Abstract
Purpose: :
Health and longevity critically depend on lifestyle and diet. This long-standing paradigm has recently been substantiated by the discovery of the mammalian SIR2 ortholog SIRT1 (Sirtuin1) at the center of the physiological effects of a calorie restriction diet. As a predominantly nuclear deacetylase, SIRT1 modifies the function of target proteins in a NAD+ - dependent manner. Known protein targets of SIRT1 include the tumor suppressor p53, NFkappaB, and histones H3 and H4. Whereas much of the attention has focused on the consequences of SIRT1/p53-interaction, surprisingly little is known about the cellular effects of SIRT1-induced histone deacetylation, which is generally associated with transcriptional repression. Using human embryonic kidney cells (HEK 293) as cellular model, we have identified endogenous DNA targets that associate with SIRT1.
Methods: :
To achieve the experimental goal, we used a modified chromatin immunoprecipitation (ChIP) approach, followed by DNA cloning. SIRT1-containing protein/DNA complexes were isolated in two parallel but independent approaches, of which one relied on a polyclonal - and the other a monoclonal antibody directed against human SIRT1. After the elimination of all protein, short (500-1000 bp) endogenous DNA fragments were ligated to DNA-adapters, amplified by PCR, and transferred into a standard DNA cloning vector. Following bacterial amplification, the cloned DNA was sequenced and identified with the "Basic Local Alignment Search Tool" (BLAST; www.ncbi.nlm.nih.gov/blast/). A selected number of identified DNA targets were verified by PCR using sequence-specific DNA-primers on unamplified ChIP DNA.
Results: :
The sequence analysis of approximately 100 clones revealed more than 30 different DNA-targets directly or indirectly associated with human SIRT1. More than 60% of the identified DNA sequences corresponded to a particular open reading frame (ORF) with the majority being intronic. Most of the genes found associated with SIRT1 fall into three main categories: Cell cycle and cancer, immune response, and neuronal phenotype.
Keywords: aging • cell survival • gene/expression