Analysis of Histones in Xenopus laevis

Abstract

Histone proteins contain epigenetic information that is encoded both in the relative abundance of core histones and variants and particularly in the post-translational modification of these proteins. We determined the presence of such variants and covalent modifications in seven tissue types of the anuran Xenopus laevis, including oocyte, egg, sperm, early embryo equivalent (pronuclei incubated in egg extract), S3 neurula cells, A6 kidney cells, and erythrocytes. We first developed a new robust method for isolating the stored, predeposition histones from oocytes and eggs via chromatography on heparin-Sepharose, whereas we isolated chromatinized histones via conventional acid extraction. We identified two previously unknown H1 isoforms (H1fx and H1B.Sp) present on sperm chromatin. . Furthermore, we subjected a subset of these histones to two-dimensional gel analysis and subsequent immunoblotting and mass spectrometry to determine the global remodeling of histone modifications that occurs as development proceeds. Overall, our observations suggest that each metazoan cell type may have a unique histone modification signature correlated with its differentiation status.

Protein Arginine Methyltransferase Prmt5-Mep50 Methylates Histones H2A and H4 and the Histone Chaperone Nucleoplasmin in Xenopus laevis Eggs

“…Our unpublished observations 4 show that the vast majority of endogenous nucleoplasmin is methylated on Arg-187. Furthermore, we previously showed that egg and pronuclear H4 is methylated on Arg-3 (7,56). Additional unpublished observations 4 show that egg and pronuclear H2A, primarily the abundant variant H2A.X-F (37), are highly methylated on Arg-3.…”

“…Dynamic changes in deposition of histone variants and alterations of post-translational modification patterns occur throughout the life of a multicellular organism and vary in each cell type (7). Furthermore, these dynamic alterations are specified on individual gene loci (8).…”

Analysis of Histones in Xenopus laevis

“…Along with our accompanying article (40), this is the first reported "life span" analysis of global histone modifications from gametic, embryonic, and somatic cell types. Our mass spectrometry analysis is mostly consistent with and complementary to the immunoblotting reported in the accompanying article (40). It also provides a thorough and more quantitative analysis of the H3 modifications than does immunoblotting alone.…”

“…Pronuclei were assembled and isolated as described in the accompanying article (40). Egg histones were purified on heparin-Sepharose, as described, whereas chromatin-bound histones from pronuclei, erythrocytes, and tissue culture cells were acidextracted as described, all in the presence of phosphatase inhibitor mixture (Roche PhosSTOP), protease inhibitor mixture (Roche Complete), and 10 mM sodium butyrate (20).…”

“…Xenopus interphase egg extract was prepared as described (19) and in the accompanying article (40). Pronuclei were assembled and isolated as described in the accompanying article (40).…”