Tip60 might be a candidate for the acetylation of hepatic carbonic anhydrase I and III in mice

Carbonic anhydrases (CAs) play a significant role in maintaining pH balance by catalyzing the conversion of carbon dioxide to bicarbonate. The regulation of pH is critical for all living organisms. Although there are many studies in the literature on the biochemical, functional, and structural features of CAs, there is not sufficient information about the epigenetic regulation of CAs. The lysine acetyltransferase TIP60 (60 kDa Tat-interactive protein) was knocked out specifically in mouse liver using the Cre/loxP system, and knockout rate was shown as 83–88% by Southern blot analysis. The impact of Tip60 on the expression of Ca1, Ca3, and Ca7 was investigated at six Zeitgeber time (ZT) points in the control and liver-specific Tip60 knockout mice (mutant) groups by real-time PCR. In the control group, while Ca1 showed the highest expression at ZT8 and ZT12, the lowest expression profile was observed at ZT0 and ZT20. Hepatic Ca1 displayed robust circadian expression. However, hepatic Ca3 exhibited almost the same level of expression at all ZT points. The highest expression of Ca7 was observed at ZT12, and the lowest expression was determined at ZT4. Furthermore, hepatic Ca7 also showed robust circadian expression. The expression of Ca1 and Ca3 significantly decreased in mutant mice at all time periods, but the expression of Ca7 used as a negative control was not affected. It was suggested for the first time that Tip60 might be considered a candidate protein in the regulation of the Ca1 and Ca3 genes, possibly by acetylation.


Introduction
Carbonic anhydrases (CAs), zinc-containing metalloenzymes that are common in living organisms, catalyze the reversible hydration of CO 2 to bicarbonate (HCO − 3 ) and proton (H + ) [1][2][3]. Although these enzymes were first discovered in hemolyzed blood, nowadays, they are known to have activity in many tissues such as the kidney, liver, brain, muscle, and bone tissues [4][5][6][7]. CAs in mice are composed of 16 different isoenzymes and play a role in various functions such as respiration, acid-base homeostasis, ion transport, bone resorption, taste preferences, ureagenesis, and gluconeogenesis [3,8,9]. Eight of these zinc-containing metalloenzymes are in the cytosol (CAI, CAII, CAIII, CAVII, CA VIII, CA X, CA XI, and CA XIII), five are transmembrane or membrane-bound (CAIV, IX, XII, XIV and XV), two are in the mitochondria (CAVA and VB), and one (CAVI) is secreted [10]. Although there are many studies in the literature on the biochemical, functional, and structural properties of the CA family, there is insufficient information on the regulation of CAs at the gene and protein levels, especially their epigenetic regulation [11].
Epigenetic mechanisms such as acetylation, phosphorylation, methylation, ubiquitination, sumoylation, and glycosylation are required to regulate gene expression and chromatin structure in mammalian cells without modulating the DNA sequence [12,13]. Protein acetylation, which refers to the covalent binding of an acetyl group to an amino 1 3 acid residue of a protein, is the most well-known along with phosphorylation [14]. The correlation between increased transcription and histone acetylation has been known for many years. Thus, acetylation regulates a number of metabolic and physiological processes by affecting protein functions, protein-DNA and protein-protein interactions, and the subcellular localization of the protein [15,16]. A recent study has reported that the members of the α-CA family, CAI (at the N-terminus), CAII (at the N-terminus, K18, K39, and K113), CAIII (at the N-terminus and K126), and CAXII (at K194), are acetylated by acetyltransferase enzymes. However, the proteins involved in this acetylation are still unknown [11].
The lysine acetyltransferase TIP60 (60 kDa Tat-interactive protein), a member of the histone acetyltransferases (HATs) protein family, has important and vital functions such as transcriptional regulation, DNA repair, cell cycle, apoptosis mechanism, cancer, the circadian system and the generation of cellular signals, both directly and indirectly [17][18][19][20]. Studies have demonstrated that the TIP60 protein is associated with many transcription factors and proto-oncogenes, such as androgen receptor, c-Myb, c-Myc, STAT3, NF-Kb, E2F1, p53, and acts as a regulator/correlator [21][22][23]. The study performed by Chen et al. showed that the c-Myb transcription factor increased Ca1 expression by binding to its promoter in mouse erythroleukemia cells [24]. However, c-Myb inactivation is required to inhibit the Ca1 gene when the division of the cell is achieved. The cell becomes cancer without this suppression. It is also known that Tip60 is a regulating factor for c-Myb [21]. This study aimed to determine whether Tip60 plays a role in the regulation of hepatic Ca1 and Ca3 predominantly expressed in the liver. To this end, liver-specific Tip60 knockout mice were generated using Cre/loxP recombination. The quantitative expression of the Ca1 and Ca3 genes at different Zeitgeber time (ZT) points were determined for both the control and knockout groups and then compared to each other. Ca7, not regulated by acetylation, was used as a negative control [11].

Liver-specific conditional knockout mouse model
To generate liver-specific Tip60 knockout mice (mutant), Tip60 floxed mice (10-12-week-old male) with loxP sites flanking exons 1 and 9 of the Tip60 gene [20] were crossed to a SACre driver mouse line resulting in the Cre-mediated deletion of Tip60 in the liver [25]. Mice had been previously backcrossed to a C57BL/6N background for at least ten generations. To delete Tip60, 10-12-week-old male mice (Tip60 fl/− ; SA +/Cre−ERT2 ) were injected daily with tamoxifen (10 mg/ml stock solution, Sigma, St. Louis, MO, USA) in corn oil for five consecutive days. The control group (Tip60 fl/ fl ;SA +/+ ) was injected with corn oil. Genotyping was performed with gene-specific primers [20,25]. Liver and kidney tissues were collected 5 days after the last injection. Genomic DNA was isolated from both tissues and analyzed by Southern blot analysis. Liver-specific Tip60 knockout mice were generated at the Max Planck Institute for Biophysical Chemistry (MPIBPC), Germany.

Southern blot analysis
Genomic DNA from liver and kidney tissues was isolated using the DNeasy tissue kit (Qiagen Inc., Valencia, CA, USA), and fifteen micrograms of the genomic DNA were digested with BamHI overnight (NEB, Ipswich, MA, USA). DNA was separated on a 0.6% agarose gel and transferred onto a Hybond-XL positively charged nylon membrane (GE Healthcare/Amersham Biosciences, Sweden). The membranes were hybridized with a 32 P -dCTP labeled radioactive double-stranded DNA probe prepared by random priming using an appropriate commercial kit according to the manufacturer's instructions (Amersham Rediprime™ II DNA Labeling System, GE Healthcare) and purified with the illustra ProbeQuant™ G-50 Micro Columns (GE Healthcare). The hybridization of the radioactive probe (100 µl) to the membrane was performed at 65 °C overnight in the presence of a hybridization buffer. The membranes were washed with 2× SSC/0.1% SDS, 1× SSC/0.1% SDS, and 0.1× SSC/0.1% SDS, at 60 °C until the excess label was removed and exposed to a sensitive X-ray film (Kodak X-Omat 1000, 1000A and 1000J Processors). Southern blot experiments were performed at the MPIBPC, Germany.

RNA extraction and first-strand cDNA synthesis
Total RNA isolation from approximately 50 mg mice liver tissues was performed using the RNeasy Lipid Tissue Mini Kit (Qiagen-74804) following the manufacturer's instructions. The concentrations and purities of RNA were measured by a spectrophotometer (Thermo Scientific, Multiskan GO, USA). RNA quality was checked by agarose gel electrophoresis and stored at − 80 °C until use. Total RNA was converted into first-strand cDNA using the SuperScript III First-Strand cDNA kit system (Invitrogen, California, USA), by utilizing random hexamers, according to the manufacturer's protocol. The resulting cDNA was diluted to 100 ng/ μl with nuclease-free water and stored at − 20 °C [29].

Primer and probe design
Primer3 software (v. 0.4.0) (http:// bioin fo. ut. ee/ prime r3-0. 4.0/) was used for the design of gene-specific primers and probes meeting the following criteria: amplicon size 75-200 bp, ≤ 3 G or C repetitions, GC content 50-65%, ≤ 4 base repetitions, melting temperature (Tm) 60 °C. Primers and probes were verified with the Blast tool (NCBI) to confirm their specificity for the desired target. Then they were synthesized and purchased from Methabion International (Martinsried, Germany). The gene symbols and GenBank ID numbers are as follows: Ca1 (Gene ID: 12346), Ca3 (Gene ID: 12350), Ca7 (Gene ID: 12354), and Actb (Gene ID: 11461). Since housekeeping genes were not affected by any of the treatments, β-actin was used as reference gene. The sequences of the specific primers of all genes are shown in Table 1.

Quantitative real-time PCR
To determine the expression levels of the Ca1, Ca3, and Ca7 genes at different ZT points, real-time PCR (qPCR) was carried out on the Rotor-Gene Q instrument (QIA-GEN, Inc., Hilden, Germany). Beta-actin was selected as a reference control gene. The qPCR reactions were carried out with 2 µl of cDNA (final concentration of 0.02 ng), 4 pmol of TaqMan probe, 8 pmol of forward and reverse primers, and 10 µl FastStart TaqMan Probe Master Mix (Roche Diagnostics GmbHCorp, Mannheim, Germany) in a final volume of 20 µl. Optimal cycling conditions were 50 °C for 2 min, 95 °C for 10 min, 45 cycles of 95 °C for 15 s, and annealing and extension at 60 °C for 1 min [30]. The expression results were analyzed using the ΔCT method [31].

Statistical analysis
Each group contained three animals, and all measurements were triplicated for each animal. Statistical analysis was performed for each experiment using one-way and twoway analysis of variance (ANOVA) with Tukey's and Bonferroni's multiple comparison tests using the Prism software 7.0 (GraphPad Software, San Diego, CA). Statistically significant changes are displayed by a symbol (*). The symbol expressions are as follows: p > 0.05 (not significant, ns); *p < 0.05 (significant); **p < 0.01 (very significant); ***p < 0.001 and ****p < 0.0001 (extremely significant).

Liver-specific Tip60 knockout mouse models
Tamoxifen-inducible Cre/lox recombination was used to create genetically modified mouse lines in C57BL/6N backgrounds for tissue-specific or conditional knockouts of Tip60 due to the lethal effect of full knockout on mice [32]. The Tip60 gene was specifically knocked out in the mouse liver using this system. The Cre-mediated deletion of Tip60 in the mouse liver tissue was shown by Southern blot analysis. In Fig. 1, Cre-mediated deletion did not occur due to the absence of the SACre-driver in Tip60 fl/− ;SA +/+ mice.
Therefore, the deletion efficiency of Tip60 was 50% in the liver. Since Tip60 fl/− ;SA +/Cre−ERT2 mice contain the SACre driver, Cre-mediated deletion was achieved and the knockout rate was shown as 83-88%. The leakage of the Cre driver was also checked in the kidney tissues of both mouse lines, and any leakage was not observed within the kidney tissues (Fig. 1).

Expression levels of the Ca1, Ca3, and Ca7 genes at different Zeitgeber time points in the control groups
The circadian expression of Ca1, Ca3, and Ca7, expressed in the liver, was investigated in mice at six different ZT points in the control group. While Ca1 showed the highest expression at ZT8 and ZT12, the lowest expression profile was observed at ZT0 and ZT20. Hepatic Ca1 displayed robust circadian expression. While hepatic Ca3 exhibited almost the same level of expression at different ZT points, it revealed no circadian expression. While the highest expression of Ca7 was observed at ZT12, the lowest expression was determined at ZT4. Furthermore, hepatic Ca7 also displayed robust circadian expression ( Table 2).

Comparison of the gene expression levels of the control and mutant groups
It has been recently reported that while CAI and CAIII are acetylated by acetyltransferase enzymes, CAVII is not acetylated [11]. However, the proteins involved in this acetylation  are still unknown. Since Tip60 is thought to be a candidate for the acetylation of Ca1 and Ca3 in the liver, the quantitative expression levels of Ca1 and Ca3 in the control and mutant groups were analyzed by qPCR. The analysis, determined that the expression amount in mutant tissues decreased significantly at all time points compared to the control groups. No change was observed in the expression of Ca7 used as a negative control (Fig. 2).

Discussion
CO 2 is one of the simplest molecules involved in important physiological processes for all life domains. It is produced as part of the metabolic process and is quickly transported in the body by the blood [33,34]. The acidity of the blood increases due to the high solubility and rapid spread of CO 2 .
Since the pH of the human body is critical to health, all organisms must develop mechanisms to control it [35,36].
CAs are an important family of enzymes that catalyze the conversion of CO 2 to HCO 3 − to regulate the pH balance of the blood [37][38][39].
Many studies have proven the significant role of CAs in physiological processes and shown that the abnormal activity levels of these enzymes are associated with various human diseases such as glaucoma, erythroleukemia, malignant brain tumors, and renal, gastric, and pancreatic carcinomas [35,40]. Furthermore, HCO 3 − , produced by carbonic anhydrases, is essential for the function of metabolic liver enzymes that perform many functions, including digestion, glycogen synthesis, manufacturing triglycerides and cholesterol, bile production, storage of many essential vitamins and minerals [41]. It also metabolizes many drugs, medications, chemicals, and natural substances. Although several CA isoforms, including CA I, CA II, CA III, CAVII, and CA IX, have been described in the liver, there is very limited information about the regulation of CAs at the gene and protein levels, especially their transcriptional regulation [4,[42][43][44].
As a result of the analysis, the Ca1, Ca3, and Ca7 genes were expressed at all ZT points in the liver tissues of the control group. It was observed that the expression of Ca1 and Ca7 reached the maximum level at ZT8 (the end of the light cycle) and ZT12 (the beginning of the dark cycle), and the Ca3 gene was expressed in high amounts at all time  Ca3 (b), and Ca7 (c) were detected by qPCR. β-actin was used as a housekeeping gene points, except for ZT16. In the control group, upon comparing the quantitative expression levels of Ca1, Ca3, and Ca7 in liver tissues, genes with the highest expression were Ca3, Ca1, and Ca7, respectively. In this respect, our findings are consistent with the "NCBI-Mouse ENCODE transcriptome data" [45].
The study performed by Chen et al. in 2006 on mouse erythroleukemia (MEL) cells revealed that the c-Myb transcription factor was bound to the promoter of the Ca1 gene and increased the expression of Ca1 for proliferation and differentiation [24]. Zhao et al. stated that TIP60, a histone acetyltransferase with activity in the cytoplasm and nucleus, belonged to the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family and was known to be responsible for acetylation in both mouse and human cells, was a regulating factor for c-Myb [21,[46][47][48]. Another study, investigated the relationship between hepatocellular carcinoma (HCC) and tumor development with the expression of CAs. The activity and protein expression of the CA family in tumor tissues were significantly lower than normal cells [49]. A recent study conducted by Di Fiore A, et al. has reported that CA I and CA III proteins are regulated by post-translational acetylation [11].
Many researchers have stated that knockout mice are widely used to better understand the biological role of specific genes, and molecular and cellular mechanisms [50][51][52]. The study carried out by Hu et al. showed that the homozygous disruption of the Tip60 gene led to early embryonic death [32]. Therefore, we generated liver-specific Tip60 knockout mice using the tamoxifen-inducible Cre/loxP system to investigate the role of Tip60 in the regulation of Ca1 and Ca3 at the gene level. Moreover, since TIP60 has been shown to play a role in the regulation of the circadian clock [20], it has been investigated whether this arrangement occurs at different ZT points. As shown in Fig. 1, the Tip60 gene knockout rate was shown between 83 and 88% by the Southern blot technique in the liver.
The knockout rate of Tip60 in the liver tissue obtained from our study is efficient and useful for further studies, as shown in the literature [53,54]. We also investigated the expression of the Ca1 and Ca3 genes at six different ZT points in the control and mutant groups. While the expression of Ca1 and Ca3 significantly decreased in the absence of Tip60 in mouse liver at all time periods (Fig. 2a, b), the expression of Ca7, used as a negative control as mentioned above, was not affected (Fig. 2c). Potter [58]. It was hypothesized that CA III was reexpressed at the later stages of the metastatic progression of HCC, and it might have a significant impact on the development of metastasis in liver cancer [39]. According to the results obtained from these two studies, it was hypothesized that while the decreased expression of Ca3 was important in the pathogenesis of HCC, increased Ca3 expression was required for metastasis after cancer formation [39]. Based on the literature data, it is thought that decreased in Ca1 and Ca3 expression in mice due to the depletion of Tip60 may lead to HCC.
In our preliminary study to show how total CA activity changes in the presence and absence of Tip60, total CA activity was measured by Wilbur and Anderson method in the control and mutant groups [59]. No statistically significant difference in total CA activity was observed in both groups (data not shown). There are 16 different isoforms of CAs in mice. It is not known which CAs activity can increase or decrease in the absence of TIP60. Although some compounds were synthesized for specific inhibition of CAs by Carta et al. [60], there are no specific inhibitors defined for CAI and CAIII. For this reason, no results could be obtained about how the enzyme activity of CAI and CAIII changes in the absence of Tip60.
In conclusion, TIP60 may be considered a candidate protein in the regulation of the Ca1 and Ca3 genes, possibly by acetylation. Moreover, our results showed that Tip60 could be a new actor in explaining the molecular mechanism of hepatocellular carcinoma. However, it is clear that more studies including in vitro and in vivo tests are needed to support this hypothesis.
Mouse handling was carried out in accordance with the German Law on Animal Welfare and was ethically approved and licensed by the Office of Consumer Protection and Food Safety of the State of Lower Saxony (License Numbers 33.11.42502-04/072/07 and 33.9-42502-04-12/0719).