Chromosomal organization of Xenopus laevis oocyte and somatic 5S rRNA genes in vivo.

Abstract

We describe the chromosomal organization of the major oocyte and somatic 5S RNA genes of Xenopus laevis in chromatin isolated from erythrocyte nuclei. Both major oocyte and somatic 5S DNA repeats are associated with nucleosomes; however, differences exist in the organization of chromatin over the oocyte and somatic 5S RNA genes. The repressed oocyte 5S RNA gene is protected from nuclease digestion by incorporation into a nucleosome, and the entire oocyte 5S DNA repeat is assembled into a loosely positioned array of nucleosomes. In contrast, the potentially active somatic 5S RNA gene is accessible to nuclease digestion, and the majority of somatic 5S RNA genes appear not to be incorporated into (38). The association of histone Hi with the oocyte genes may be involved in actually establishing repression (28, 49). However, the relationship between the organization of the oocyte genes into nucleosomes, the presence or absence of histone Hi within nucleosomal DNA containing the oocyte genes, and the repressed state of the oocyte genes in vivo has not yet been resolved.In contrast to the existing analyses of the chromosomal organization of the major oocyte 5S RNA genes, no comparable study of the somatic 5S RNA genes has appeared. In this report, we have examined the chromosomal organization of both the oocyte and the somatic 5S RNA genes in erythrocytes. We find that the oocyte genes are repressed and that the whole oocyte 5S DNA repeat is organized into a positioned array of nucleosomes. Consistent with earlier work (21, 56), the internal promoter elements of the major oocyte 5S RNA gene are incorporated into a nucleosome.* Corresponding author.Disruption of this chromatin organization occurs following the removal of histone Hi. This facilitates the activation of transcription of 5S RNA genes in a complete transcription extract. Readdition of histone Hi to this active chromatin selectively represses transcription of the oocyte genes and alters the chromatin structure over the oocyte genes. In contrast, the somatic 5S DNA repeats are assembled into arrays of nucleosomes that do not appear to have any uniform organization with respect to DNA sequence. We present evidence for a region around the somatic 5S RNA gene that is accessible to nucleases and for the association of transcription factors with both the internal control region (ICR) and the 5' flanking region of this gene. MATERIALS AND METHODSPreparation of chromatin. X. laevis females were purchased from Xenopus I. Blood was collected from the heart into 1 x STM (90 mM NaCl, 10 mM Tris, 2 mM MgCI2, pH 7.4)-heparin (10 U/ml) on ice. Erythrocytes were washed twice in the same buffer by centrifugation at 600 x g for 5 min (26,56). Hepatocytes were isolated from Xenopus liver (39).Nuclei were prepared from erythrocytes and hepatocytes by established methods (23,50). Cells were Dounce homogenized, and the nuclei were resuspended in 50% glycerol-50 mM Tris HCI (pH 7.5)-100 mM KCI-5 mM MgCl2-2 mM dithiothreitol (DTT)-0.5 mM phenylmethylsulfonyl fluoride (PMSF)-0.1% ...

Specific regulation of Xenopus chromosomal 5S rRNA gene transcription in vivo by histone H1.

“…Removal of histone H1 from somatic cell chromatin allows transcription factors to gain access to the oocyte type 5S rRNA genes. The oocyte 5S rRNA genes in somatic cells in vivo are known to be packaged into nucleosomes (Gottesfeld and Bloomer 1980;Young and Carroll 1983) and the positioning of histone-DNA contacts appears dependent on histone H1 (Chipev and Wolffe 1992). Sliding of nucleosomes following H1 removal may allow transcription factors access to the oocyte 5S rRNA genes.…”

“…Sliding of nucleosomes following H1 removal may allow transcription factors access to the oocyte 5S rRNA genes. The addition of histone HI to chromatin deficient in the protein in vitro can cause the selective repression of oocyte 5S rRNA genes and the displacement of transcription factors (Wolffe 1989b;Chipev and Wolffe 1992).…”

“…Histone H1 prefers the prior assembly of histone octamers onto DNA for efficient incorporation into chromatin (Hayes and Wolffe 1993) and will direct the formation of nucleosomal arrays on the oocyte 5S rRNA genes (Chipev and Wolffe 1992). Association of histone octamers with the oocyte 5S rRNA genes are necessary for the selective repression of oocyte 5S rRNA genes within genomic DNA in vitro (Wolffe 1989b).…”

“…In vivo, the overexpression of linker histones in somatic cells leads to cell cycle arrest and the reversible inhibition of DNA replication (Sun et al 1989;Aubert et al 1991). In contrast to these general inhibitory effects, evidence for a specific role for histone H1 in gene expression comes from experiments in Xenopus, where removal of histone H1 from the chromosomal chromatin of somatic cells selectively allows transcription factor access to the oocyte 5S rRNA genes (Schlissel and Brown 1984;Wolffe 1989a, b;Chipev and Wolffe 1992).…”

“…Incorporation of a single molecule of linker histone into the nucleosome stabilizes the interaction of 166 bp of DNA with the histones (Simpson 1978;Allan et al 1980). Linker histones may also influence the positioning of nucleosomes (Meersseman et al 1991;Chipev and Wolffe 1992). Distinct variants of linker histones are expressed during the development of several organisms, including vertebrates (Poccia 1986).…”