Rolle der Acetylase-Aktivität des transkriptionellen Ko-Regulators p300 in der Differenzierung
Final Report Abstract
Reversible acetylation of proteins by acetyltransferases (HATs) and deacetylases (HDACs) regulates many physiological processes. Changes in protein acetylation occur in certain pathological conditions, in particular cancer, and drugs that reestablish the balance between HATs and HDACs, in particular inhibitors of HDACs, constitute one of the most promising groups of drugs. One of at least 15 HATs in mammals is the p300 protein. To identify physiological and pathological processes that are regulated by p300 acetyltransferase activity, we have generated mice that carry a conditional, dominant negatively acting point mutant allele of p300 enconding a protein that specifically lacks acetyltransferase activity but retains all other biochemical functions of wildtype p300. Mice expressing the acetyltransferase-deficient version of p300 exclusively in Blymphocytes die prematurely with full penetrance. The mice develop an autoimmune disease similar to systemic lupus erythematosus in its pathological manifestations such as splenomegaly, glomerulonephritis, vasculitis, deposition of immune complexes, and production of autoantibodies against double-stranded DMA. Aged mice show a severe reduction of transitional and marginal zone B-cells and generate aberrant mature B-cells. These B-cells show diminished proliferation in response to stimulation of the B-cell receptor, but respond normally to other stimuli. Yet, the mice mount a normal primary immune response against a T-dependent antigen. In contrast, the memory response is impaired. In addition, serum immunoglobulin levels, in particular lgG2b, are increased. Therefore, p300 via ist acetyltransferase-activity is a suppressor of autoimmunity. These findings have clinical implications as they support the concept of treating lupus with inhibitors of protein deacetylases, and point to B-cells as a''critical target of these drugs. To identify the critical substrates of p300 acetyltransferase activity in B-cells we have analysed in primary naive B-cells of different p300 status, histone acetylation at the promoters of genes whose expression based on cDNA microarray analyses and quantitative RT-PCR is affected by acetyltransferase-deficient p300. These experiments did not provide evidence for changes in histone acetylation in B-cells expressing mutant p300, suggesting that acetylation of non-histone proteins is impaired in these cells. To identify these substrates we have generated clones of immortal mature B-cells that are homozygous for the mutant allele of p300. These cells will now provide unlimited material for biochemical and global proteomics approaches. In addition, our results provide proof of principle that our experimental approach permits the identification of p300 acetyltransferase-dependent functions of p300 in vivo. We now employ the same strategy to address the role of p300-dependent protein acetylation in other processes such as tumor suppression. Studies such as the one reported here will be instrumental in optimising therapies that are based on drugs that target HDACs and HATs, respectively.
