The Role of histone and non-histone proteins in nuclear function

In eukaryotic cells, DNA, which contains the genetic information of the organism, is complexed with histones, a group of small, basic proteins of distinctive amino acid composition. Since the DNA of eukaryotes is mostly repressed, it was suggested by Stedman and Stedman, (1950, 1951) that the histones function as gene inhibitors. Unfortunately, the limited heterogeneity and comparative lack of tissue and species specificity (Panyim et al, 1971) of the histones is inadequate to explain the relative restriction of DNA which is found. Two observations have suggested answers to the problem of selectivity. On the one hand, non-histone proteins are extremely heterogeneous (e.g. Yeoman et al, 1973) and show tissue and species specificity (Dravid and Burdman, 1968; Platz et al, 1970). They are present in high concentration in the chromatin of metabolically active tissues, and there is some evidence that they are responsible for specificity of transcription from chromatin (Paul and Gilmour, 1968). The other relevant observation is that histones are subject to post-transcriptional chemical micromodifications in vivo. The main modifications are methylation (Murray, 1963), acetylation (Allfrey et al,. 1964), thiolation (Ord and Stocken, 1966) and phosphorylation (Ord and Stocken, 1966). Acetylation of histones correlates in general with RNA synthesis, while phosphorylation, and perhaps thiolation, correlate with DNA synthesis and cell division. Current opinion is that modifications of histones are concerned with general changes in the state of the nucleus, while the non-histones include specific genetic repressers. It is known also that many non-histone proteins are subject to micromodification in vivo.
In order to study nuclear proteins, the first requirement is to separate single species of protein. In spite of the distinctive characteristics of the histones, they frequently contain non-histone contaminants (Johns, 1964; Murray, 1965). Although the degree of contamination is not high, it is likely to be significant, particularly in studies of phosphorylation, because of the high phosphorus content of non-histone proteins (Langan, 1968). Since the greatest amount of phosphorylation is observed in the histone F1 fraction (Ord and Stocken, 1966) the first objective of this study was to obtain a pure sample of histone F1, and to characterize its non-histone contaminants. Consequently, non-histone proteins which coextracted with histone F1 were separated from the histone on DEAE Cellulose, and the major component, P1, was purified. It was shown by SDS gel electrophoresis to be a small protein, of molecular weight 20,000 daltons, and to have an N-terminal glycine residue. It contains a high proportion of lysine, and acidic residues. It has a cysteine residue which allows it to form dimers through a disulphide bond. In the oxidised form, it forms complexes with histone F1. Both F1 and P1 have covalently bound phosphate in vivo, and both are substrates for cytoplasmic protein kinase in vitro, although P1 is less effective than F1 in this respect. Both F1 and P1 are partially extracted from nuclei with 0.35M salt, P1 being more loosely bound.
The comparatively loose binding of both F1 and P1 to chromatin is such as to allow these proteins to be involved in control of chromatin state (see Comings and Tack, 1973). The redox-dependent complex formation between P1 and F1 is such that P1 can 'relay' the redox state of the cell to produce an effect on the histone.
Now that a pure preparation of histone F1 is available, it is possible to examine the nature of the bound phosphate. In addition to the previously described phosphoserine -(Ord and Stocken, 1966), covalently bound ADPribose is also found. The bound ADPribose is stable to neutral hydroxylamine, RNAse and alkaline phosphatase, but is liberated by alkali and venom phosphodiesterase. Enzymic hydrolysis of the protein yields ADPribose monomer bound to the phosphate group of phosphoserine as the major nucleotide product. This product gives phosphoserine and ADPribose on mild acid hydrolysis, with a ratio for serine : adenine : ribose : phosphate of 1 : 1 : 2 : 3. It is proposed that the structure of the product is 1-"(0-serylphosphoryl)-ADPribose. Peptides bearing the nucleotide, isolated from a tryptic digest of histone F1, contained a number of acidic amino acid residues. In resting rat liver, more than 90% of the phosphate bound to histone F1 was present as nucleotide.
The presence of ADPribose bound to nuclear protein after incubation of nuclei in vitro with labelled NAD+ has been noted previously (Nishizuka et al, 1968). In this study, ADPribose was found bound to non-histone proteins in saline, acid and urea-saline nuclear extracts. In each case the acceptor protein appears to be a formally acidic non-histone protein with a high serine and glycine content, which forms aggregates readily. The ADPri"bose is attached by two types of bond, one of which is labile to neutral hydroxylamine.
A peptide of molecular weight about 3,000 daltons is also an acceptor of ADPribose. This compound is most actively synthesised in growing tissues. The nature of its peptide-nucleotide bond is not known.
In perchloric acid extracts from nuclei of resting liver, a large proportion of the histone F1 is aggregated around the ADPribose derivative of the histone. When the cell passes from Go to G1, after partial hepatectomy, F1 accepts phosphate as phosphoserine and the proportion of aggregated material is rapidly reduced.
It is proposed that the aggregation observed in vitro reflects the function of the ADPribosyl derivative of the histone in vivo, namely to maintain Go chromatin in a condensed form.
Increased phosphate contents of histones F2a1, F2a2 and F2b are found at 4 and 13 hours after partial hepatectomy, at times corresponding to the peaks of cAMP levels in liver. The phosphoserine contents of histone F1 and protein P1 are raised to a considerable degree during the period of DNA synthesis. Histone F3 is not phosphorylated prior to mitosis. Both the phosphoserine and ADPribose levels of the non-histone proteins are higher in S phase nuclei than in Go. An increase in synthesis of the small ADPribose - accepting peptide is observed at early times after partial hepatectomy, which continues until mid S phase.
These results are in keeping with a model in which histones F2 and F3 maintain the secondary and tertiary structure of chromatin, and histone F1 is concerned with higher order structure. Major alterations of chromatin structure are brought about by micromodifications of histones, particularly by phosphorylation. The exact manner in which various non-histone proteins control the function of the nucleus and the details of chromatin structure remain to be elucidated. ADPribosyl derivatives of nuclear proteins would appear to be concerned with broad aspects of nuclear function.