Altered histone H1 stoichiometry and an absence of nucleosome positioning on transfected DNA.
The packaging of DNA with histones to form chromatin represents an important and powerful mechanism to regulate gene expression. Critical aspects of chromatin-specific contributions to gene regulation have been revealed by the comparison of the activities from DNA regulatory elements examined both as transiently transfected reporters and stably integrated reporters organized as chromatin. Using the mouse mammary tumor virus (MMTV) promoter as a model, we probed the structural differences between transiently transfected and stably integrated DNA templates. We demonstrated that all four core histones and the linker histone (H1) are associated with the transient template. However, whereas the core histones were present at a similar stoichiometry between the transient and the stable templates, we found that linker histone H1 molecules are fewer on the transient template. By using supercoiling assay, we show that the transient template shows intermediate levels of nucleosomal assembly. Overexpression of H1 resulted in repression of MMTV transcriptional activity and reduced accessibility to restriction endonucleases on the transient MMTV promoter. However, the addition of exogenous H1 failed to impose a normal chromatin structure on the transient template as measured by micrococcal nuclease digestion pattern. Thus, our results suggest that while transiently transfected DNA acquires a full complement of core histones, the underrepresentation of H1 on the transient template is indicative of structural differences between the two templates that may underlie the differences in the mechanisms of activation of the two templates.
D000818:Animals; D042002:Chromatin Assembly and Disassembly / Q000502:physiology*; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D008324:Mammary Tumor Virus, Mouse / Q000502:physiology*; D051379:Mice; D008836:Micrococcal Nuclease / Q000737:chemistry; D009707:Nucleosomes / Q000737:chemistry / Q000378:metabolism*; D020218:Response Elements / Q000502:physiology*; D014158:Transcription, Genetic / Q000502:physiology; D014162:Transfection; D016662:Virus Integration / Q000502:physiology
D004279:DNA, Viral; D006657:Histones; D009707:Nucleosomes; D008836:Micrococcal Nuclease