![]() laevis ( Dimitrov et al., 1994 Dworkin-Rastl et al., 1994). Thus, our data are consistent with previous studies indicating that multiple embryonic H1 variants exist in X. Purification of H1 from egg extract pooled from ∼20 different frogs yielded four bands that were confirmed by Western blot analysis and mass spectrometry to be embryonic H1 ( Fig. 1 C), and alkaline phosphatase treatment did not alter the migration of H1 bands (not depicted). Heterogeneity did not result from cell cycle–dependent phosphorylation, as no differences in H1 migration were detected between interphase and mitosis ( Fig. A variable number of distinct H1 bands (1–4) ranging in molecular mass from 35 to 40 kD were detected by SDS-PAGE and Western blots of different extract preparations ( Fig. We generated a polyclonal antibody against recombinant B4 (hereafter referred to as H1) that specifically decorated interphase and metaphase chromatin by immunofluorescence ( Fig. laevis eggs are stockpiled with a maternal histone H1 variant known as B4 that functions as the linker histone in early embryonic cell divisions until the midblastula transition ( Dworkin-Rastl et al., 1994). However, the assay conditions that were applied, although frequently used to study mitotic chromosome condensation, failed to support essential features of normal genome propagation, including spindle assembly, chromosome replication, and kinetochore assembly. Although linker histone knockouts disrupted chromosome compaction in Tetrahymena thermophila ( Shen et al., 1995), the dispensability of H1 for establishing chromosome structure in vertebrates was suggested by experiments in Xenopus laevis, as cytostatic factor (CSF)–arrested metaphase egg extracts that had been depleted of embryonic linker histone H1 supported the normal morphological condensation of unreplicated sperm chromatids ( Ohsumi et al., 1993). Mice lacking multiple H1 subtypes die by midgestation, but the cause is unknown ( Fan et al., 2003). Rather, functional studies of H1 and related proteins in multiple systems to date have indicated a role for linker histones in regulatory processes, including gene expression, chromatin accessibility, homologous recombination, and apoptosis (for review see Harvey and Downs, 2004). Although classic structural studies signified an important role for H1 in overall chromatin organization ( Thoma and Koller, 1977 Thoma et al., 1979), a definitive role for H1 in generating vertebrate mitotic chromosome architecture has not been established. Linker histone H1 was once hypothesized to be an important determinant of the mitotic chromosome structure because it can stabilize the compaction of nucleosomes into a 30-nm chromatin fiber, and its hyperphosphorylation is a hallmark of mitosis in many cell types ( Boggs et al., 2000 Hansen, 2002). Thus, our results indicate that linker histone H1 plays an important role in the structure and function of vertebrate chromosomes in mitosis. Histone H1 depletion did not significantly affect the recruitment of known structural or functional chromosomal components such as condensins or chromokinesins, suggesting that the loss of H1 affects chromosome architecture directly. Although functional kinetochores assembled, aligned, and exhibited poleward movement, long and tangled chromosome arms could not be segregated in anaphase. Immunodepletion of histone H1 caused the assembly of aberrant elongated chromosomes that extended off the metaphase plate and outside the perimeter of the spindle. We have investigated the role of embryonic linker histone H1 during mitosis in Xenopus laevis egg extracts. During cell division, condensation and resolution of chromosome arms and the assembly of a functional kinetochore at the centromere of each sister chromatid are essential steps for accurate segregation of the genome by the mitotic spindle, yet the contribution of individual chromatin proteins to these processes is poorly understood. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |