Cell-specific expression of aquaporin-5 (Aqp5) in alveolar epithelium is directed by GATA6/Sp1 via histone acetylation

Epigenetic regulation of differentiation-related genes is poorly understood. We previously reported that transcription factors GATA6 and Sp1 interact with and activate the rat proximal 358-bp promoter/enhancer (p358P/E) of lung alveolar epithelial type I (AT1) cell-specific gene aquaporin-5 (Aqp5). In this study, we found that histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) increased AQP5 expression and Sp1-mediated transcription of p358P/E. HDAC3 overexpression inhibited Sp1-mediated Aqp5 activation, while HDAC3 knockdown augmented AQP5 protein expression. Knockdown of GATA6 or transcriptional co-activator/histone acetyltransferase p300 decreased AQP5 expression, while p300 overexpression enhanced p358P/E activation by GATA6 and Sp1. GATA6 overexpression, SAHA treatment or HDAC3 knockdown increased histone H3 (H3) but not histone H4 (H4) acetylation within the homologous p358P/E region of mouse Aqp5. HDAC3 binds to Sp1 and HDAC3 knockdown increased interaction of GATA6/Sp1, GATA6/p300 and Sp1/p300. These results indicate that GATA6 and HDAC3 control Aqp5 transcription via modulation of H3 acetylation/deacetylation, respectively, through competition for binding to Sp1, and suggest that p300 modulates acetylation and/or interacts with GATA6/Sp1 to regulate Aqp5 transcription. Cooperative interactions among transcription factors and histone modifications regulate Aqp5 expression during alveolar epithelial cell transdifferentiation, suggesting that HDAC inhibitors may enhance repair by promoting acquisition of AT1 cell phenotype.

express AT2 cell markers in response to partial pneumonectomy 15 , indicating considerable phenotypic plasticity. Elucidation of molecular mechanisms that regulate cell-specific gene expression accompanying phenotypic transitions between AT2 and AT1 cells is important to understand how alveolar epithelial cells (AEC) are maintained and regenerate following injury.
Epigenetic modulation has been implicated in transcriptional regulation of tissue-specific gene expression and cell differentiation in eukaryotic cells, but evaluation of these mechanisms in the context of AT2 and AT1 cell-specific gene expression has been limited. In this regard, interaction of the homeodomain transcription factor NKX2.1 (also known as thyroid transcription factor-1 (TTF1)) with ATP-dependent chromatin remodeling protein BRG1 at the unmethylated Sftpb promoter increased Sftpb expression, which correlated with increased H3K4 trimethylation. These observations suggest that NKX2.1 cooperates with proteins mediating both DNA methylation and histone modification to regulate Sftpb 16 . In fetal lung, Sftpa activation by cyclic AMP (cAMP) and cytokines (e.g., interleukin-1α (IL-1α)) is mediated by increased NKX2.1, CREB (cAMP-response element-binding protein)-binding protein (CBP) and NF-κB binding to the TTF-1-binding element (TBE) of the Sftpa promoter, which is correlated with increased H3K9 acetylation, decreased dimethylation of H3K9 and decreased histone deacetylase (HDAC) binding to the TBE. In contrast, Sftpa inhibition by glucocorticoids is associated with enhanced glucocorticoid receptor (GR) and HDAC binding to the promoter and decreased H3 acetylation 17,18 . Additionally, activation of T1α is regulated by methylation at a key Sp1 site in the proximal promoter 19 .
AQP5 is a water channel protein that is highly expressed in lung, salivary gland and lacrimal gland 20,21 . In the rat lung, AQP5 is exclusively expressed on the apical membrane of AT1 (and not AT2) cells 20,22 . AQP5 expression in human salivary gland is regulated by acetylation of histone H4 23 and DNA methylation 24 . In rat lung, activation of Aqp5 in AT1 cells is associated with increased Sp1 binding to the hypomethylated proximal Aqp5 promoter 25 . However, a role for histone modifications in regulation of Aqp5 expression in AEC has not been reported. We previously showed that AQP5 expression is controlled by the zinc-finger transcription factor GATA6, involving interactions with Sp1 at the rat 358 bp proximal promoter/enhancer region (p358P/E) 26 . In the current study, we examined the contributions of histone modifications to GATA6/Sp1-mediated Aqp5 transactivation. Our results demonstrate that GATA6 increases H3 acetylation, while HDAC3 causes H3 deacetylation at the proximal Aqp5 promoter/enhancer, leading to activation and repression of Aqp5 transcription, respectively. These data suggest that epigenetic regulation of Aqp5 transcription involves competition of GATA6 and HDAC3 for binding to Sp1. Furthermore, we demonstrate a role for p300 in GATA6/Sp1 activation of Aqp5 transcription, possibly via modulation of H3 acetylation and/or GATA6 and Sp1 activity.

Results
Histone acetylation/deacetylation is involved in Aqp5 gene regulation. We previously identified a rat proximal Aqp5 promoter/enhancer fragment, p358P/E (−358 bp relative to the transcription start site (TSS)), containing multiple putative Sp1 sites, and showed that GATA6 activates this region via interactions with Sp1 26 . Since Sp1 binds to both p300, which has histone acetylase activity, and HDACs 27-29 , we hypothesized that modification of histone acetylation is involved in GATA6/Sp1-mediated Aqp5 gene expression. To test this hypothesis, mouse lung epithelial (MLE-15) cells were treated with HDAC inhibitor SAHA. As shown in a representative western blot (Fig. 1A), SAHA (1 µM) increased AQP5 protein expression in MLE-15 cells by 203 ± 14.7% at 24 h (Fig. 1B). To further investigate whether SAHA regulates Aqp5 expression at the transcriptional level, we performed transient transfection assays in MLE-15 and NIH3T3 cells in which GATA6 and Sp1 have been shown to regulate Aqp5 transcriptional activity 26 . In both MLE-15 and NIH3T3 cells, SAHA increased p358P/E transcriptional activity in a dose-dependent manner (Fig. 1C,D), implicating histone acetylation/deacetylation in regulation of AQP5 expression, although luciferase (Luc) activity declined at higher doses in NIH3T3 cells. Cell viability assays showed no toxicity of SAHA (1 µM) treatment for 24 h in both NIH3T3 and MLE-15 cells (Fig. 1E,F). Furthermore, SAHA significantly increased Sp1-mediated -358-Aqp5-Luc promoter activity in NIH3T3 cells (Fig. 1G), suggesting that Sp1 binding with HDACs at the p358P/E is involved in responses to SAHA. SAHA did not further increase GATA6-or GATA6/Sp1-mediated activation (Fig. 1G), perhaps because either GATA6 binding to Sp1 interferes with binding of HDACs to Sp1 or GATA6/Sp1 binding inhibited HDAC activity 30,31 . To further investigate if SAHA treatment affects histone acetylation at the Aqp5 promoter/enhancer region, we performed qPCR following chromatin immunoprecipitation (ChIP) in MLE-15 cells treated with SAHA using antibodies against the N-terminal portion of acetylated H3 and H4. There was enrichment of H3 acetylation (Fig. 1H) but decreased H4 acetylation (Fig. 1I) at the Aqp5 promoter/enhancer region homologous to rat p358P/E compared to DMSO control, indicating that SAHA induction of Aqp5 expression involves increased H3 acetylation (but decreased H4 acetylation) at the proximal Aqp5 promoter/enhancer region. HDAC3 inhibits Aqp5 gene expression. Sp1 has been shown to interact with HDAC1, 2 and 3 in regulation of gene expression 27,29,32 . To investigate which HDAC is involved in regulation of Sp1 activation of Aqp5 promoter/enhancer activity, -358-Aqp5-Luc was co-transfected with Sp1 and increasing amounts of HDAC1, 2 and 3 expression vectors or empty pCDNA3 vector in NIH3T3 cells. HDAC2 (Fig. 2B) and HDAC3 (Fig. 2C), but not HDAC1 ( Fig. 2A), inhibited Sp1-activated -358-Aqp5-Luc transcription. However, at higher doses, HDAC3 did not show an inhibitory effect, possibly due to HDCA3 inhibition of an unknown repressor of Aqp5 at higher concentrations or dependence of HDAC3 activity on its interaction with a corepressor (e.g., SMRT) or a serine/ threonine protein phosphatase, PP4 33 . We then knocked down HDAC2 and HDAC3 alone or in combination (Fig. 2D) using Hdac2 small interfering RNA (siRNA) and Hdac3 short hairpin RNA (shRNA). Knockdown of HDAC3 ( Fig. 2E) but not HDAC2 (Fig. 2F) increased AQP5 protein expression (Fig. 2G). These data suggest that, while both HDAC2 and HDAC3 can inhibit Aqp5 transcription, compensatory effects from other HDACs following HDAC2 reduction likely exist. Additionally, HDAC2 may increase translation of some proteins by promoting sumoylation of eukaryotic translation initiation factor 4E (eIF4E), independently of its deacetylase activity 34 which might in turn affect translation and therefore levels of AQP5 protein, resulting in no net change in AQP5 protein expression. To examine whether HDAC3 inhibition of Aqp5 transcription is associated with H3 deacetylation at the −358 bp proximal Aqp5 promoter/enhancer region, ChIP was performed using chromatin harvested from MLE-15 cells with/without HDAC3 knockdown, followed by pull-down with anti-acetyl-H3 antibody (Ab). qPCR following ChIP demonstrates enrichment of H3 acetylation at the Aqp5 promoter/enhancer region in cells with HDAC3 knockdown compared to control shRNA (Fig. 2H), confirming that HDAC3 regulation of Aqp5 expression involves changes in H3 acetylation at the proximal Aqp5 promoter/enhancer region. . Additionally, ChIP with anti-acetyl-H3 Ab demonstrated enrichment of H3 acetylation at the Aqp5 promoter/enhancer region following GATA6 overexpression compared to control (Fig. 3E). These data suggest that increased H3 acetylation at the proximal Aqp5 promoter/enhancer contributes to GATA6-mediated activation of Aqp5 transcription. Precise mechanisms whereby GATA6 modulates H3 acetylation are unknown but, since SAHA does not affect GATA-6 activation of Aqp5-Luc, include the possibilities that GATA6 interaction with Sp1 decreases HDAC3 binding to Sp1 and/or GATA6 binding to the Sp1/HDAC3 complex inhibits HDAC3 activity. In addition, as shown further below, recruitment of transcriptional coactivators with histone acetyltransferase (HAT) activity (e.g., p300) may modulate acetylation. p300 but not CBP enhances GATA6/Sp1-mediated Aqp5 transcription. p300 and CBP are highly homologous transcriptional coactivators that possess intrinsic HAT activity 35,36 . It has been reported that p300/ CBP increase H3 acetylation at promoter binding sites for Sp1 in transforming growth factor-β (TGFβ) target genes p21 and plasminogen activator inhibitor-1 (PAI-1) to activate transcription in mesangial cells 37 . Transient transfections were performed in NIH3T3 cells to determine if p300 or CBP are involved in modulation of Aqp5 transcription by GATA6/Sp1. Neither p300 nor CBP alone had an effect on p358P/E activity (Fig. 4A). However, p300 but not CBP further augmented GATA6- (Fig. 4B) and Sp1- (Fig. 4C) mediated transcriptional activation of Aqp5-Luc. Consistent with results showing that knockdown of p300 decreases AQP5 expression in rat AEC in primary culture 38 , knockdown of p300 in MLE-15 cells with shRNA ( Fig. 4D,F,G) significantly decreased Aqp5 mRNA (Fig. 4E) and AQP5 protein expression (Fig. 4F,H). These findings suggest that p300, either through its intrinsic HAT activity or function as a transcriptional co-activator, regulates Aqp5 transactivation through interactions with GATA6 and/or Sp1, and suggest a mechanism whereby GATA6 may regulate acetylation of the Aqp5 proximal promoter/enhancer via recruitment of p300.
HDAC3 competes with GATA6 for binding to Sp1. To further examine if HDAC3 binds to Sp1 and if HDAC3 affects GATA6/Sp1 binding, co-immunoprecipitation (co-IP) was performed using protein lysates from MLE-15 cells with/without HDAC3 knockdown. As shown in Fig. 5A, HDAC3 binds to Sp1 as expected, and this interaction decreases following HDAC3 knockdown (Fig. 5A). HDAC3 knockdown also increased GATA6/ Sp1 interaction (Fig. 5B), supporting the suggestion that HDAC3 competes with GATA6 for binding to Sp1. Additionally, co-IP showed that knockdown of HDAC3 increased the interaction of both GATA6 (Fig. 5B) and Sp1 (Fig. 5C) with p300, suggesting that GATA6 further recruits p300, perhaps due to increased affinity of the GATA6/Sp1 complex for p300. Based on the findings in this study, we suggest a model (Fig. 5D) where increased expression levels of GATA6 leads to increased GATA6/Sp1 interaction, decreased binding of HDAC3 to Sp1 and increased histone acetylation, contributing to Aqp5 promoter activation. Furthermore, the findings indicate that p300, either through its intrinsic HAT activity and/or functioning as a transcriptional co-activator, regulates Aqp5 transactivation through interactions with GATA6 and Sp1, and suggest a mechanism whereby GATA6 may regulate acetylation of the Aqp5 proximal promoter/enhancer via recruitment of p300.

Discussion
In the distal lung, AQP5 is specifically expressed in AT1 cells and plays an important role in alveolar homeostasis 21,39,40 . The role of epigenetic mechanisms, especially histone modifications, in regulation of AT1 cell-specific expression of Aqp5 is not completely understood. In this study, we found that HDAC inhibitor SAHA increases expression of AQP5 and that HDAC3 inhibits Sp1-mediated transcriptional activation of Aqp5, which is associated with H3 acetylation/deacetylation, respectively, at the proximal promoter/enhancer. We demonstrated that GATA6 activation of Aqp5 transcription involves H3 acetylation at the proximal promoter/enhancer and that p300 augments Aqp5 activation by GATA6 and Sp1. We further provide evidence that HDAC3 binds to Sp1 and competes with GATA6/Sp1 binding, and that HDAC3 inhibition of Aqp5 transcription is associated with H3 deacetylation. These data suggest that H3 acetylation/deacetylation is involved in regulation of statistical analysis for comparison with control. *p < 0.05 compared to column 5 (Sp1 with DMSO). ChIP with anti-acetyl-H3 (H3Ac) and anti-acetyl-H4 (H4Ac) Abs demonstrates enrichment of H3 acetylation (H) and decreased H4 acetylation (I) at the Aqp5 promoter/enhancer region homologous to the proximal 358-bp of the rat Aqp5 promoter following SAHA treatment (1 µM, 24 h) in MLE-15 cells (n = 3, *p < 0.05). ChIP efficiency was calculated relative to untreated cells precipitated with H3Ac and H4Ac Ab, respectively, which was set as 1. Rabbit IgG pull-down is used as control. differentiation-related Aqp5 gene expression concomitant with GATA6/Sp1/p300 and/or Sp1/HDAC binding at the proximal promoter/enhancer. Although SAHA decreased H4 acetylation at the Aqp5 promotor and a decrease  Luciferase assays were performed 48 h after transfection. p300 and CBP have no effect on Aqp5-Luc activity (A), while p300 (but not CBP) augments both GATA6-(B) and Sp1-(C) mediated Aqp5 transcriptional activity. Protein concentration was used to normalize samples for transfection efficiency (n = 3, *p < 0.05 compared to GATA6 or Sp1 only). qRT-PCR shows knockdown of p300 mRNA using two different shRNAs (V2LMN_102133 indicated as p300-1 and V2LMN_90586 indicated as p300-2) (D) decreases Aqp5 mRNA expression following p300 knockdown (E) (n = 4, *p < 0.05 compared to non-silencing shRNA control (NS shRNA)). WB shows knockdown of p300 (F,G) decreases AQP5 protein expression (F,H). (n = 4, *p < 0.05 compared to NS shRNA control).
in H4 acetylation is known to be associated with gene activation 41,42 , the mechanisms underlying decreased H4 acetylation as a result of SAHA treatment and how this might contribute to increased Aqp5 gene expression remain unknown and will require future studies. Nevertheless, our data are consistent with previous findings that the Sp family of transcription factors (e.g., Sp1 and Sp3) regulates cell-specific gene expression by recruiting HDACs or proteins with HAT activity (e.g., p300 43,44 ) to target gene promoters 37,45 .
GATA6 is a key transcription factor that regulates organogenesis and epithelial cell differentiation in the lung 46 . GATA6 is the only GATA family member expressed in the distal epithelium of the developing lung, where it plays an important role in lung branching and AEC differentiation 46,47 . GATA6 regulates expression of AT2 cell-specific genes (e.g., Sftpc 48 , Sftpa 49 and Nkx2.1 50 ) while also regulating activity of the promoter/enhancer of the AT1 cell-specific gene Aqp5 51 . Overexpression of dominant negative GATA6 in transgenic mice led to impaired AT1 cell development and reduced AQP5 expression in vivo 51 . In our in vitro rat AEC culture model, expression of GATA6 increases concurrent with increases in AQP5 during AT2 to AT1 cell transdifferentiation. We have previously shown that GATA6 mediates activation of Aqp5, largely through interactions with Sp1 at the proximal promoter/enhancer region, further supporting a role for GATA6 in regulation of AT1 cell differentiation 26 . In this study, we demonstrate that GATA6 regulation of the AT1 cell-specific gene Aqp5 is associated with increased histone acetylation. Our data suggest that GATA6/Sp1 interaction alters acetylation by interfering with HDAC3/Sp1 binding and recruiting p300 43,44 . Aqp5 transcriptional regulation and involvement of HDAC3, GATA6, and p300: HDAC3 and GATA6 regulate Sp1-mediated Aqp5 transcription via H3 deacetylation and acetylation, respectively, at the proximal promoter/ enhancer. Mechanisms underlying GATA6-dependent H3 acetylation at the Aqp5 enhancer/promoter might involve competition between GATA6 and HDAC3 for binding to Sp1, as well as recruitment of histone acetylase p300 to the Sp1/GATA6 complex. In addition to effects on H3 acetylation, p300 might also modulate GATA6 and/or Sp1 activity. p300 is a transcriptional coactivator that does not directly bind to DNA. It encompasses different domains that enable p300 to regulate cell-specific gene expression by interacting with various transcription factors as an adaptor 52 . Additionally, the HAT domain of p300 catalyzes acetylation of promoter-bound histones, leading to chromatin opening and gene activation 36 . p300 can also acetylate transcription factors such as NF-κB 53 , Smad 37 , p53 54 and Sp1 43,44,55 , as well as GATA family members GATA1 56 and GATA4 57 , to modulate their transcriptional activity via changes in DNA binding activity, protein stability and interactions with other transcription factors. It will be important to elucidate if p300 mediates activation of Aqp5 transcription by GATA6/Sp1 through histone acetylation, direct acetylation of GATA6/Sp1 or a combination thereof or independent of its acetylation activity. We propose a model (Fig. 5) in which GATA6 activates Aqp5 transcription by interfering with HDAC3 binding to Sp1 leading to increased H3 acetylation, as well as recruitment of p300 that may acetylate H3 through its HAT activity and/or serve as a transcriptional coactivator.
Interestingly, p300 but not CBP is involved in GATA6/Sp1-mediated Aqp5 transcription. Despite high homology between these coactivators, they differentially regulate cell function, with CBP/β-catenin interaction implicated in progenitor cell maintenance and p300/β-catenin promoting differentiation 58,59 . We recently showed that p300, but not CBP, promotes AT2 to AT1 cell transdifferentiation through interaction with β-catenin 38 . Our data confirm previous findings that p300, but not CBP, plays a role in activation of AT1 cell-specific gene Aqp5. However, involvement of β-catenin in Aqp5 gene activation mediated by the GATA6/Sp1/p300 complex reported here will require further investigation.
Our data strongly suggest that correct spatial and temporal regulation of Aqp5 transcription via specific transcription factor interactions and histone modifications is important to maintain AT1 cell-specific gene expression. Altered epigenetic modifications and transcription factor binding on the Aqp5 promoter may contribute to disease pathogenesis and impair AT1 cell recovery following injury. HDAC inhibitors may therefore be beneficial to promote re-epithelialization of the lung in response to injury.

Co-immunoprecipitation (Co-IP).
Total protein lysate (70 µg in 200 µl RIPA buffer) from MLE-15 cells transduced for 48 hours with lentivirus expressing Hdac3 or non-silencing shRNA was used for each IP. Thirty µl of cross-linked HDAC3, Sp1, GATA6 Ab or corresponding IgG was added to each sample and incubated in a rotator overnight at 4 °C. After washes in RIPA buffer, immunoprecipitated proteins were eluted in Laemmli sample loading buffer and boiled for 15 min. Samples were then subjected to western analysis for HDAC3, GATA6, Sp1 and p300 (#SC 584, Santa Cruz Biotechnology).

Statistical analysis.
Data are shown as mean ± SEM, where (n) is the number of observations. We performed two-way ANOVA, t-tests and z-tests for ratiometric data to determine significance. P < 0.05 was considered significant.