and green dots indicating upregulation and downregulation, respectively. MNI coordinates at cross-hairs are indicated. The heat map reported in the bottom part of (A) shows NGB expression in dissected substructures. (B): Heat map showing coexpression of TFs and NGB in specific neuroanatomic regions. The rows legend is indicated on the left part of the panel. The TFs are selected on the basis of NGB expression (Z-Score > 1). Columns represent TFs ordered left to right by decreasing Pearson coefficient computed respect NGB expression. (C): Heat map representing Pearson coefficient of the most correlated/anti-correlated TFs (absolute coefficient > 0.4) with NGB expression considering the data provided by both NGB probes. Expression levels are reported as Z-scores.
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retina than in the brain, where it may help to control the high metabolic demands required for vision and protection against oxidative stress derived from external exposure. In retinal cells, the prevalent subcellular localization of NGB is in mitochondria, in agreement with the reported role of NGB in mitochondrial metabolism and integrity (10–13). To identify TFs potentially involved in NGB regulation, we explored coexpression profiles in neuroanatomically defined regions. We analyzed the two available experimental datasets provided by the AHBA and we integrated them, obtaining a Pearson coefficient between their expression values of 0.8995, suggesting highly reproducible results. In the first step, we selected a number of specific NGB expressing human brain areas and then we searched for TFs coexpression. The correlation data between NGB and TFs expression are graphically represented as heat maps (Fig. 1B). Considering the subregional location, we found high correlation of NGB expression with estrogen receptor alpha (ESR1) in ventromedial hypothalamic nucleus, supramammillary nucleus, emboliform nucleus, globus pallidus and lateral nucleus. In supramammillary nucleus, paraventricular nucleus of hypothalamus and supraoptic neurons, SIM1 is coexpressed with NGB, consistent with the expression pattern in the mouse (14). SIM1 is involved in food intake and energy expenditure (15) in accordance with a possible role of NGB in maintaining energy homeostasis in neurons. In the neurogenic subventricular zone in adult rat brain SOX-3 and SOX-4, which are involved in the maintenance of progenitor neuronal cells, are coexpressed with NGB, coherent with a role of NGB in neurogenesis, migration and differentiation (16,17). Correlative evidence presented in Fig. 1 finds only limited support from the experimental data available on NGB transcriptional regulation, which is restricted to the promoter region (2549 to 16). This region is well conserved among human, mouse and rat and is characterized by the presence of binding sites for several TFs such as members of the Nuclear Factor NF-jB (NFjB) family (p65, p50, cRel), Egr1, Sp1 and CREB, which regulate basal NGB expression via specific interactions with the mouse promoter. NFkB, Sp1 and HypoxiaInducible Factor 1 (HIF-1) are required for NGB overexpression in hypoxic conditions. However, there is no consensus sequence for HIF-1 in NGB promoter, and it has been suggested that HIF-1 forms a complex with NFkB (18–21). To investigate functional genomic elements that can contribute to NGB transcriptional regulation, we explored the ENCODE Project database (http://genome.ucsc.edu/ENCODE/). We focused on a 6-kbp region containing the gene locus of NGB (Chromosome 14: 77,731,826–77,737,655). Genomic and epigenetic features of NGB gene locus are available in the ENCODE database, generated from experiments in astrocytes, glial and neuroblastoma cell lines. These include SK-N-SH and SH-SY5Y, SK-N-MC, SK-N-SH_RA (retinoic acid [RA]-differentiated neuroblastoma), NH-A, HA-sp, HAc astrocytes (Fig. 2). To better understand the regulation of NGB, we analyzed DNasehypersensitive sites in the NGB locus, corresponding to regions
48
accessible to transcriptional regulatory complexes, commonly defined as open chromatin state. In ENCODE Open Chromatin by DNase-Duke University and Open Chromatin by DNaseWashington University, a very evident peak is visible in the first NGB intron, which is present in diverse cell types, as shown in Fig. 2. To understand the function of these open chromatin regions, we explored histone modifications known to be associated with transcriptionally active or regulatory regions, such as H3K4me3/2, H3K36me3, H3K27ac or to silenced, heterochromatic regions, such as H3K27me3 and H3K9me3. There are only few studies on histone modifications in neuronal cell lines and astrocytes (ENCODE Histone Modifications ChIP-SeqUniversity of Washington). A weak H3K27me3 signal is present only in SK-N-SH cells treated with RA, suggesting that this DNase-hypersensitive site is in poised status and these regulatory regions are used in response to specific stimuli. In RNASeq assay, a high signal is present in the first intron region in undifferentiated cells, which decreases upon RA-induced differentiation. These transcripts are most likely noncoding eRNAs (RNA transcribed from enhancers). Altogether, these data indicate that the NGB locus is silenced in RA-differentiated cells. In accordance with the fact that NGB expression is very low in all cell lines studied, no active H3K4me3 histone mark is observed on the NGB promoter. In correspondence with the area of the first intron, positive to DNAse analysis, the binding of several TFs was observed by Chromatin Immunoprecipitation followed by high-troughtput sequencing (ChIP-Seq) in these cell lines. In addition, this region displays a positive signal of H3K27 acetylation and p300 coactivator. All together, these data suggest that NGB first intron contains an enhancer in a preprogrammed poised status. It should be stressed, however, that enhancer-bound transcriptional complexes can regulate genes in a distanceindependent fashion, even jumping over intervening genes (22). Taking into consideration this evidence, we can assume that this enhancer may either regulate NGB promoter activity or form long-range interactions with other regions, in basal normal culture conditions of the cell types in investigation. Interestingly, next to this enhancer there are two Single-Nucleotide Polymorphisms (SNPs) (rs10133981 and rs7149300) associated with Alzheimer’s disease, suggesting that this genomic region plays a central role in the NGB regulation in pathological conditions. A single base change within a TFs binding sequence can impair TF association and therefore inhibit biological functions, as described in the case of allelespecific activity (23). Predictive analysis of the consensus sequences that can be recognized by various TFs in NGB promoter was already reported and some TFs have been experimentally validated by chromatin immunoprecipitation, for example, NFkB and Sp1 (20,24). Figure 3 shows the results we obtained using matrixscan algorithms (Jaspar CORE 2009, NHRScan) reported in top and bottom panels, respectively. We discovered different TFBSs in the regions indicated as A (promoter) and B (putative enhancer). In the NGB promoter region, we found putative
Genomic Lens On Neuroglobin Transcription
Fig 2
Integration at NGB locus of ENCODE high-throughput experiments performed in astrocytes and neuronal cell lines. Respect to NGB locus at top, black horizontal bars A and B refer to gene promoter and candidate enhancer regions, respectively. From top to bottom, each row reports the genomic coverage obtained by RNA-Seq, DNAse-Seq and ChIP-Seq against different histone modifications and TFs. Letters on the right column indicate the cell line analyzed, row by row. Sample information is reported at bottom. The image was generated using WashU Epigenome Browser (http://epigenomegateway.wustl.edu).
response elements for Peroxisome Proliferator-Activated Receptor c (PPARc) and retinoid X receptor (RXR), Zinc Finger Protein, X-linked (ZFX), Nuclear Transcription Factor Y alpha (NFYA), Transcriptional enhancer domain 1 (TEAD1), which was also detected in the AHBA analysis (Fig. 1C) as the TF showing the highest correlation with NGB. PPARc and RXR are involved,
Cutrupi et al.
together with NGB, in Alzheimer’s disease experimental models, and therefore they may act as NGB modulators (3). Recent evidence indicates that NGB expression is induced by estradiol in neuroblastoma cells and primary astrocytes (25,26), building up a possible mechanistic link between the known neuroprotective effect of estrogen and NGB action.
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Fig 3
Predictive analysis of TFBSs at NGB promoter (A) and the candidate intronic enhancer (B). Top panels report the most enriched predictions obtained using Jaspar website (matrix score > 10). Bottom panels report nuclear receptor BSs predicted by NHRScan with their enrichment expressed as Viterbi probability. ER6 and ER8: everted repeat with 6-length and 8-length bp spacing between repeats, respectively; IR0: inverted repeat with no spacing between repeats.
Indeed, a significant correlation between NGB and ERa expression in the hypothalamic regions exists, indicating a possible direct regulation of NGB transcription by ERa. Both NHRScan, which finds putative BSs for nuclear receptors based on the presence of direct, inverted or everted repeats, and other algorithms confirm the presence of partially conserved nuclear receptor response elements in both the promoter and putative enhancer regions. However, binding of ERs in these regions awaits direct demonstration. It should be stressed that both ERa and ERb may regulate NGB transcription not only directly, but also indirectly, by interacting with other TFs bound to their DNA elements, for example, Sp1, as demonstrated previously (27). In this article, we provide a hypothesis on NGB regulation, by integrating published experimental data with bioinformatic analysis. In physiological conditions, NGB is highly expressed in the hypothalamus, which is a key integrative center in the brain, consisting of many different cell types required for a variety of functions including homeostasis, reproduction, stress response, social and cognitive behavior. During neurogenesis, the fine tuning of NGB may contribute to support high cellular
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growth. In the retina, NGB localizes in optic nerve glial cells for ensuring visual information transmission to the brain and protects against oxidative stress induced by direct exposure to the extracellular milieu. Under basal condition, NGB may act as modulator of cell energy balance, whereas it will regulate mitochondrial activity under stress conditions. The molecular mechanisms leading to neuronal differentiation and specialization in the different hypothalamic areas are poorly understood. The convergent expression of NGB and specific TFs in defined neuroanatomic regions may help identifying molecular mechanisms linked to specific biological functions. In pathological conditions, NGB protects cells against oxidative stress. It is well demonstrated that NGB has a neuroprotective role in hypoxia condition, such as stroke and Alzheimer’s disease. NGB is expressed at higher levels in neurons than astrocytes and glial cells in hypoxia condition-adapted mammals (28), suggesting that cell type-specific NGB may increase upon low O2 concentration in restricted locations. We focused our analysis on basal coexpression of NGB with TFs in specific brain regions. We observed that TFs potentially involved in NGB regulation, such as ESR1, SIM1,
Genomic Lens On Neuroglobin Transcription
SOX-3, SOX-4 and PPARc, are coherent with NGB role in cell metabolism and neuroprotection. The integration of genomic, epigenetic and experimental data represents a step forward to understand the mechanisms of NGB transcriptional regulation, which rely on complex networks of regulatory proteins acting in concert to provide cell-specific functions.
Acknowledgements The authors thank Silvia De Marchis and Stefano Gotti, NICO Neuroscience Institute Cavalieri Ottolenghi for discussions. This work was supported by grants from Italian MURST (PRIN Grant20109MXHMR_005) to M.D.B.
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