Enrichment of ubiquitinated histone H2A in a low salt extract of micrococcal nuclease-digested myotube nuclei.
We recently demonstrated that ubiquitinated histone H2A (uH2A) declines 2.5-fold during terminal skeletal muscle differentiation, coincident with reductions in transcriptional activity (Wunsch, A. M., Haas, A. L., and Lough, J. (1987) Dev. Biol. 119, 85-93). To assess whether this indicates an association of uH2A with transcriptionally active genes, we have used micrococcal nuclease digestion and salt extraction to fractionate myotube nuclei. An oligonucleosomal fraction obtained by micrococcal nuclease digestion and extraction in low salt (100 mM NaCl) comprising only 25% of the nuclear DNA contained 90% of the total uH2A, as revealed by Western blotting. Further fractionation of this 100 mM salt extract by sucrose gradient centrifugation revealed that virtually all of the uH2A was localized in monomer to heptamer-sized oligonucleosomes. A second ubiquitinated species of 57-kDa (u57) was also localized in the 100 mM salt extract. In contrast, an 18-kD band (u18) was associated with fractions that were resistant to micrococcal nuclease digestion and salt extraction. Although micrococcal nuclease recognized a unique structural feature of active myotube chromatin, as evidenced by the appearance of hybridized skeletal alpha-actin sequences as a smear rather than the nucleosomal ladder exhibited by inactive and bulk sequences, neither the skeletal alpha-actin gene nor the inactive alpha D-globin gene was exclusively localized in the 100 mM salt fraction. Moreover, further fractionation of the 100 mM salt extract on a sucrose gradient failed to separate active from inactive genes. These findings suggest that uH2A is localized in a fraction of myotube chromatin which, although nuclease-sensitive and relatively soluble, is not enriched in active or inactive genes.
Parlow
M H
MH
Department of Anatomy, Medical College of Wisconsin, Milwaukee 53226.
Haas
A L
AL
Lough
J
J
D000818:Animals; D015139:Blotting, Southern; D015153:Blotting, Western; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000032:analysis*; D002478:Cells, Cultured; D002642:Chick Embryo; D002843:Chromatin / Q000648:ultrastructure; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D006657:Histones / Q000302:isolation & purification*; D008836:Micrococcal Nuclease; D009132:Muscles / Q000032:analysis; D009693:Nucleic Acid Hybridization; D009994:Osmolar Concentration; D014452:Ubiquitins / Q000302:isolation & purification*
D002843:Chromatin; D006657:Histones; D014452:Ubiquitins; D004247:DNA; D008836:Micrococcal Nuclease