Functional analysis of the mouse CBP gene in the adult central nervous system

Zhang, Zuwen (2002). Functional analysis of the mouse CBP gene in the adult central nervous system. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000fd2c

Abstract

CBP (CREB-binding protein) was originally characterized as a transcriptional coactivator of CREB (cAMP response element-binding protein) which is implicated in many processes including the formation of long-term memory. CBP itself is regulated by nuclear Ca²⁺ and Ca²⁺/Calmodulin kinase IV, suggesting a crucial role in synaptic plasticity. Mutations in human CBP gene are associated with the Rubinstein-Taybi syndrome (RTS) characterized by mental retardation and patterning abnormalities. These features suggest that CBP plays a central role in cAMP-mediated gene expression, which in turn is implicated in the formation of long-term memory.

The expression patterns of CBP and its homologue p300 were analyzed. Both are expressed in most of forebrain areas, with particularly high expression in the hippocampus. Parts of the mouse CBP gene were characterized. Six exons encode the CREB-binding domain (CBD). Two gene targeting experiments were performed to generate mouse lines with mutations in the CBP gene. The first was a deletion of the CBD exon 2, resulting in a frameshift and, thus, a truncated CBP protein (CBP^stop523). In the second experiment, a point mutation was aimed to be introduced in the CBD exon 5 (CBP^Tyr658Ala). After germline transmission, three mouse lines were obtained from CBP^stop523 allele: CBD2^+/-, a general deletion of the CBD exon 2; CBD2.floxed, which will be crossed with different Cre mouse lines for tissue-specific mutants; and CBD2.floxed.neo, which might be a hypomorphic allele due to the Neo cassette. For the construct CBP^Tyr658Ala none of the chimeras gave germline transmission, as apparently the splicing of the inverted allele was incorrect, possibly leading to strong reduction of vitality. Meantime, six CBP mutants were generated to elucidate functional domains of CBP in vitro: a deletion mutation CBPACBD2-5; CBPllOOaa, which was reported in mutant mice displaying RTS-like phenotypes; and a point mutation CBP658Ala, which was shown to be critical for interaction with phospho-CREB. The other three mutants retain different sizes of truncated N-terminal CBP and similar proteins were observed in RTS. Both CBP658Ala and CBPACBD2-5 interferred with the cAMP pathway, but not with nuclear receptor-mediated functions. RTS mutants showed strong inhibitory effects on the cAMP pathway.