Enrichment of ubiquitinated histone H2A in a low salt extract of micrococcal nuclease-digested myotube nuclei. J Biol Chem 1990 May 05;265(13):7507-12
Date
05/05/1990Pubmed ID
2159002Scopus ID
2-s2.0-0025344438 (requires institutional sign-in at Scopus site) 18 CitationsAbstract
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.
Author List
Parlow MH, Haas AL, Lough JAuthor
John W. Lough PhD Professor in the Cell Biology, Neurobiology and Anatomy department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsBlotting, Southern
Blotting, Western
Cell Fractionation
Cell Nucleus
Cells, Cultured
Chick Embryo
Chromatin
DNA
Histones
Micrococcal Nuclease
Muscles
Nucleic Acid Hybridization
Osmolar Concentration
Ubiquitins