Msb

DNA from

14-day erythroblasts

DNA from MSB

Nonhistone Proteins Provide a Structural Scaffold for Long Chromatin Loops

Although histones are the predominant proteins in chromosomes, nonhistone proteins are also involved in organizing chromosome structure. Electron micrographs of histone-depleted metaphase chromosomes from HeLa cells reveal long loops of DNA anchored to a chromosome scaffold composed of nonhistone proteins (Figure 10-23). This scaffold has the shape of the metaphase chromosome and persists even when the DNA is digested by nucleases. As depicted schematically in Figure 10-24, loops of the 30-nm chromatin fiber a few megabases in length have been proposed to associate with a flexible chromosome scaffold, yielding an extended form characteristic of chromosomes during interphase. Folding of the scaffold has been proposed to produce the highly condensed structure characteristic of metaphase chromosomes. But the geometry of scaffold folding in metaphase chromosomes has not yet been determined.

▲ EXPERIMENTAL FIGURE 10-22 Nontranscribed genes are less susceptible to DNase I digestion than active genes.

Chick embryo erythroblasts at 14 days actively synthesize globin, whereas cultured undifferentiated MSB cells do not. (a) Nuclei from each type of cell were isolated and exposed to increasing concentrations of DNase I. The nuclear DNA was then extracted and treated with the restriction enzyme BamHI, which cleaves the DNA around the globin sequence and normally releases a 4.6-kb globin fragment. (b) The DNase I- and BamHI-digested DNA was subjected to Southern blot analysis with a probe of labeled cloned adult globin DNA, which hybridizes to the 4.6-kb BamHI fragment. If the globin gene is susceptible to the initial DNase digestion, it would be cleaved repeatedly and would not be expected to show this fragment. As seen in the Southern blot, the transcriptionally active DNA from the 14-day globin-synthesizing cells was sensitive to DNase I digestion, indicated by the absence of the 4.6-kb band at higher nuclease concentrations. In contrast, the inactive DNA from MSB cells was resistant to digestion. These results suggest that the inactive DNA is in a more condensed form of chromatin in which the globin gene is shielded from DNase digestion. [See J. Stalder et al., 1980, Cell 19:973; photograph courtesy of H. Weintraub.]

Chapter 11, the control of acetylation of histone N-termini in specific chromosomal regions is thought to contribute to gene control by regulating the strength of the interaction of histones with DNA and the folding of chromatin into condensed structures. Genes in condensed, folded regions of chromatin are inaccessible to RNA polymerase and other proteins required for transcription.

▲ EXPERIMENTAL FIGURE 10-23 An electron micrograph of a histone-depleted metaphase chromosome reveals the scaffold around which the DNA is organized. The long loops of DNA are visible extending from the nonhistone protein scaffold (the dark structure). The scaffold shape reflects that of the metaphase chromosome itself. The chromosome was prepared from HeLa cells by treatment with a mild detergent. [From J. R. Paulson and U. K. Laemmli, 1977, Cell 12:817. Copyright 1977 MIT.]

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