Nucleosomes are translationally positioned on the active allele and rotationally positioned on the inactive allele of the HPRT promoter

Differential chromatin structure is one of the hallmarks distinguishing act ive and inactive genes. For the X-linked human hypoxanthine phosphoribosylt ransferase gene (HPRT), this difference in chromatin structure is evident i n the differential general DNase I sensitivity and hypersensitivity of the promoter regions on active versus inactive X chromosomes. Here we character ize the nucleosomal organization responsible for the differential chromatin structure of the active and inactive HPRT promoters. The micrococcal nucle ase digestion pattern of chromatin from the active allele in permeabilized cells reveals an ordered array of translationally positioned nucleosomes in the promoter region except over a 350-bp region that is either nucleosome free or contains structurally altered nucleosomes. This 350-bp region inclu des the entire minimal promoter and all of the multiple transcription initi ation sites of the HPRT gene. It also encompasses all of the transcription factor binding sites identified by either dimethyl sulfate or DNase I in vi vo footprinting of the active allele. In contrast, analysis of the inactive HPRT promoter reveals no hypersensitivity to either DNase I or a micrococc al nuclease and no translational positioning of nucleosomes. Although nucle osomes on the inactive promoter are not translationally positioned, high-re solution DNase I cleavage analysis of permeabilized cells indicates that nu cleosomes are rotationally positioned over a region of at least 210 by on t he inactive promoter, which coincides with the 350-bp nuclease-hypersensiti ve region on the active allele, including the entire minimal promoter. This rotational positioning of nucleosomes is not observed on the active promot er. These results suggest a model in which the silencing of the HPRT promot er during X chromosome inactivation involves remodeling a transcriptionally competent, translationally positioned nucleosomal array into a transcripti onally repressed architecture consisting of rotationally but not translatio nally positioned nucleosomal arrays.