Control elements within the PWS/AS imprinting box and their function in the imprinting process

Abstract

A cluster of imprinted genes on human chromosome 15q11-q13 (the PWS/AS domain) and its ortholog on mouse chromosome 7c is believed to be regulated by an imprinting control center. Although minideletions in this region in Angelman syndrome (AS) and Prader-Willi syndrome (PWS) patients revealed that two elements, shortest deletion regions of overlap in AS families and PWS families (AS-SRO and PWS-SRO), respectively, constitute the IC, the molecular mechanism that governs this regional control remains obscure. To understand how this imprinting center works, a mouse model was sought. The striking similarity between the human and mouse sequences allowed the generation of a minitransgene (AS-SMP) composed of AS-SRO and the Snrpn minimal promoter (SMP) the mouse ortholog of PWS-SRO. This minitransgene carries out, in a highly reliable and reproducible manner, all steps of the imprinting process. In an attempt to decipher the molecular mechanism of the imprinting process, we generated and tested for imprinting five minitransgenes based on AS-SMP, in which various parts of the 160 bp SMP were deleted. These experiments revealed a set of five cis elements that carry out the various steps of the imprinting process. This set includes: (i). two copies of a de novo methylation signal (DNS) that establish the maternal imprint during oogenesis; (ii). an allele discrimination signal that establishes the paternal imprint; and (iii). two elements that act together to maintain the paternal imprint. Two functionally redundant sets of the five elements were found on the respective endogenous mouse sequence explaining the previously published contradictory results of targeted deletion experiments. Together with the fact that all five elements bind specific proteins that are presumably the factors acting in trans in the imprinting process, our observations set the stage for a comprehensive study of the molecular mechanism involved in the control of the imprinting process.