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Am J Physiol Cell Physiol 291: C1029-C1037, 2006. First published July 5, 2006; doi:10.1152/ajpcell.00059.2006
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GROWTH, DIFFERENTIATION, AND APOPTOSIS

Shuttling of HDAC5 in H9C2 cells regulates YY1 function through CaMKIV/PKD and PP2A

Carmen C. Sucharov,1 Stephen Langer,2 Michael Bristow,1 and Leslie Leinwand2

1Division of Cardiology, School of Medicine, University of Colorado Health Sciences Center, Denver, Colorado; and 2Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado

Submitted 7 February 2006 ; accepted in final form 3 May 2006


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
YY1 is a transcription factor that can activate or repress transcription of a variety of genes and is involved in several developmental processes. YY1 is a repressor of transcription in differentiated H9C2 cells and in neonatal cardiac myocytes but an activator of transcription in undifferentiated H9C2 cells. We now present a detailed analysis of the functional domains of YY1 when it is acting as a repressor or an activator and identify the mechanism whereby its function is regulated in the differentiation of H9C2 cells. We show that histone deacetylase 5 (HDAC5) is localized to the cytoplasm in undifferentiated H9C2 cells and that this localization is dependent on Ca2+/calmodulin-dependent kinase IV (CaMKIV) and/or protein kinase D (PKD). In differentiated cells, HDAC5 is nuclear and interacts with YY1. Finally, we show that HDAC5 localization in differentiated cells is dependent on phosphatase 2A (PP2A). Our results suggest that a signaling mechanism that involves CaMKIV/PKD and PP2A controls YY1 function through regulation of HDAC5 and is important in the maintenance of muscle differentiation.

differentiation

YY1 IS A UBIQUITOUSLY expressed, highly conserved transcription factor that has been implicated in a variety of cellular processes, including development, where it can function as a polycomb protein (1). Through its interactions with other proteins, YY1 can repress, activate, or initiate transcription from various promoters (for a review, see Ref. 33). Previous work has suggested that YY1 functions primarily as a repressor and that its activator function is dependent on a conformational change that alters its repression domain (4, 13, 14). YY1 has also been shown to displace transcriptional activators from promoters, resulting in transcriptional repression (15, 33). Posttranslational modifications of YY1, including acetylation and deacetylation, also play roles in its various functions (38). Acetylation of histones results in derepression of transcription due to an opening of the chromatin structure, whereas deacetylation results in transcription repression. YY1 has been shown to interact with class I histone deacetylases (HDACs) 1, 2, and 3 in HeLa cells (38), and this may be a mechanism of gene regulation shared by YY1 and histones. There are three classes of HDACs (I, II, and III) based on their homology with three structurally and biochemically distinct yeast HDACs (reviewed in Refs. 24 and 25). Muscle cells express all three HDAC classes (40). Localization of class II HDACs is regulated by phosphorylation; they translocate to the cytoplasm when phosphorylated, which results in transcription derepression (22).

In muscle cells, YY1 has been shown to repress most muscle-specific genes tested with the exception of the BNP promoter (2, 6, 29). We have recently shown that YY1 represses gene expression and promoter activity of the human and rat {alpha}-myosin heavy chain ({alpha}-MyHC) gene in cardiac myocytes (21, 32). Interestingly, we showed that repression is dependent on a region of the protein known to interact with HDACs (21, 32). We also showed that YY1 activates transcription of the {alpha}-MyHC promoter in undifferentiated H9C2 cells and that it represses transcription of the same promoter in differentiated H9C2 cells (14). H9C2 cells are derived from a rat atrial embryonic tumor and have characteristics of both skeletal and cardiac muscles (26), and differentiation with retinoic acid (RA) results in the activation of a more cardiac phenotype (11, 26). Here, we show that the different domains of YY1 have different functions depending on the cell type and the differentiation state of the cell. We also show that YY1 and HDAC5 act in concert to repress the activity of the {alpha}-MyHC promoter in H9C2 cells. Our results show that HDAC5 is largely cytoplasmic in undifferentiated H9C2 cells and its localization is dependent on Ca2+/calmodulin-dependent kinase IV (CaMKIV) and/or protein kinase D (PKD). We also show that HDAC5 migrates to the nucleus in differentiated cells and that this migration is dependent on phosphatase 2A (PP2A). More importantly, YY1 interacts with HDAC5 only in differentiated cells, and its function as a repressor is dependent on colocalization with HDAC5 in the nucleus, suggesting that YY1's interaction with HDAC5 is necessary for its function as a repressor in muscle cells and possibly for maintaining a terminal differentiated state of the cells.


   METHODS
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RESULTS
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Antibodies. YY1 (SC-7341X) and GAPDH (SC-20357) antibodies were purchased from Santa Cruz Biotech. Flag antibody (F3165) was purchased from Sigma. The horseradish peroxidase (115-035-146) anti-mouse antibody was purchased from Jackson Laboratories. Alexa Fluor 594 (A11032) anti-mouse antibody was purchased from Molecular Probes.

Plasmid construct. The –454/+32-bp fragment of the human {alpha}-MyHC promoter was cloned into the pGL3 basic vector (Promega). For the cotransfection experiments with GAL4-YY1 cDNA construct, the YY1 binding site in the {alpha}-MyHC promoter was substituted by a GAL4 binding site. The YY1 expression construct and all GAL4-YY1 deletions constructs were a gift from Dr. Michael Atchison (University of Pennsylvania). The YY1 construct containing the 170–200 deletion was a gift from Dr. Ed Seto (University of Florida). The DNA constructs were purified with the Qiagen method. The HDAC-Flag adenovirus construct was a gift from Dr. Tim McKinsey (Myogen), and the YY1 adenovirus construct was a gift from Dr. Aristidis Moustakas (Ludwig Institute of Cancer Research).

Cell culture and transfection. Neonatal rat ventricular myocytes (NRVM) were prepared according to the method described by Waspe et al. (35). Briefly, 150,000 cells/well were plated in 12-well tissue culture plates coated with gelatin. Eighteen hours later, the medium was changed to MEM supplemented with Hanks' salt and L-glutamine. HEPES (20 mM; pH 7.5), penicillin, vitamin B12, BSA, insulin, and transferrin were added to the medium. Transfections were carried out by the Fugene 6 (Roche) method according to manufacturer's recommendations; 0.75 µl of Fugene/0.25 µg of plasmid DNA were transfected in each well. In the cotransfection experiments, the total amount of DNA was kept constant by the addition of a plasmid containing the cytomegalovirus promoter not driving the expression of any gene. H9C2 cells were maintained according to American Type Culture Collection recommendations. Transfection in H9C2 cells was done by the Fugene method; 0.18 µl of Fugene/0.06 µg of DNA were transfected in each well on a 24-well plate. All transfection experiments were done in triplicates and were repeated 6–10 times. RA differentiation of H9C2 cells was done by treatment of the cells with 10 nM RA everyday for 5–7 days in medium with 1% FBS. Infection of H9C2 cells was done at an MOI of 20 pfu/cell. All transfection experiments were repeated at least three times with triplicates in each experiment.

Nuclear and cytoplasmic fractionation. Nuclear and cytoplasmic fractionations were performed with the NE-PER kit (Pierce) according to the manufacturer's recommendation.

Western blots. Western blots were performed essentially as described (32). YY1 or Flag antibody was diluted 1:1,000 in 1x TBS (20 mM Tris, 500 mM NaCl, pH 7.5) containing 3% BSA and 0.1% Tween 20 and incubated with the blot overnight at 4°C. The mouse secondary antibody conjugated to horseradish peroxidase was diluted 1:5,000 in 1x TBS containing 5% low-fat dry milk and 0.1% Tween 20 and incubated with the blot for 1 h at room temperature.

Immunoprecipitation/immunobloting. Immunoprecipitation experiments were done with YY1 and Flag antibodies. Experiments were done according to Santa Cruz Biotech recommendations with minor modifications and as described previously (31). After four washes with 1x RIPA 1640 buffer (5), the sample was incubated with 2–3x packed volume of 2x sample buffer (Bio-Rad) and incubated at room temperature for 30 min. beta-Mercaptoethanol was added to the supernatant after centrifugation, and samples were loaded without boiling. Western experiments were done as described above.

Immunofluorescence. Immunofluorescence of H9C2 cells was done according to Harrison et al. (8). Cells were washed with TBS-Tween 20 (TBST) and fixed with 10% formaldehyde for 20 min. Cells were again washed with TBST and incubated with 1% BSA in TBST for 1 h followed by 1-h incubation with a 1:500 dilution of the Flag antibody. Cells were then washed with TBST and incubated with a 1:1,000 dilution of Alexa 594 anti-mouse antibody and 2 µg/ml Hoechst staining for 1 h. Cells were washed three times with TBST and one time with water and sequentially covered with mounting solution (Southern Biotech) and glass coverslips. Images were captured at a x20 magnification with a fluorescence microscope (Nikon E800) equipped with a digital camera (Zeis AxioCam) and Zeis Axiovision version 3.0.6.36 [EC] imaging software.

Statistical analysis. All analyses were performed using ANOVA, with a P < 0.05 in a two-tailed distribution considered to be statistically significant. All statistical analyses are a result of comparisons to control experiments described in the text and defined as 100%.


   RESULTS
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 METHODS
 RESULTS
DISCUSSION
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YY1 functional domains have diverse activity in different cell types. To understand whether the function of the previously mapped domains of YY1 varies according to cell type and to YY1 function within specific cells, undifferentiated and differentiated H9C2 cells and NRVMs were cotransfected with the {alpha}-MyHC promoter linked to luciferase and various YY1 deletion constructs. In Fig. 1B, we show that these constructs were equally expressed in undifferentiated H9C2 cells. YY1 is a repressor of {alpha}-MyHC promoter activity in RA-differentiated H9C2 cells and in NRVMs but an activator in undifferentiated cells (32). Because YY1 interacts with the {alpha}-MyHC promoter and because it differentially regulates its promoter activity depending on the differentiated state of the cell, {alpha}-MyHC is a unique reporter to determine YY1 function in the cells. Various groups have described several distinct functional domains of YY1 (reviewed in Ref. 33), and the function of these domains can vary according to the cell type or promoter region. To identify the domains responsible for the YY1 function, NRVMs and differentiated H9C2 cells (where YY1 is a repressor) and undifferentiated H9C2 cells (where YY1 is an activator) were transfected with wild type YY1 (1–414) or various deletion constructs and with the {alpha}-MyHC promoter. Because some of the YY1 deletion constructs lack the DNA binding domain, the YY1 constructs were tagged with a GAL4 binding domain, with the exception of the YY1 170–200 deletion construct. Therefore, for the transfection experiments with the YY1-GAL4 constructs, the {alpha}-MyHC promoter construct used had the YY1 binding site substituted by a GAL4 binding site. Transfection experiments with the YY1 170–200 deletion construct were done with the wild-type {alpha}-MyHC promoter construct. The NH2 terminus of the protein has been described as an activation domain by some groups and a repression domain by another group (33). As shown in Fig. 1A, transfection of a construct in which the NH2-terminal region had been deleted (deletion 16–100) results in reduced activation of the {alpha}-MyHC-luciferase reporter in undifferentiated cells, suggesting that this region is necessary for YY1 to function as an activator under these conditions. Interestingly, however, deletion of this region does not significantly alter the repressive activity of YY1 in NRVMs. The COOH-terminal region of the protein has also been shown to function as an activator or repressor of transcription. Our results show that deletion of the COOH terminus (construct 1–256) results in a loss of repression in NRVMs and a reduced or loss of activation in undifferentiated cells, suggesting that the COOH-terminal region can function as a repressor or activator. In fact, this region can interact with both histone acetyltransferases (HATs) and HDACs. The DNA binding domain of YY1 was deleted in this construct, and, to ensure DNA binding, it was tagged with a GAL4 DNA binding domain. The {alpha}-MyHC promoter construct used in these studies had the YY1 binding site substituted by a GAL4 binding site. In addition, the deletion of the 170–200 region of the protein functions only to alter the repressive function of YY1 (32). This region has been shown to interact with HDACs. The fact that YY1 cannot repress transcription when this region is deleted suggests that interaction with HDACs is necessary for YY1 to function as a transcription repressor. These data suggest that the cellular context and the factors that interact with specific domains of YY1 are essential for its proper function as a repressor or activator. Determination of domains that mediate YY1-HDAC5 interactions will provide valuable information to better understand the mechanisms regulating their activities.


Figure 1
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  		Fig. 1. Analysis of YY1 functional domains in neonatal rat ventricular myocytes (NRVMs) and differentiated and undifferentiated H9C2 cells. A: H9C2 cells and NRVMs were transfected with {alpha}-myosin heavy chain ({alpha}-MyHC) promoter construct and various YY1 deletion constructs as shown. Undifferentiated H9C2 cells and NRVMs were harvested 48 h posttransfection. Differentiated H9C2 cells were harvested 5 days after retinoic acid (RA) treatment. Luciferase assay for each construct was normalized to transfection of the {alpha}-MyHC promoter with an empty vector. The control experiment is defined as 100%. B: expression of each construct in H9C2 cells. Samples were subjected to Western blot procedure with either the GAL4 antibody (1–3) or the FLAG antibody (4). Generation and migration of these constructs were described by others (3, 38).

 
HDAC5 cellular localization varies depending on the differentiated state of the cells. Class II HDACs are expressed in muscle cells and are regulated by phosphorylation, making them an attractive candidate for cell-specific differential regulation of YY1 activity. Phosphorylated class II HDACs undergo nuclear export, and this results in transcriptional derepression (22). Because YY1 is a transcriptional repressor only in differentiated cells, it is possible that HDAC5 is only nuclear in these cells. To test this hypothesis, nuclear and cytoplasmic fractions of HDAC5-Flag-infected differentiated and undifferentiated H9C2 cells were prepared and submitted to Western blot experiments. As shown in Fig. 2A, HDAC5 is mostly cytoplasmic in undifferentiated H9C2 cells but migrates to the nucleus in differentiated cells. We next tested whether the endogenous protein also had its localization changed in response to RA treatment. As shown in Fig. 2B, endogenous HDAC5 is also mostly cytoplasmic in undifferentiated cells but nuclear in differentiated cells. Similarly, immunofluorescence of differentiated H9C2 cells showed virtually the same results (Fig. 2C). This difference in HDAC5 localization correlates with the activity of YY1 in these cells. In undifferentiated cells, HDAC5 is cytoplasmic, and YY1 in those cells is an activator of transcription. Conversely, in differentiated cells, HDAC5 is nuclear and YY1 is a repressor of transcription. In contrast, YY1 localization does not change according to the differentiated state of the cells, and it is always nuclear (data not shown)


Figure 2
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  		Fig. 2. Histone deacetylase 5 (HDAC5) is mostly cytoplasmic in undifferentiated H9C2 cells and migrates to the nucleus in differentiated H9C2 cells. A: H9C2 cells were infected with the YY1 and HDAC5-FLAG viruses and harvested 48 h postinfection or treated with RA for 5 days and harvested. Cells were fractionated in nuclear and cytoplasmic fractions, and both fractions were submitted to Western blot experiments with the FLAG antibody. A GAPDH antibody was used as a loading control for the cytoplasmic fraction, whereas the Sp1 antibody was used as a control for the nuclear fraction. Lanes 1 and 3, undifferentiated H9C2 cells; lanes 2 and 4, differentiated H9C2 cells. B: Western blot of endogenous HDAC5 in undifferentiated and differentiated H9C2 cells. Lanes 1 and 3, undifferentiated H9C2 cells; lanes 2 and 4, differentiated H9C2 cells. Bottom: results of 3 independent experiments. C: differentiated and undifferentiated HDAC5-FLAG-infected cells were submitted to immunofluorescence experiments using the FLAG antibody. Nuclei were defined with Hoechst staining. Right: merging of FLAG and Hoechst staining.

 
HDAC5 interacts with YY1 in differentiated H9C2 cells. From the transfection results, the 174–200 region of YY1 is important for its repressive function in both differentiated H9C2 cells and NRVMs. This region is also known to interact with class I HDACs in HeLa cells (38). As described, YY1 and HDAC5 colocalize in differentiated cells, where YY1 is a repressor. To determine whether YY1 interacts with the class II HDAC5, H9C2 cells were infected with the HDAC5-Flag and YY1-GFP viruses for 48 h or infected on day 1 and differentiated with RA for 5 days followed by nuclear and cytoplasmic fractionation and immunoprecipitation of the nuclear and cytoplasmic fractions. In a control experiment, cells were infected with the HDAC5-Flag virus and a GFP virus. As shown in Fig. 3, HDAC5 interacts with YY1 only in differentiated cells, suggesting that this is the mechanism that results in YY1-repressive activity in these cells. Immunoprecipitation was specific because no protein was detected in the HDAC5-Flag/GFP experiment.


Figure 3
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  		Fig. 3. YY1 interacts with HDAC5 in differentiated H9C2 cells. H9C2 cells were infected with the YY1 and HDAC5-FLAG or GFP and HDAC5-FLAG viruses and harvested 48 h postinfection or treated with RA for 5 days and harvested. Cells were fractionated in nuclear and cytoplasmic fractions, and nuclear fractions were submitted to immunoprecipitation experiments. A: immunoprecipitated (IP) with YY1 antibody and Western blot with Flag antibody. Lane 1, undifferentiated H9C2 cells; lane 2, differentiated H9C2 cells; lane 3, GFP and HDAC5-FLAG infection in differentiated H9C2 cells. B: IP with FLAG antibody and Western blot with YY1 antibody. Lane 1, undifferentiated H9C2 cells; lane 2, differentiated cells; lane 3, GFP and HDAC5-FLAG infection in differentiated H9C2 cells.

 
HDAC5 cytoplasmic localization in undifferentiated cells is dependent on CaMKIV/PKD. HDAC5 cytoplasmic localization has been shown to be dependent on CaMKIV, Ca2+/calmodulin-dependent kinase II (CaMKII), and protein kinase C (PKC) or PKD (18, 22, 34). H9C2 cells express both CaMKII and CaMKIV (9, 37). To test the mechanism of HDAC5 cytoplasmic localization in undifferentiated H9C2 cells, HDAC5-Flag-infected cells were treated for 2 h with 1 µM of the general kinase inhibitor K252A (39), 2 µM of the CaMKII inhibitor KN-93 (12, 28), 10 µM of the PKC inhibitor Bis I, and 10 µM of the PKD inhibitor Gö-6976 (34). As shown in Fig. 4A, treatment with the PKC inhibitor Bis I failed to change HDAC5 localization. Treatment with the CaMKII inhibitor KN-93 resulted in partial localization of HDAC5 to the cytoplasm. Treatment with the general kinase inhibitor K252A and the PKD inhibitor Gö-6976 resulted in nuclear localization of HDAC5. K252A is a general kinase inhibitor that can inhibit CaMKIV and PKD. Both proteins have been shown to phosphorylate HDAC5, and they are both present in H9C2 cells (data not shown). Therefore, these results suggest that HDAC5 phosphorylation and cytoplasmic localization can be largely dependent on CaMKIV or PKD.


Figure 4
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  		Fig. 4. HDAC5 cytoplasmic localization in undifferentiated cells is dependent on CaMKIV. A: H9C2 cells were infected with the HDAC5-FLAG viruses and treated with K252A, Bis I, KN-93, or Gö-6976 for 2 h. Cells were submitted to immunofluorescence experiments using the FLAG antibody. Nuclei were defined with Hoechst staining. Right panels: merging of FLAG and Hoechst staining. Control shown in Fig. 2B. B: undifferentiated H9C2 cells were transfected with the {alpha}-MyHC promoter linked to luciferase or the cytomegalovirus (CMV)-luciferase (luc) control. Cells were treated with K252A for 48 h, harvested, and assayed for luciferase activity. The results were normalized to {alpha}-MyHC-luc promoter activity, defined as 100%. C: undifferentiated H9C2 cells were transfected with the {alpha}-MyHC promoter linked to luciferase and the YY1 cDNA. Cells were treated with K252A for 48 h, harvested, and assayed for luciferase activity. The results were normalized to {alpha}-MyHC-luc promoter activity, defined as 100%.

 
Inhibition of CaMKIV/PKD results in change in YY1 function in undifferentiated cells. To show a correlation between HDAC5 localization and YY1 function, undifferentiated H9C2 cells were cotransfected with the {alpha}-MyHC promoter and YY1 cDNA. Cells were then treated with the kinase inhibitor K252A. As shown in Fig. 4B, treatment with K252A results in repression of {alpha}-MyHC promoter activity and in reduced YY1 function as a transcriptional activator (Fig. 4C). {alpha}-MyHC inhibition in response to K252A treatment is specific because this treatment resulted in upregulation of a control SV40-luciferase construct.

PP2A inhibition results in HDAC5 cytoplasmic localization in differentiated cells. PP2A levels are increased in differentiated H9C2 cells, and PP2A has been shown to dephosphorylate CaMKIV, inhibiting its activity (37, 39). To determine whether HDAC5 nuclear localization in differentiated cells is dependent on PP2A, cells were treated with the PP2A inhibitor okadaic acid for 2 h after the differentiation process. As shown in Fig. 5A, PP2A inhibition results in HDAC5 cytoplasmic localization in differentiated cells, suggesting that this is a PP2A-dependent mechanism.


Figure 5
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  		Fig. 5. HDAC5 nuclear localization in differentiated cells is dependent on phosphatase 2A (PP2A). A: H9C2 cells were infected with the HDAC5-FLAG viruses and treated with RA and okadaic acid for 5 days. Cells were submitted to immunofluorescence experiments using the FLAG antibody. Nuclei were defined with Hoechst staining. Right: merging of FLAG and Hoechst staining. Control shown in Fig. 2B. B: undifferentiated H9C2 cells were transfected with the {alpha}-MyHC promoter linked to luciferase and the PP2A cDNA; 48 h posttransfection, cells were harvested and assayed for luciferase activity. The results were normalized to {alpha}-MyHC-luc promoter activity, defined as 100%.

 
Overexpression of PP2A results in a change of YY1 function in undifferentiated cells. Because inhibition of PP2A results in HDAC5 cytoplasmic localization, we next sought to determine whether overexpression of PP2A in undifferentiated cells results in a change in YY1 function to transcription repressor in these cells. As shown in Fig. 5B, overexpression of PP2A results in decreased YY1 function as a transactivator of the {alpha}-MyHC promoter.


   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
YY1 functional domains. Various groups have studied the functional domains of YY1, and their results are somewhat contradictory. Some of these studies were done in a context in which YY1 is an activator of transcription (3, 4), whereas in others YY1 was a repressor of transcription (14, 17). Because we had the advantage of having a single cell line in which YY1 functions as a repressor or activator of transcription depending on the differentiated state of the cells, we analyzed the function of these different domains in the two differentiated states of the cells and in NRVMs. Our results are in agreement with most groups with respect to the activation domain being localized to the NH2 terminus of the protein (3, 4, 14, 17). However, our data indicate that this region is only important in a cell state in which YY1 is an activator. Once cells are differentiated to a "muscle" phenotype, the repressive function of YY1 overcomes its activator function and deletion of the NH2 terminus does not change the repressive activity of the protein.

Also, in agreement with several published studies, the COOH terminus of the protein functions as a transcriptional repressor in NRVMs (3, 4, 14, 17). Upon deletion of the distal HDAC binding domain (YY1, 1–256), a derepression is observed. Unlike other studies, our results showed that deletion of the 257–414 region of YY1 results in deactivation and derepression, suggesting that, in cardiac and skeletal muscle cells, this region has both an activator and a repressor function. As shown previously (38), this region is both acetylated by pCAF and deacetylated by HDACs, and it is possible that in different cell types this region functions as an activator or repressor of transcription. Our results show that the various domains of YY1 have different functions, depending on the cell type, and may shed some light on further dissecting the function of this transcription factor.

YY1 and cellular differentiation. Recently, YY1 has been identified as a polycomb group (PcG) protein and has been shown to be involved in development (1). During the development and differentiation process, there is a fundamental mechanistic need to maintain key transcription patterns throughout the development and lifetime of an organism. PcG proteins are an essential component of the maintenance of transcription repression in development and differentiation. PcG proteins can repress transcription by generating a chromatin structure that is refractory to gene expression (reviewed in Ref. 16). PcG proteins are well characterized in Drosophila, and YY1 is the first mammalian factor identified to have a PcG function. This suggests that YY1 may be a crucial protein for the proper regulation of development in the mammalian system.

HDAC and the control of muscle differentiation. Class I and II HDACs are expressed in undifferentiated myoblasts, and they interact with transcription factors known to be important for muscle differentiation, thereby playing a role in repression of differentiation (20, 30). In undifferentiated myoblasts, class II HDACs are nuclear, resulting in transcriptional repression. The inhibitory action of class II HDACs can be overcome by myogenic stimulatory signals. These signals result in the phosphorylation of class II HDAC and nuclear export (7, 19, 22, 23, 27). Interestingly, recent work has shown that, in the muscle cell line C2C12, HDAC4 is nuclear in the undifferentiated stage, translocates to the cytoplasm during differentiation, and returns to the nucleus once fusion has occurred (27). It is proposed that reentry of HDAC4 into the nucleus after myotube fusion may serve to inhibit a subset of promoters involved in early myogenesis, therefore establishing a terminally differentiated state (27). Terminally differentiated cardiac myocytes show a similar localization of class II HDACs with HDAC4 and HDAC5 being nuclear in those cells. Interestingly, our results show that a similar regulation of HDAC localization occurs in differentiated H9C2 cells, suggesting that these cells may have reached terminal differentiation. Localization of HDAC5 in undifferentiated H9C2 cells is cytoplasmic, and in differentiated cells it is nuclear. Here, we showed that either CaMKIV or PKD is the likely kinase responsible for HDAC5 cytoplasmic localization in H9C2 cells. Our results show that the general kinase inhibitor K252A completely blocks cytoplasmic localization of HDAC5 and that KN-93, a CaMKII inhibitor that has a minor inhibitory effect on CaMKIV (10), partially blocked HDAC5 cytoplasmic localization, suggesting that this is a CaMKIV- and/or PKD-dependent mechanism. PKD is generally thought to be activated by PKC (34). Treatment of the cells with the PKC inhibitor Bis I did not block HDAC5 cytoplasmic localization, arguing against a PKD-mediated effect. However, as shown by Vega et al. (34), inhibition of PKC by Bis I does not always block cytoplasmic localization of HDAC5 in NRVMs. In fact, as shown in Fig. 4A, inhibition of PKD with the PKD specific inhibitor, Gö-6976, did prevent HDAC5 nuclear export, suggesting a PKC-independent effect. Because K252A can inhibit both CaMKIV and PKD and because both proteins have been shown to phosphorylate HDAC5, we cannot determine whether cytoplasmic localization of HDAC5 in undifferentiated cells is dependent on CaMKIV or PKD or whether both contribute to HDAC5 cytoplasmic localization. We also showed that PP2A is involved in HDAC5 nuclear localization in differentiated cells. PP2A levels are increased in differentiated cells (11), and PP2A promotes CaMKIV dephosphorylation (36, 37). We cannot determine whether HDAC5 nuclear localization in differentiated cells is a direct consequence of PP2A dephosphorylation of HDAC5 or whether it is a consequence of CaMKIV inactivation. Functionally, PP2A overexpression affects YY1 transcriptional activity in undifferentiated cells, suggesting that increased PP2A levels are a determinant factor in YY1 function in differentiated cells. We propose that YY1 interaction with HDAC5, and its function as a repressor in differentiated cells, is a mechanism by which terminal differentiation is maintained. In summary, our results suggest that YY1/HDAC5 interaction is an important component of maintenance of muscle differentiation.


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This work was possible with the support of National Heart, Lung, and Blood Institute Grants 2R01 HL-48013 and RO1 HL-56510.


   ACKNOWLEDGMENTS
 
We thank Dr. Tim McKinsey for the HDAC5-Flag virus and for all the very helpful discussions. We also thank Dr. Michael Atchison and Dr. Ed Seto for the YY1 deletion constructs. Finally, we thank Dr. Aristidis Moustakas for the YY1-GFP virus.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. C. Sucharov, Division of Cardiology, School of Medicine, Univ. of Colorado Health Sciences Center, Denver, CO 80262 (e-mail: kika.sucharov{at}uchsc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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