Genome exposure and regulation in mammalian cells

A method of measurement of exposed DNA (i.e. hypersensitive to DNnse I hydr olysis) as opposed to sequestered (hydrolysis resistant) DNA in isolated nu clei of mammalian cells is described While cell cultures exhibit some diffe rences in behavior from day to day, the general pattern of exposed and sequ estered DNA is satisfactorily reproducible and agrees with results previous ly obtained by other methods. The general pattern of DNA hydrolysis exhibit ed by ail cells tested consists of a curve which at first rises sharply wit h increasing DNnse I, and then becomes almost horizontal, indicating that r oughly about half of the nuclear DNA is highly sequestered. In 4 cases were transformed cells (Raszip6, CHO, HL60 and PC12) were compared, each with i ts more normal homolog (3T3, and the reverse transformed versions of CHO, H L60 and PC12, achieved by dibutyryl cyclic AMP [DBcAMP], retinoic acid and nerve growth factor [NGF] respectively), the transformed form displayed les s genome exposure than the nontransformed form at every DNase I dose tested When Ca++ was excluded from the hydrolysis medium in both the Raszip6-3T3 and the CHO-DBcAMP systems, the normal cell forms lost their increased expo sure reverting to that of the transformed forms. Therefore Ca++ appears nec essary for maintenance of the DNA in the more highly exposed state characte ristic of the nontransformed phenotype. LiCl increases the DNA exposure of all transformed cells tested Dextran sulfate and heparin each can increase the DNA exposure of several different cancers. Colcemid prevents the increa se of exposure of CHO by DBcAMP but it most be administered before or simul taneously with the latter compound Measurements on mouse biopsies reveal la rge differences in exposure in different normal tissues, Thus, the exposure from adult liver cells was greater than that of adult brain, bur both feta l liver and fetal brain had significantly greater exposure than their adult counterparts. Exposure in normal human fibroblasts as revealed by in situ nick translation reveals a nuclear distribution pattern around the peripher y, around the nucleoli and in punctate positions in the nuclear interior in parts of both S and G1 phases of the cell cycle. The same exposure pattern is duplicated by the pattern of DNA synthesis in S cells. It would appear that these nuclear regions represent positions of special activity. The pre viously proposed theory of genome regulation in mammalian cells is supporte d by these findings. The theory proposes that: a) gene activity requires ex posure of the given locus followed by action of transcription factors on th e exposed genes; b) the fiber system of the cell (cytoskeleton, nuclear fib ers, and extracellular fibers) are required for normal exposure; c) active sites for gene expression and replication consist of the nuclear periphery where differentiation genes particularly are exposed; the nucleoli where at least some housekeeping genes am exposed; and possibly also punctate regio ns in the interior; d) noncoding sequences play a critical role in genome r egulation, possibly including the transport of loci to be activated to appr opriate exposure transcriptional and replicating locations. Cancer cells ha ve lost specific differentiation gene activities, at least sometimes becaus e of mutation of appropriate exposure genes; at least some protooncogenes a nd tumor suppressor genes are responsible for exposure and transport of spe cific differentiation gene loci to their appropriate exposure sires in the nucleus and for inducing exposure.