Regulation of mouse histone deacetylase 1 expression

Reversible histone acetylation induces changes in the chromatin structure and thereby modulates gene expression in eukaryotic cells. Histone acetyltransferases and histone deacetylases control the dynamic acetylation of core histones. Histone deacetylase 1 (HDAC1) is a nuclear protein that belongs to a growing family of evolutionary conserved enzymes catalyzing the removal of acetyl residues from histones and other proteins. HDAC1-mediated transcriptional regulation in manimalian cells was shown to be involved in a variety of biological processes including proliferation, cell cycle progression, differentiation and development. Previously we have identified HDAC1 as a growth factor-inducible protein in mouse T cells. HDAC 1 mRNA and protein expression are significantly increased in proliferating cells. Recent results from our group show that both overexpression and lack of HDAC1 lead to impaired cell cycle progression indicating that a tight control of HDAC1 activity might be crucial for unrestricted proliferation of mammalian cells. Additional data from our laboratory also suggest that the expression of HDAC1 is regulated in an auto-regulatory loop. Given the involvement of HDAC1 in several biological regulatory circuits, it is important to understand how the expression of this enzyme is modulated in mammalian cells. In this project we will analyze these regulatory mechanisms in more detail. We will characterize the elements within the HDAC1 promoter required for transcriptional activity and growth factor-dependent regulation. Furthermore we win identify the transcription factors which regulate the expression of HDAC1 in mammalian cells during the cell cycle entry. The second part of the project deals with a potential autoregulatory mechanism, that modulates HDAC1 expression in the presence of high cellular HDAC activity. HDAC1 levels in resting fibroblasts are strongly induced by inhibition of HDAC activity. The activation of HDAC1 is enhanced by activation of the MAP and SAP kinase pathways suggesting a cooperative regulation by acetylation and phosphorylation. We will examine which elements within the HDAC1 gene are required for induction by either of the two signals. Identification of putative binding sites necessary for regulation of the HDAC1 promoter will allow for characterization of the corresponding transcription factor complexes and their function. Finally we will test by chromatin immunoprecipitation if particular regions of the HDAC1 gene are hyperacetylated upon activation and if the kinase pathways directly affect histone phosphorylation within the HDAC1 locus.

In this project we analyzed how the packaging of our genetic material affects the expression of mammalian genes. The DNA is organized as chromatin with the nucleosome as basic unit that consists of histones with two turns of DNA wrapped around. Modification of histones by acetylation induces the opening of chromatin and increases the accessibility for factors required for gene expression. Phosphorylation of histones is linked at one hand to cell division and on the other hand to the activation of immediate early genes. By using the histone deacetylase 1 (HDAC1) gene as model system we examined the effect of histone acetylation and phosphorylation for the activation of this late inducible gene. We identified SP1 and NF-Y as the transcription factors required for the activation of the HDAC1 gene. Acetylation of histones associated with the regulatory region of the HDAC1 gene was found to be linked to transcriptional induction. Recruitment of HDAC1 to the promoter of its own gene and the resulting down regulation of HDAC1 expression allows the feedback regulation, when intracellular HDAC1 levels are high. In addition, phosphorylation of histones at the HDAC1 promoter occurred in proliferating cells, but not in resting cells. Phosphorylation and acetylation cooperated in the activation of the HDAC1 gene. While the acetylation mark reflects the balance between acetylating and deacetylating enzymes, the phosphorylation mark is regulated by growth factor signals. Furthermore, we have analyzed the role of histone acetylation in the regulation of the CDK inhibitor gene p21/WAF1. We showed that the tumor suppressor p53 and HDAC1 were antagonistic regulators of the p21 gene. Both proteins compete for binding to the transcription factor SP1 and thereby for association with the p21 promoter. HDAC1 represses p21 expression in tumor cells thereby enabling unrestricted proliferation. Finally, we investigated the regulation of the thymidine kinase/ kynurenine formamidase gene cluster by growth factors. Thymidine kinase expression that is linked to proliferation was found to correlate with histone acetylation, while transcription of the kynurenine formamidase gene in resting cells is accompanied by histone hypoacetylation. This is the rare example of a mammalian gene whose expression is linked to reduced acetylation of its promoter. Taken together, our data show how histone modifications such as acetylation and phosphorylation allow integrating different signal into the regulatory network that controls mammalian gene expression.