We have recently demonstrated that torsional stress is responsible for maintaining the DNase I sensitive conformation of active chromatin (Cell 39, 469). Gamma irradiation of chick red blood cells or inhibition of the topoisomerase II using novobiocin, both lead to reversal of DNase I sensitivity as assayed by dot blot hybridization to globin gene probes. We will reverse the active conformation and then transcribe isolated nuclei to determine whether elongation or initiation of transcription is blocked. We will do Southern hybridization on reversed active chromatin to see if the dinucleosome repeat characteristic of inactive chromatin is regenerated. We wish to establish an ATP dependent system for reversible generation of active chromatin in vitro. To determine whether topologically independent torsionally stressed domains are congruent with DNase I sensitivity domains, we will carry out gamma irradiation target size analysis as well as DNase I mapping. Finally, we will identify the sites of nuclear scaffold attachment flanking these domains. We have shown that an enzyme with many properties of topoisomerase II is bound at enhancer-like sequences within hypersensitive sites based on SDS, Mg++ induced double stranded cleavaga assays on nuclei. We propose to establish definitively that the enzyme is topoisomerase II using immuno precipitation of cleaved protein DNA complexes followed by Southern hybridization. We will then examine the association of topoisomerase II with known enhancers in the immunoglobulin genes to determine if this association is tissue specific. Finally, we will attempt to carry out sequence specific factor dependent assembly of topoisomerase II with DNA in vitro. We have found that a brief treatment of cells with novobiocin (5 min.) initiates a complex series of events; first topoisomerase II inhibition, then reversal of DNase I sensitivity and finally cleavage of the genome in an unprecedented manner to yield short oligonucleosomal fragments derived primarily from within actively transcribed genes. This cleavage reaction represents a perturbed, but physiologically significant process since it takes place only within intact, healthy cells following drug treament and not in isolated nuclei or cells poisoned with dinitrophenol. We are going to characterize this reaction and attempt to reproduce it in vitro order to determine its relationship to regulation of transcription. Finally, we will investigate a similar phenomenon which occurs in cells infected by certain adenovirus mutants.
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|Brawley, J V; Martinson, H G (1992) HMG proteins 14 and 17 become cross-linked to the globular domain of histone H3 near the nucleosome core particle dyad. Biochemistry 31:364-70|
|Lois, R; Freeman, L; Villeponteau, B et al. (1990) Active beta-globin gene transcription occurs in methylated, DNase I-resistant chromatin of nonerythroid chicken cells. Mol Cell Biol 10:16-27|
|Lundell, M; Martinson, H G (1989) The DNase I sensitive state of ""active"" globin gene chromatin resists trypsin treatments which disrupt chromatin higher order structure. Biochemistry 28:9757-65|
|Villeponteau, B; Martinson, H G (1987) Gamma rays and bleomycin nick DNA and reverse the DNase I sensitivity of beta-globin gene chromatin in vivo. Mol Cell Biol 7:1917-24|