Rubinstein-Taybi syndrome lymphoblastoid cells show oncreased sensitivity to oxidative DNA damage

The Rubinstein-Taybi syndrome (RSTS) is a genetic disorder associated with defects in postnatal growth, microcephaly, intellectual disability and increased risk of tumors. The exact molecular etiology of RSTS is not clearly understood; however it is widely accepted that RSTS is associated with mutations and chromosomal rearrangements (microdeletions and translocations) with breakpoints at 16p13.3, where the gene encoding the cAMP Response Element-Binding Protein (CREBBP or CBP) is located. About 60% of RSTS individuals carry a heterozygous mutation/deletion of CREBBP, while a small percentage (~3%) of RSTS are caused by mutations in the EP300 gene (located at 22q13.2) encoding for p300. CREBBP and p300 are highly homologous proteins with lysine acetyl transferase (KAT) activity, and play a key role in transcription regulation. However, they have also distinct cellular functions and cannot always replace one another. Both proteins are involved in other aspects of DNA metabolism, including cell cycle checkpoints (by acetylating p53), and DNA repair (by acetylating factors involved in nucleotide (NER) and base excision repair (BER) mechanisms, such as XPG, FEN-1, DNA polymerase ? (pol ?), some DNA glycosylases (e.g. TDG, NEIL2, OGG1), as well as PARP-1, and WRN and RECQL4 helicases. In agreement with these findings, we have recently shown that depletion of both CBP and p300 by RNA interference, significantly impairs NER. Thus, deficiency in CBP and/or p300 may result in genome instability and cancerogenesis, thereby explaining, at least in part, the higher incidence of malignancies observed in RSTS patients. In order to study the involvement of CBP and p300 in response to DNA damage, and in particular in DNA repair, we have analyzed lymphoblastoid cell lines derived from RSTS patients carrying monoallelic deletion, or mutations of CREBBP gene. Flow cytometry and BrdU labeling has been used to assess proliferative characteristics of these cell lines. Western blotting analysis has shown, as expected, a reduced expression of CBP, and in one case also of p300 protein in RSTS lymphoblasts, as compared to the lymphoblastoid cell lines from normal donors. No significant differences were observed in the protein expression of PCNA, and other related DNA repair proteins, such as XP proteins (A-G), PARP-1, XRCC1 and DNA pol ?. Despite a similar proliferation rate, RSTS cells showed signs of histone H2AX phosphorylation, suggesting the presence of endogenous DNA damage. In addition, all RSTS cell lines tested were more sensitive to treatment with the oxidative agent KBrO3. Experiments have been performed to assess the recruitment of XRCC1 and DNA pol beta to DNA damage sites, and the efficiency of DNA repair is being analysed by the Comet test. Preliminary results suggest a different and delayed kinetics in the repair of oxidative lesions in RSTS cells; however, further investigations are required in order to fully characterize the behaviour of these cell lines.