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The transcriptional corepressor CtBP2 is essential for proper development of the nervous system. The factor exerts its repression by interacting in complexes ...
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Research Article

Oxygen-dependent acetylation and dimerization of the corepressor CtBP2 in neural stem cells Esra Karaca, Jakub Lewicki, Ola Hermansonn Linnaeus Center in Developmental Biology for Regenerative Medicine (DBRM), Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

article information

abstract

Article Chronology:

The transcriptional corepressor CtBP2 is essential for proper development of the nervous system.

Received 9 October 2014

The factor exerts its repression by interacting in complexes with chromatin-modifying factors

Accepted 15 October 2014

such as histone deacetylases (HDAC) 1/2 and the histone demethylase LSD1/KDM1. Notably, the histone acetyl transferase p300 acetylates CtBP2 and this is an important regulatory event of the

Keywords:

activity and subcellular localization of the protein. We recently demonstrated an essential role for

NSCs

CtBPs as sensors of microenvironmental oxygen levels influencing the differentiation potential of

Cortex

neural stem cells (NSCs), but it is not known whether oxygen levels influence the acetylation

Neural development

levels of CtBP factors. Here we show by using proximity ligation assay (PLA) that CtBP2 ace-

Hypoxia

tylation levels increased significantly in undifferentiated, proliferating NSCs under hypoxic

Sirt1

conditions. CtBP2 interacted with the class III HDAC Sirt1 but this interaction was unaltered in

p53

hypoxic conditions, and treatment with the Sirt1 inhibitor Ex527 did not result in any significant change in total CtBP2 acetylation levels. Instead, we revealed a significant decrease in PLA signal representing CtBP2 dimerization in NSCs under hypoxic conditions, negatively correlating with the acetylation levels. Our results suggest that microenvironmental oxygen levels influence the dimerization and acetylation levels, and thereby the activity, of CtBP2 in proliferating NSCs. & 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NCND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Introduction The development of the mammalian cortex is a complicated process involving many cellular events, including proliferation, differentiation, cell death, senescence, autophagy, migration, and axon pathfinding, and these events are controlled by secreted signaling factors, intrinsic signaling pathways, and ultimately transcriptional regulators of gene expression [1–3]. In recent years, the strong influence of the microenvironment on the state of undifferentiated neural progenitors has been acknowledged [4]. Hence, in addition to external signaling factors such as FGFs, BMPs,

neurotrophic factors, IL-6 related cytokines and hormones such as retinoic acid and thyroid hormone to mention a few, the importance of extracellular matrix, stiffness, roughness, oxygen levels, and the spatial variation in such influences creating gradients to be interpreted by the progenitor cell have been highlighted [5–7]. Thus, to achieve a greater understanding of neural differentiation and thereby provide the basis for development of new tools for diagnosis, prognosis, and treatment of neurodevelopmental and neurological disorders, a broad knowledge of the mechanisms underlying the integration of these signals in the progenitor cell nucleus is required.

n

Corresponding author. E-mail address: [email protected] (O. Hermanson).

http://dx.doi.org/10.1016/j.yexcr.2014.10.013 0014-4827/& 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Please cite this article as: E. Karaca, et al., Oxygen-dependent acetylation and dimerization of the corepressor CtBP2 in neural stem cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.10.013

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The transcriptional regulators CtBP1 and CtBP2 are particularly interesting in this context. Identified in cancer research and later shown to be essential for nervous system development, CtBP1/2 has been shown to be members of transcriptional corepressor complexes together with class I histone deacetylases (HDACs)

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HDAC1/2 and histone H3K4 demethylases such as LSD1/KDM1 [8–10]. CtBP1 has further been associated with CoREST and there by REST, a major repressor of neuronal genes, and it has been demonstrated that the CtBP complex contains the enzymatic constituents to mediate coordinated deacetylation and methylation

Fig. 1 – CtBP2 and Sirt1 interaction in the nuclei of neural stem cells. (A) Micrograph depicting proliferating neural stem cells (NSCs) after proximity ligation assay (PLA) experiments demonstrating CtBP2 and Sirt1 are located in close proximity with predominantly nuclear localization. (B) Colocalization of DAPI and PLA signal from cross-sectional view in 3D rendering of Z-stacks. (C) Micrographs depicting decreased CtBP2–Sirt1 PLA signal (red) following siRNA knockdown of CtBP2, Sirt1, and CtBP2þSirt1 (F-actin green, DAPI blue). (D–F) Quantifications of CtBP2–Sirt1 PLA (D), CtBP2 single PLA (E), and Sirt1 single PLA (F) following siRNA knockdown of CtBP2 and/or Sirt1. Data are presented as mean PLA signal per cell7S.E.M (n¼ 3). Scale bar ¼10 lm. Please cite this article as: E. Karaca, et al., Oxygen-dependent acetylation and dimerization of the corepressor CtBP2 in neural stem cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.10.013

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of lysine 9 residues on histone H3 [11]. CtBP1/2 have also been shown to play major roles in regulating differentiation potential, epithelial-to-mesenchymal transition (EMT), and cell survival [12,13]. The activities of CtBP1/2 have been shown to be regulated by various mechanisms. CtBP1/2 are sensitive to NAD/NADH levels, implying a regulation by metabolic state and oxygen levels, and we recently showed that CtBPs function as sensors of microenvironmental oxygen levels in the presence of BMP, directly influencing the differentiation potential of neural stem cells [14]. CtBP2 has further been shown to be acetylated by the major histone acetyl transferase (HAT) p300 [15,16], and this modification strongly influences the subcellular localization and thus the repression exerted by the factor. Less is however known of a CtBP1/2 deacetylase or whether microenvironmental oxygen levels influence acetylation levels and transcriptional activity of CtBP1/2. Another transcriptional regulator that is influenced by the NAD/ NADH ratio is the class III histone deacetylase Sirt1. A number of targets for p300 HAT activity have also been shown to be targets for Sirt1 deacetylase activity, including the tumor suppressor p53 [17]. Notably, we and others have shown that Sirt1 can act as a repressor of neuronal differentiation in neural stem cells [18–20], however see also [21], and it has been demonstrated that Sirt1 gene expression can be regulated by CtBP1 [22], but it is not known whether Sirt1 can interact directly with or deacetylate CtBP2. To enable studies of protein interactions and modifications in neural stem cells derived from the mammalian embryonic cortex (NSCs), we employed proximity ligation assay (PLA) in this system [23,24]. We aimed at investigating whether i) total acetylation levels of CtBP2 were influenced by oxygen levels, ii) the NAD/ NADHþ-sensitive deacetylase Sirt1 could interact with CtBP2, iii) CtBP2 is a target of Sirt1, and iv) the dimerization of CtBP2 could be affected by oxygen levels, and/or correlated with the total acetylation levels of CtBP2. Using PLA, we have found that the total acetylation levels of CtBP2 are altered under hypoxic conditions in NSCs and that this modification showed a negative correlation with dimerization. We further demonstrate that CtBP2 interact with Sirt1 in NSCs but no evidence was found to establish that CtBP2 is a bonafide Sirt1 target. We suggest that CtBP2 acetylation and

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dimerization are altered by microenvironmental oxygen levels in NSCs, and that these events may be critical for CtBP2-dependent events of neural differentiation and development.

Fig. 2 – Increased CtBP2 total acetylation levels in NSCs after metabolic changes. (A) NADH/NADþ assay demonstrating an upregulation of NADH/NADþ ratio when NSCs are grown in 1% oxygen, and a downregulation of the ratio after 2DG treatment. Data are presented as mean7S.E.M. (n¼3; t ¼7.05 (hypoxia), t ¼ 1.84 (2DG)). (B) Micrographs depicting PLA signal after using an antibody specific for acetylated lysines in combination with CtBP2 antibody (CtBP2-Ac) following normoxia, hypoxia, and 2DG treatments. (C) Quantification of CtBP2-Ac-dependent PLA signal showed increased acetylation of CtBP2 under hypoxic conditions. Data are shown as mean PLA signal count per cell7S.E.M. (n¼ 5; t¼ 2.98 (hypoxia), t ¼2.67 (2DG)). (D) Micrographs depicting PLA product after detection of CtBP2-Sirt1 interaction under normoxia, hypoxia, and 2DG treatments. (E) Quantification of PLA signal after investigating CtBP2–Sirt1 interaction revealed no significant changes after hypoxia or 2DG treatments compared to normoxia (n ¼5; t ¼0.27 (hypoxia), t¼ 0.99 (2DG)). Differences between means were analyzed using one-way ANOVA followed by Bonferroni's test. All data are presented as mean7S.E.M. (npo0.5, nnnpo0.001). Scale bar ¼10 lm. Please cite this article as: E. Karaca, et al., Oxygen-dependent acetylation and dimerization of the corepressor CtBP2 in neural stem cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.10.013

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Materials and methods Cell culture

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siRNA knockdown For siRNA-mediated knockdown of CtBP2 and Sirt1, Amaxa NSC Nucleofection kit (Lonza) was used according to manufacturer's

The isolation and expansion of neural stem cells derived from embryonic rat cortex was essentially performed as previously described [25,26]. Cortical neural stem cells (NSC) were dissected from E15.5 rat embryos and seeded in DMEM/F12 enriched with N2 supplement on coated plates. NSCs were mechanically split using HANKS and used for experiments at passage 1 and 2 (P1 and P2). Cells were fed FGF2 (10 ng/ml, RD, 233-FB) every day and the media was changed every other day. For metabolic treatments cells were grown in hypoxia chamber (1% oxygen) or treated with 5 mM 2DG (2-Deoxy-D-glucose) (Sigma, D6134) overnight. In order to inhibit Sirt1 cells were treated with 1 mM Ex527 (Tocris, 2780) overnight or 5 mM Ex527 for 5 h. To study effects of H2O2 induced DNA damage on p53 and CtBP2 acetylation NSCs were treated with 500 mM of H2O2 for 30 min and 1 h.

Proximity ligation assay (PLA) For detection of protein–protein interactions and total amount of proteins Proximity Ligation Assay kit (DuoLink/Sigma; DUO92101) was used. NSCs were grown on glass coverslips, fixed in 4% PFA for 10 min followed by 15 min permeabilization in 0.1% Triton X-100. PLA was carried out according to manufacturer's manual using kit reagents. The probe and antibody concentrations were optimized for each assay. F-actin counterstaining was carried out following the last wash of PLA using Alexa488-Phalloidin (Molecular Probes). Cells were mounted in DuoLink mounting medium and imaged with fluorescent microscopy. Images were sharpened using image deconvolution software Autoquant and analyzed with ImageJ. The primary antibodies used for PLA assays were targeted to overlapping regions at the C-terminus of human CtBP2: CtBP2 mouse monoclonal antibody targeted to mouse CtBP2 amino acids 361–445 (BD Biosciences, 612044, 1:200), CtBP2 rabbit monoclonal antibody targeted to human CtBP2 amino acids 415–445 (Epitomics/Abcam; ab128871, 1:200). Sirt1 rabbit polyclonal antibody (Santa Cruz, sc-15404, 1:50), panAcetylated lysine antibody (Cell Signaling, 9441, 1:200), p53 mouse monoclonal (Cell Signaling, 2524, 1:200).

Fig. 3 – No significant effects of Sirt1 inhibition on total CtBP2 acetylation levels. (A) Quantification of CtBP2-Ac-dependent PLA product. (B) Quantification of PLA product elicited by CtBP2–Sirt1 antibodies following treatment with Sirt1 inhibitor Ex527 in combination with normoxia and hypoxia. The effect of Ex527 treatment was analyzed using two-way ANOVA followed by t-test for CtBP2-Acetyl PLA. The signal after CtBP2–Sirt1 PLA was not significantly influenced by metabolic changes or Ex527 treatment according to two-way ANOVA test (n¼3). (C) Quantification of CtBP2-Ac-dependent PLA signal in NSCs in response to siRNA knockdown of Sirt1 and CtBP2. All data are presented as mean PLA count per cell7S.E.M. (n¼ 3; n po0.05). Scale bar¼ 10 lm. Please cite this article as: E. Karaca, et al., Oxygen-dependent acetylation and dimerization of the corepressor CtBP2 in neural stem cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.10.013

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optimized protocol. Both CtBP2 and Sirt1 siRNA were obtained from Dharmacon (Accell and ON-TARGETplus siRNA). Following nucleofection, NSCs were seeded in 35 mm plates in N2 media under proliferative conditions. The results were analyzed 48 h later by RT-qPCR.

RT-qPCR RNA was isolated from cells using Qiagen RNeasy mini kit. 400 ng of RNA was used for RT-PCR carried out with Applied Biosystems High Capacity cDNA Reverse Transcription kit. Q-PCR was performed with Invitrogen's Platinum SYBR Green mix according to manufacturer's manuals.

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Hypoxic conditions alter the acetylation levels of CtBP2 but do not affect the close localization to Sirt1 As mentioned above, CtBP2 has been demonstrated to be acetylated by the HAT p300 [15] and a number of proteins acetylated by p300 are in parallel substrates for the class III histone deacetylase Sirt1, including the tumor suppressor protein p53 [17]. Since we recently reported that the activity of CtBP proteins are influenced by microenvironmental oxygen levels, and since CtBP2 was found in close proximity to Sirt1, we asked whether CtBP2 acetylation could be influenced by changes in oxygen levels and further whether the

NADH/NADþ Assay In order to detect free NADH and NADþ inside the cells under normoxic and hypoxic conditions we used Abcam NAD/NADH assay kit (ab65348). After rinsing with PBS, 2  106 cells were pelleted and the assay was carried out according to manufacturer's protocol.

Immunoblotting For immunoblotting experiments, the Bio-Rad Mini-Protean 3 system was used. Electrophoresis and transfer steps were carried out according to manufacturer's protocols. 30 mg protein was loaded per lane. Following transfer to nitrocellulose membranes, the blot was blocked and probed using Li–Cor reagents and visualized with Odyssey infra-red system. The antibodies used for western blot were: CtBP2 rabbit monoclonal (Abcam/Epitomics, ab128871, 1:2000), Sirt1 rabbit polyclonal (Santa Cruz, sc-15404, 1:100) and Beta-actin mouse monoclonal (Sigma, A5441, 1:4000).

Results and discussion CtBP2 and SIRT1 are located in close proximity in undifferentiated, proliferating neural stem cells We speculated whether Sirt1 could directly interact with CtBPs, in particular CtBP2 due to its verified role in the development of the forebrain [27]. To test this hypothesis, we employed proximity ligation assay (PLA). PLA enables the detection of interactions between endogenous proteins within 40 nm distance to each other [28]. In addition, as PLA results in a fluorescent signal detectable by conventional microscopy, it is further possible to analyze the subcellular localization of the putative interaction. This approach revealed that CtBP2 and Sirt1 are in close proximity in NSCs (Fig. 1A). The signal was predominantly nuclear, and this nuclear localization of the PLA signal was confirmed in a crosssectional view of a 3D image (Fig. 1B). To control for the specificity of the PLA signal, siRNA knockdown of CtBP2 and Sirt1 in parallel experiments were performed. The amount of PLA signal per cell was reduced 50–60% (in nuclei, control levels (mean7S.E.M.): 11.1370.51; after siRNA: 5.2471.01 (siCtBP2), 6.9571.26 (siSirt1), 4.6571.24 (siCtBP2þsiSirt1)) following siRNA knockdown with nucleofection efficiency being typically around 50% (Figs. 1C–F). These results indicate that CtBP2 and Sirt1 are located in close proximity in the nuclei of proliferating NSCs, suggesting either a direct interaction or co-localization to the same nuclear complex.

Fig. 4 – Total amount of CtBP2 and Sirt1 comparing single PLA with immunoblotting. Quantifications of the total amount of CtBP2 (A) and Sirt1 (B) detected by single PLA under normoxia, hypoxia, 2DG, and Ex527 treatments. The means of nuclear and cytoplasmic PLA signals were compared separately using oneway ANOVA followed by Bonferroni's test. No significant changes were detected. (C) Immunoblotting analysis of CtBP2 and Sirt1 levels in whole cell lysates of NSCs grown under the same conditions as in A and B.

Please cite this article as: E. Karaca, et al., Oxygen-dependent acetylation and dimerization of the corepressor CtBP2 in neural stem cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.10.013

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close proximity of CtBP2 and Sirt1 was affected under hypoxic conditions. As both Sirt1 and CtBP2 are influenced by NADH/NADþ levels, we first investigated the effect of metabolic changes on the total acetylation levels of CtBP2. NADH/NADþ ratio was increased approximately 3-fold when cells were incubated in 1% oxygen (0.1170.0073 (control) versus 0.3070.039 (hypoxic)), and reduced E40% upon treatment of NSCs with 2-Deoxy-D-glucose (2DG; 0.06670.016), which acts as a glycolytic inhibitor (Fig. 2A). Previously, hypoxic conditions have been shown to inhibit Sirt1 whereas treatment with 2DG increases the activity, directly or indirectly [22]. Upon hypoxia treatment or treatment with 2DG, the total acetylation levels of nuclear CtBP2 was increased approximately 50% as assessed by PLA signal count per cell (16.96 versus 25.83 and 24.89, respectively) in NSCs as detected by PLA (Figs. 2B and C). However, analyses of the putative CtBP2– Sirt1 interaction in response to the metabolic stimuli demonstrated that the PLA signal did not change significantly with hypoxia or 2DG treatments (Figs. 2D and E). These results indicate that total CtBP2 acetylation levels are altered under hypoxic conditions and after 2DG treatment in NSCs, but that these conditions do not affect the close proximity between CtBP2 and Sirt1.

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(Fig. 3C), Sirt1 siRNA resulted in a small decrease in CtBP2 acetylation that failed to reach statistical significance (Fig. 3C). To exclude whether changes in protein levels were influencing the results, we compared the total amount of CtBP2 and Sirt1

The oxygen-dependent alteration in total CtBP2 acetylation levels seems to be independent of Sirt1 activity Although the close proximity of CtBP2 and Sirt1 in NSCs was unaltered under hypoxic conditions, this experiment did not rule out the possibility that CtBP2 could be targeted by Sirt1 deacetylase activity. Therefore, we treated the NSCs with the specific Sirt1 inhibitor Ex527 to investigate whether Sirt1 could be a regulator of total CtBP2 acetylation under hypoxic conditions. However, Ex527 did not show any significant effect on the total acetylation levels of CtBP2 nor in the proximity of CtBP2 and Sirt1, either in combination with normoxia or hypoxia (Figs. 3A and B). Accordingly, whereas CtBP2 siRNA treatment expectedly caused a 3-fold drop in CtBP2 acetylation as assessed by the PLA signal Fig. 5 – DNA damage induced p53 acetylation can be confirmed by PLA. (A) Images of p53-Acetylated lysine (p53-Ac) PLA following exposure to hydrogen peroxide for 30 min or 1 h. (B) Quantification of p53-Ac PLA shows significant increase of p53 acetylation upon 1 h H2O2 treatment. According to two-way ANOVA Sirt1 inhibitor Ex527 did not have a significant effect. H2O2 induced p53 acetylation was analyzed using one-way ANOVA followed by Bonferroni's test. (n¼ 5, t ¼ 1.6 (H2O2 30 min), t ¼3.9 (H2O2 1 h)). (C) Images of CtBP2-Ac-elicited PLA signal under treatment of H2O2 for 30 min or 1 h. (D) Quantification of CtBP2-Ac PLA shows significant decrease of CtBP2 total acetylation levels upon H2O2 treatment. According to two-way ANOVA Ex527 did not have a significant effect. Effect of H2O2 was analyzed with one-way ANOVA followed by Bonferroni's test. (n¼3, t ¼ 7.1 (H2O2 30 min), t ¼7.7 (H2O2 1 h)) E) CtBP2 single PLA to compare total levels of CtBP2 upon H2O2 treatment. 2-way ANOVA showed no significant effect of Ex527 or H2O2 treatments on CtBP2 levels. (Scale bar¼ 10 lM.) (npo0.05, nnpo0.01). All data are presented as meanþs.e.m. Please cite this article as: E. Karaca, et al., Oxygen-dependent acetylation and dimerization of the corepressor CtBP2 in neural stem cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.10.013

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Fig. 6 – Alteration of CtBP2 homodimerization in proliferating NSCs in response to metabolic changes. (A) Micrograph depicting the PLA product in NSCs demonstrating CtBP2 homodimers following normoxia, hypoxia, and 2DG treatments. (B) Quantification of the PLA signal representing CtBP2 homodimers unveiled a significant decrease upon hypoxic treatment. Means were compared using one-way ANOVA followed by Bonferroni's test. Data are presented as mean PLA signal count per cell7S.E.M. (n¼ 4; t¼ 4.5 (hypoxia), t¼ 2.3 (2-DG)). (C) Quantification of the PLA product representing CtBP2 homodimers after CtBP2 RNA knockdown as a control for the specificity of the PLA signal. Sirt1 siRNA knockdown had no significant effect on CtBP2 homodimerization. Error bars are S.E.M., n¼3; nnpo0.01. Scale bar¼ 10 lm). proteins under different treatments with a single PLA approach. This was pursued by using one primary antibody and complementary probes targeted against the species of the primary antibody to provide a quantitative measure of protein amount and location. These experiments confirmed that there were no significant differences in CtBP2 and Sirt1 single PLA levels under normoxia, hypoxia, or 2DG or Ex527 treatments (Figs. 4A and B). Sirt1 showed a slight variation in cytoplasmic location but this observation did not reach statistical significance (Fig. 4B). These results using the PLA approach were confirmed using immunoblotting (Fig. 4C). The tumor suppressor p53 is a well-established target of Sirt1 [17]. To verify the negative results regarding the hypothetical regulation of CtBP2 by Sirt1, as well as the altered acetylation levels of CtBP2 under hypoxic conditions using PLA, we tested whether we could detect the established increase in acetylation of p53 by the PLA method by using DNA damaging agents. Treatment of NSCs with H2O2 resulted in a significant increase in acetylation of p53 in NSCs (Figs. 5A and B). Interestingly, total acetylation levels of CtBP2 in NSCs were significantly decreased by treatment with H2O2 as assessed by quantification of PLA signal per cell (17.39 versus 10.34 (30 min) and 9.66 (1 h)) but not by Ex527 (Figs. 5C

and D) while the levels of total CtBP2 were unaltered by the same treatment (Fig. 5E). In summary, these observations suggest that CtBP2 is not a target of Sirt1 but rather that the two proteins are interacting in a cell context-dependent manner in NSCs.

Alteration of dimerization in hypoxic conditions correlates negatively with the total acetylation levels of CtBP2 Next, we asked whether hypoxic conditions could influence the dimerization of CtBP2. We therefore performed PLA using two primary antibodies that were targeted to overlapping regions at the C-terminus of CtBP2. Importantly, under hypoxic conditions, the PLA signal for CtBP2 homodimer showed a small but reproducible decrease (19.83 versus 15.2; po0.01; Figs. 6A and B). In accordance with previous experiments, CtBP2 siRNA caused a E75% decrease in CtBP2 homodimer PLA signal whereas siRNA against Sirt1 was not effective (Fig. 6C). Although we here show that PLA is a useful tool for analyzing protein interactions, enzymatic modifications, and homodimerizations in NSCs, there are some caveats to be discussed. For example, it should be noted that this approach with PLA cannot distinguish

Please cite this article as: E. Karaca, et al., Oxygen-dependent acetylation and dimerization of the corepressor CtBP2 in neural stem cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.10.013

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dimers from higher order oligomerization such as tetramers, which CtBP proteins are suggested to form based on its crystal structure [29]. Further, it should be emphasized that our analysis of CtBP2 acetylation was aimed at elucidating total acetylation levels. Changes in single lysine residues may therefore escape our detection, which is important to keep in mind when interpreting the negative results regarding the role for Sirt1. We conclude that microenvironmental oxygen levels influence the total acetylation levels and homodimerization of CtBP2. We further show that CtBP2 are in close proximity to, and thus likely interact in a complex with, the deacetylase Sirt1, but this interaction does not seem to play a critical role for the alteration in total acetylation of CtBP2 under hypoxic conditions.

Acknowledgments We thank the Hermanson lab members for discussions, and Katarina Gradin for experimental assistance. This work was supported by Karolinska Institutet (KID and Department of Neuroscience, KI, Sweden) to E.K. The Swedish Cancer Society, VR-MH (project grant and DBRM), KAW (CLICK), KI TEMA, and the Swedish Childhood Cancer Foundation (to O.H.).

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Please cite this article as: E. Karaca, et al., Oxygen-dependent acetylation and dimerization of the corepressor CtBP2 in neural stem cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.10.013