Lysine-rich histones and the selective digestion of the globin gene in avian red blood cells

Abstract

Deoxyribonuclease I (DNase I) is known to preferentially digest the adult globin gene sequences in avian red blood cells. We have investigated the contribution of histones H1 and H5 in maintaining the nuclease-sensitive structure about the globin genes. When the lysine-rich histones H1 and H5 were selectively removed from avian red blood cell nuclei, the rate of digestion with DNase I increased several fold. However, the globin genes in H1-and H5-depleted nuclei were still selectively digested. Since histone H1 is necessary for the higher order folding of the nucleosomes, these data suggest that DNase I recognizes an aspect of structural heterogeneity within each core particle rather than some higher order packaging of the nucleosome cores.

The effect of salt extraction on the structure of transcriptionally active genes; evidence for a DNAseI-sensitive structure which could be dependent on chromatin structure at levels higher than the 30 nm fibre

“…Since low pH extraction used to extract Hi and H5 from nuclei leaves unaltered the DNAseI-sensitive structure of active genes, one may have predicted that low pH extraction would leave HMG14 and 17 still bound to the chromatin. Villeponteau et al [3] did not examine this but we now report that this prediction is not fulfilled when the proteins extracted from nuclei at low pH are examined. We find that HMG14 and 17 (together with Hi and H5) are quantitatively extracted from nuclei at low pH.…”

“…Extraction of nuclei at pH 3 prior to DNAseI digestion was carried out by a modification of the method of Villeponteau et al [3], which is based on the methods devised by Lawson and Cole [9] to extract lysine-rich histones. Nuclei were washed twice with phosphate-citrate pH 3 buffer (10nM NaH2PO4, 10mM sodium citrate, 2mM MgC12, 2mM CaC12, pH 3) and then twice (or in some experiments three times) with phosphate-citrate pH 3 buffer containing either 0.3M NaCl or 0.6M NaCl, as described by Villeponteau et al [3]. The extracted nuclei were then washed several times with RSB buffer until the pH of the nuclear suspension returned to neutral.…”

“…Lawson and Cole [9] and Villeponteau et al [3] have shown that saltextraction of nuclei at pH3 is a useful method for removing lysine-rich histones without causing clumping or lysis of the nuclei. Despite the low pH used the nucleosomal structure of the chromatin appears to remain unimpaired judging from the normal micrococcal nuclease and DNAseI cutting patterns seen when low pH extracted nuclei are digested with these enzymes.…”

“…These studies did not investigate the fate of the HM4G proteins in these extractions. Figure 1 shows an electrophoretic analysis of the proteins extracted from 16 day chick embryro red blood cell nuclei with 0.3M NaCl (pH3) by the method of Villeponteau et al [3], and an analysis of the PCA-soluble proteins remaining on the chromatin after these extractions. 0.3M NaCl pH3 extracts Hi, HMG14, HMG17, some H5, some HMG1 and a number of other unidentified proteins (lanes 1,2,3).…”

“…Results have been presented to suggest that histone acetylation or, more recently, that the HMG proteins are responsible for inducing the DNAseI-sensitive conformation of active genes (Reviewed in [2]). Villeponteau et al [3] studied the effect of higher order chromatin structures on DNAseI-sensitivity by removing Hi and H5 from red blood cells by low pH extraction of nuclei and asked whether this removal affected the DNAseI-sensitivity of the globin gene. Removal of Hi and H5 was found to have negligible effect on the overall DNAseI-sensitivity of the globin genes as assayed by solution hybridization of DNA samples from nuclei digested until 20% of the DNA was rendered acidsoluble.…”