IN-VIVO OCCUPANCY OF HISTONE GENE PROXIMAL PROMOTER ELEMENTS REFLECTSGENE COPY NUMBER-DEPENDENT TITRATABLE TRANSACTIVATION FACTORS AND CROSS-SPECIES COMPATIBILITY OF REGULATORY SEQUENCES

To assess systematically the structural and functional aspects of hist one gene transcription within a chromosomal context, we stably integra ted an extensive set of human histone H4 gene constructs into mouse C1 27 cells. Levels of expression were determined by S-1 nuclease protect ion assays for multiple mouse monoclonal cell lines containing these h uman H4 genes. For each cell line, we quantitated the number of integr ated human H4 genes by Southern blot analysis. The results indicate th at the expression of the human H4 gene is in part copy number dependen t at low gene dosages. However, the level of expression varies among d ifferent cell lines containing similar numbers of copies of the same H 4 gene construct. This result suggests that position-dependent chromos omal integration effects contribute to H4 gene transcription, consiste nt with the roles of long-range gene organization and nuclear architec ture in gene regulation. At high copy number, the level of human H4 ge ne expression per copy decreased, and endogenous mouse H4 mRNA levels were also reduced. Furthermore, in vivo occupancy at the human H4 gene immediate 5' regulatory elements, as defined by genomic fingerprintin g, showed copy number-dependent protein/DNA interactions. Hence, human and mouse H4 genes compete for titratable transcription factors in a cellular environment. Taken together, these results indicate cross-spe cies compatibility and suggest limited representation in vivo of the f actors I involved in regulating histone H4 gene transcription. (C) 199 5 Wiley-Liss, Inc.