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

Abstract

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 Ca 2ϩ /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 ␣-myosin heavy chain (␣-MyHC) gene in cardiac myocytes (21, 32). Interestingly, we showed that repression is dependent on a region of the protein known to interact with...

YY1 Protects Cardiac Myocytes from Pathologic Hypertrophy by Interacting with HDAC5

“…However, the factors that mediate the affinity of YY1 for HDAC5 would also be critical for the proper regulation of development in the mammalian system. In experiments with undifferentiated and differentiated H9C2 cells, YY1 acts as a transcription repressor only in differentiated cells (Sucharov et al, 2006). Last, we show that HDAC5 is necessary for YY1 function as a transcription repressor of the fetal isoforms in cardiac cells.…”

“…As shown in Figure 8A, only the YY1 174 -200 deletion construct was unable to interact with HDAC5 in COS cells. Previous work with these YY1 mutants showed that this deletion construct had the unique ability to activate promoters in cells where YY1 is known to be a repressor of transcription (Sucharov et al, 2006). Using Amaxa's nucleofection technology, the YY1 174 -200 deletion construct was transfected into NRVMs, resulting in the up-regulation of all fetal mRNAs tested.…”

Nitric Oxide Modulates Chromatin Folding in Human Endothelial Cells via Protein Phosphatase 2A Activation and Class II Histone Deacetylases Nuclear Shuttling

“…Class II HDACs are nuclear in their unphosphorylated form 22 and a physical association between HDAC5 and PP2A has also been reported. 23 According to these evidences, we demonstrated that HDAC4 (and HDAC5, not shown) failed to localize to the nucleus of ECs in the presence of small-t antigen ( Figure 4B), a well known inhibitor of PP2A, 24 which also counteracted NO-induced increase of cellular phosphatase activity ( Figure 4A). Moreover, we show that PP2A was bound to HDAC4 and HDAC5 in a multiprotein complex, by using 293 cells stably expressing an HDAC4-Flag fusion protein ( Figure 5).…”

“…It has been recently demonstrated that CaMKIV and the protein phosphatase PP2A play a role in regulating HDAC5 subcellular localization, 23 hence we hypothesized that a PP2A-related activity was involved in NO-dependent HDAC4 nuclear shuttling. To this aim, HUVECs were infected with an adenovirus encoding for the viral small-t antigen oncoprotein, a well known PP2A inhibitor, 24 and after 72 hours were treated for 1 to 4 hours with DETA/NO.…”

Class IIa histone deacetylases: conducting development and differentiation

“…In addition, results from our laboratory and others have revealed that another cellular phosphatase, PP2A, stably associates with and constitutively dephosphorylates class IIa HDACs in vivo Paroni et al, 2008). Accordingly, PP2A participates in the regulation of class IIa HDAC subcellular localization and directly impacts on their biological functions (Illi et al, 2008;Martin et al, 2008;Paroni et al, 2008;Sucharov et al, 2006). The sequential and/or coordinated actions of these multiple protein kinases and phosphatases would constitute a tightly regulated mechanism allowing appropriate, rapid, and reversible expression of class IIa HDAC target genes in response to specific developmental signals.…”