Non-Histone Protein And Gene Regulation
Introduction
it has been known for a long time that the chromosomal DNA of eukaryotes is associated with a large quantity of histone proteins. Through the role of histones in packaging DNA into nucleosomes has been well estabilished in recent years, the functional significance of the non-histone proteins is not nearly so clear. Our limited understanding of this latter group of proteins is least in part due to heterogeneity of the fraction, the relative insolubility of many of its components, and the limited availability of in vitro assays for chromosomal protein function which accurately reflect the in vivo situation. In spite of the these limitation, however, it has become gradually apperent that amongst the non-histone chromosomal proteins are macromolecules involved in chromatin structure, in catalysing nuclear reactions, and perhaps in determining wheter or not spesific genetic squences are avaliable for transcription. In this chapter, we will summarize the properties of this diverse class of proteins which have led to the above generalization, placing an emphasis on the relantionship between genomic structure and the regulation of gene expression.
General properties of non-histone proteins
Among the general characteristics which a group of molecules would be expected to exhibit if they were involved in regulating gene expression are heterogeneity, tissue and species specificity, variations which correlate with changes in gene activity, and sequence-specific interactions with DNA. In the following sections on properties of non-histone proteins, it well become apparent that these molecules not only meet the above criteria, but also exhibit several other attributes which fruther reinforce the nation that they play a role in the regulation of chromatin structure and function.
Heterogeneity and tissue/species specificity of non-histone chromosomal proteins
With present levels of resolution attainable by two-dimensional gel elctrophoresis, the non-histone proteins in chromatin have been shown to contain at least several hundred distinct species16,70. This level of complexity rivals that of cytoplasmic proteins, and contracsts markedly with the comparative lack of heterogenety of the histones.
The questions of the tissue specivity of non-histone proteins has been extensively investigated by the both electrophoresis and immunological methods. Both approaches demonstrate the existence of tissue specificity in the non-histone protein fractions, thought it is also clear that most tissues share with each other a common set of major non-histone proteins. Differences in extraction techniques and the limited resolving power of one-dimensional electrophoresis have led to some disagreement as to how extensive a variation exists in non-histone proteins between tissue. However, the introductions of high resolution two dimensional electrophoresis systems has led to the conclusion that as many as half the non-histone chromosomal proteins of Friend and HeLa cells differ from each other. This degree of divergence is less than that observed for the cytoplasmic proteins (there quarters of which differ), indicating that many of the non-histone chromosomal proteins may be under relatively strict evolutionary conservation. The tissue and/or species-specific chromosomal proteins presumably include those involved in the control of cell-specific gene expression, whereas the remainder of the proteins may be conserved because they have to interact with DNA or other molecules in general processes such as DNA replication, transcription, and the maintenance of chromosomal structure. One subclass of non-histone proteins which has little tissue or species specificity is the high mobility group (HMG) proteins, agroup of molecules whose role in chromatin structure will be discussed later.
Quantitative variations in non-histone chromosomal proteins content
Although histone are present in chromatin from most tissue at about a 1 : 1 (w/w) ratio to DNA, the proportion of non-histone proteins in chromatin from different tissue varies considerably. Most tissue have a non-histone protein DNA rato around 1.09, but this ratio varies from as low as 0.1 in certain types of sperm to as high as 9 in the slime mould. The quantity of non-histone proteins in chromatin generally varies with the biological state of cell, with increased non-histone protein content correlating with increased levels of RNA synthesis, vice versa.
Non-histone chromosomal proteins of’active’and’inactive’chromatin
Quantitative variation in the non-histone chromosomal protein content of active and inactive tissue suggests that chromatin template activity is influenced by the non-histone chromosomal proteins. This hypothesis is fruther supported by the finding that when chromatin is fractionated into ‘active’ and ‘inactive’ components, the non-histone protein to DNA ratio is generally greater in the active fraction. Electrophoretic analysis has revealed both qualitative and quantitative difference between the non-histone proteins from the different chromatin fractions. Certain spesific non-histone proteins with defined enzymes activities, such as nuclear protein kinase, have been found to be preferentially localized in the active fractions. These result indicate that some non-histone proteins may be involved in transcriptional activation and/or mainternance of a diffuse chromatin strucutre, whereas others may be involved in the inhibition of transcriptional activity or in the condensation of chromatin structure.
Changes in non-histone chromosomal proteins with modifications in gene activity
A regultory role for components of the histone chromosomal protein fraction is suggested by the changes which occur in these macromolecules in associations with modifications in gene expression. For example, alterations in non-histone proteins have been observed following the stimulation of resting cells proliferate, during differentiation and development, after hormone and drug stimulation, and during malignant transformation. Some of the many experimental systems in which such observations have been made will be summarized below. For detailed reference, the reader is reference 87.
Change in non-histone chromosomal proteins associated with cell poliferation
An increase in the rate of non-histone chromosomal proteins synthesis has been observed in many different system where non-diving cells are stimulated to profolirate, e.g. salivary glands stimulated by isoproterenol, human fibroblasts stimulated by serum, lymphocytes stimulated by phytohaemaggutinin, concanavalin A, leucoagglutinin, or anti imunoglobulin liver regeneration following partial hepatectomy, and refeeding of starved Physarum polycephalum and Tetrahymena. Electrophoretic analysis has revelated that, in many cases, a change in the overall quantity of non-histone chromosomal proteins can be accounted for by an increase in the levels of a view spesific proteins. The increase in synthesis of spesific in non-histone chromosomal proteins which occours when cells are stimulated to divide precedes increases in RNA and DNA synthesis, and so it is reasonable to postulate that these proteins are involved in the control of gene transcription at the onset of cell proliferation. However, the levels of some non-histone proteins decrease when cells are stimulated to divide ; these may be involved in the maintenance of the quiescent state.
Changes in non-histone chromosomal proteins during development and differentiation
During the course of development and differentation, some batteries of genes are activated, while others are repressed; thus, developing and defferentiating systems provide good opportunities for studying the molecules involved in controlling selective gene expression. Quantitative and qualitative stage-specific changes in non-histone chromosomal proteins have been observed in sea urchin embryos, Xenopus laevis tadpoles, Oncopeltus (milkweed bug) embryos, and during the development of chick oviduct and embryonic red blood cells. Alterations in the complement of non-histone chromosomal proteins have also been observed during the differentiation of pollen from Hipeastrum belladonna, during the conversion of lymphoid spleen to an erythroid organ, and in the dimethyl suphoxide-stimulated erythroid differentiation of Friend cells. In all above instances, changes in the non-histone chromosomal proteins in a cell and the RNA synthetic capacity of that cells is provided by the reduction in amount and the loss of spesific non-histone proteins observed during condensation of chromatin and concomitant repression of RNA synthesis which occours during spermatogenesis and the maturation of erythroid cells. In contrast in the above findings, some groups have detected only minimal changes in the complement of non-histone chromosomal proteins during prostaglandin and cAMP-induced differentation of neuroblastoma cells, during normal or hydrocortisome-induced embryonic development of the neural retina, and during normal development of various brain tissues. Thus, gross changes in non-histone proteins do not necessarily accompany the process of cells differentiation.
Changes in non-histone chromosomal proteins associated with gene activation by horomones and drugs
Many hormones and drugs are now known to alter the expression of spesific genes in their target tissues. In several of these cases, e.g. insulin, aldostereno, ecdysone, testosterone, cortisone and phenobarbitone, increases in the synthesis of non-histone proteins have been found to accompany the induction prosess. The changes which occour in the non-histone protein fraction are both hormone effects and target tissue spesific. In the case of the insect hormone ecdysone, one can directly observe the accumulation of spesific non-histone proteins in the puffed regions of polytene chromosomes after hormone administration32. In several instances, controls have been carried out to demonstrate the specificity of hormone effects on non-histone proteins. For example, the changes in non-histone proteins which occour in prostate gland in response to testosteron are prevented when the anti-androgen, cyproterone acetate, is administered together with testosterone in vivo59. In addition to finding that hormones induce alterations in the synthesis and accumulation of non-histone proteins, there is a large body of evidence which suggest that components of the non-histone protein in fraction may play a direct role in the mechanism of steroid hormone binding to chromatin83,91.
Changes in non-histone chromosomal proteins associated with maglinant
Anothe finding which supports the hypothesis that the non-histone chromosomal proteins are involved in the control of gene expression is that they undergo quantitative and qualitative changes during maglinant transformation, some such changes being evident within hours of treatment of normal tissue with carcinogens. The possible involvement of non-histone chromosomal proteins as mediators of aberrant gene expression in neoplastic cells has been discussed in recent review86, to which the reader is referred for detailed reference. In brief, changes in the levels and/or rates of synthesis non-histone chromosomal proteins have been observed in cancers of the breast, liver, colon, brain, prostate, and lymohocytes. Differences between the non-histone proteins of normal and neoplatic tissues have been confirmed by immunological techniques including complement fixation, enzyme-linked immunoassay and immunohistochemestry. A particularly interesting observation made in Busch’s laboratory is that a protein which apperas in lymphocytes only when they become leukaemic apparently corresponds to a protein present in hepatomas, but not in normal liver101,102. This protein may be involved in some general aspects of carcinogenesis, such as the perturbation of replicative control. Non-histone chromosomal proteins also seem to be altered during viral transformation of cells. It has been shown by immunological techniques that the non-histone proteins of SV40-transformed WI-38 human fibroblast differ from those of untransformed cells. Gel electrophoresis and radioactive labeling studies have also demonstrated differences in levels and rates of synthesis of spesific non-histone proteins in SV40-transformed and untransformed W-38 cells and mouse embryo fibroblast, as well as in Rous sarcoma virus infected chick embrio fibroblast.
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