Abstract
Basic proteins in nuclei and nucleosomes at different stages of development in Arbacia punctulata sea urchins were analyzed directly by in situ protamine release of chromosomal proteins into Triton/acid/urea-polyacrylamide gels. The predominant protein band in the H4 region of 2-cell through 64-cell stage embryos migrates with the mobility expected for diacetylated histone H4 (i.e. H4aa), whereas after blastulation (approximately 300 cells) the predominant H4 species is the unmodified form, H4O. In early embryos this H4aa band is highly labeled in vivo with [3H]acetic acid. The ratio of H4aa:H4O is more than 20-fold greater at the rapidly dividing 2-cell stage than at pluteus stage. This is true for both newly synthesized H4 labeled with [3H]lysine and total H4 (stained). Enhanced acetylation is also found in nucleosomes. The relative amount of this acetylated H4 species correlates roughly with the rate of cell doubling during early embryogenesis, and decreases as the average nucleosomal repeat increases. The results are indicative of a dynamically changing chromatin structure through development, as well as an intimate role of diacetylated histone H4 in the maturation of newly replicated chromatin.
Highlights
We have analyzed the basic nuclear proteins of sea urchin embryos at various stages of development with Triton/acid/ urea-polyacrylamide gel electrophoresis [26, 27] to determine the levels of histone acetylation duringdevelopment
The results are indicative of a dynamically changing important to distinguisbhetween the different stateosf acetychromatin structure through development, as well as lation, with the diacetylaftoerdm of H4 involved in replicating an intimate role of diacetylated histoneH4 in the mat- the chromatin
Histone acetylation has been the subject of intensive research over the past 20 years, its function remains controversial(e.g.see Refs. 6 and
Summary
Eggs of the sea urchinArbacia punctulatawere obtained by shedding induced either by injection of 0.5 M KC1 or by direct removal of gonadal tissue of scissor-opened females. [3H]Acetate labeling was performed by growing embryos from mid 1-cell stage tomid 8-cell stage in sea water containin[g3H] acetic acid (10 mCi/ZO ml of highly concentrated embryos) supplemented with 1 mM glutamicacid and 1 mM aspartic acid.Fluoro-. Triton/acid/urea-polyacrylamide gel electrophoresis was performed in the presence of 6 mM Triton X-100 and 8 M urea with the in situ protamine release method as described by Richards and Shaw [27] This method avoids the loss of histones commonly associated with acid extraction and allows their direct and quantitativeanalysis. Micrococcal nuclease digestions and isolation of nucleosomes on sucrose gradients were performed as described by Shaw ct a/. ( 3 3 )
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