Regulation of Histone Deacetylase 4 Expression by the SP Family of Transcription Factors

Abstract

Histone deacetylases mediate critical cellular functions but relatively little is known about mechanisms controlling their expression, including expression of HDAC4, a class II HDAC implicated in the modulation of cellular differentiation and viability. Endogenous HDAC4 mRNA, protein levels and promoter activity were all readily repressed by mithramycin, suggesting regulation by GC-rich DNA sequences. We validated consensus binding sites for Sp1/Sp3 transcription factors in the HDAC4 promoter through truncation studies and targeted mutagenesis. Specific and functional binding by Sp1/Sp3 at these sites was confirmed with chromatin immunoprecipitation (ChIP) and electromobility shift assays (EMSA). Cotransfection of either Sp1 or Sp3 with a reporter driven by the HDAC4 promoter led to high activities in SL2 insect cells (which lack endogenous Sp1/Sp3). In human cells, restored expression of Sp1 and Sp3 up-regulated HDAC4 protein levels, whereas levels were decreased by RNA-interference-mediated knockdown of either protein. Finally, variable levels of Sp1 were in concordance with that of HDAC4 in a number of human tissues and cancer cell lines. These studies together characterize for the first time the activity of the HDAC4 promoter, through which Sp1 and Sp3 modulates expression of HDAC4 and which may contribute to tissue or cell-line-specific expression of HDAC4. INTRODUCTIONHistone deacetylases (HDACs) all share the ability to deacetylate specific lysines in the tail residues of the core histones, generally resulting in the compaction of chromatin, transcriptional repression, and silencing. There has been an explosion of interest in recent years in the HDACs, because of the panoply of critical cellular functions that have now been linked to HDACs (Grozinger and Schreiber, 2002;Verdin et al., 2003;Sengupta and Seto, 2004;Yang and Gregoire, 2005) .HDACs in general lack DNA binding activity, and appear to mediate their activities as part of large multiprotein complexes (such as the NuRD, Sin3, and CoREST complexes; Hassig et al., 1997;Laherty et al., 1997;Zhang et al., 1997Zhang et al., , 1998, which include non-HDAC proteins and other unique polypeptides that together modulate histone deacetylase activity. An example of the regulation of HDAC activity mediated by protein-protein interactions relates to the SMRT/ N-CoR complex. The silencing mediator of retinoid and thyroid receptor (SMRT) and nuclear receptor corepressor (N-CoR) are nuclear receptor corepressors that bind and enhance the HDAC activity of HDAC3 (Alland et al., 1997;Heinzel et al., 1997;Wen et al., 2000;Guenther et al., 2001;. In contrast to the effects on HDAC3, the deacetylase activity of the class II HDACs (HDAC4, 5, 7, and 9) are not enhanced by binding to the SMRT/N-CoR complex, suggesting that the class II HDACs may recruit enzymatically active HDAC3-SMRT/N-coR complexes for their functional effects (Fischle et al., 2002). Regulation of the expression of class II HDACs may therefore influence the overall function of such multiprotein complexe...

HDAC4 Promotes Growth of Colon Cancer Cells via Repression of p21

“…Because class IIa HDACs have been reported to be expressed in a tissue-specific manner (Wang et al, 1999;Liu et al, 2006), we first confirmed the expression of HDAC4 in normal intestinal epithelium. Although considerable variability in HDAC4 protein expression was observed among a panel of different mouse tissues, robust HDAC4 expression was detected in the small intestine and colon ( Figure 1A).…”

“…Mithramycin is an established inhibitor of the binding of transcription factors, such as Sp1, to GC-rich elements (Liu et al, 2006). As shown in Figure 9A, siHDAC4 was unable to stimulate p21 expression in the presence of mithramycin.…”

“…HCT116 cells were treated for 48 h with NT or siHDAC4 (both 100 nM), with and without concomitant addition of 5 g/ml cycloheximide for a subsequent 24 h. functionally inhibited. Mithramycin is an established inhibitor of the binding of transcription factors, such as Sp1, to GC-rich elements (Liu et al, 2006). As shown in Figure 9A, siHDAC4 was unable to stimulate p21 expression in the presence of mithramycin.…”

HDAC4 Represses Matrix Metalloproteinase-13 Transcription in Osteoblastic Cells, and Parathyroid Hormone Controls This Repression

“…Interestingly, we have found that PTH induces HDAC4 gene expression and enzymatic activity in osteoblastic cells. The rat HDAC4 promoter has not been identified yet, but we have conducted homology searches of the human HDAC4 promoter that Liu et al (35) have reported. The human HDAC4 promoter has GC-rich DNA sequences, which bind Sp-1/Sp-3.…”

Transcriptional regulation of the human CD97 promoter by Sp1/Sp3 in smooth muscle cells

“…To get an indication whether the GC-rich sites influence CD97 transcriptional regulation, we treated cell cultures with mithramycin, a cell-permeable agent that binds to GC-rich DNA sequences and that is frequently used to explore the sequence dependency of DNA-binding 11 factors (Liu et al, 2006). Since CD97 promoter activities as well as mRNA expression levels were decreased by mithramycin in SMCs, we studied the GC-sites in more detail using reporter gene assays as well as gelshift and ChIP analysis.…”