Robert H. Nicolas

An improved large scale fractionation of high mobility group non-histone chromatin proteins

1. Methodology is presented for the large scale preparation and fractionation of high mobility group proteins from calf thymus chromatin. The total high mobility group protein from approx. 1 kg calf thymus tissue can be separated into five fractions by CM-Sephadex C25 ion-exchange chromatography. High mobility group proteins 1 and 2 comprise two fo the fractions. From a third fraction two more chromatin proteins, protein 3 and 17, can be isolated by trichloroacetic acid precipitation and CM-cellulose chromatography at pH 5.5. 2. The four proteins thus purified are lysine-rich proteins. Proteins 1 and 2 are additionally characterised by their high contents of acidic amino acids, as described previously (Goodwin, G. H. and Johns, E. W. (1973) Eur. J. Biochem. 40, 215-219). Proteins 3 and 17, having lower contents of acidic amino acids, are basic proteins similar to the histones. All four proteins exhibit single N-terminal amino acids; glycine is the N-terminal group of proteins 1, 2 and 3; protein 17 has a proline N-terminal amino acid. The proteins are not highly phosphorylated nor are they associated with appreciable quantities of nucleic acid.

The isolation of the high mobility group non-histone chromosomal protein HMG 14

Chromatin contains a group of non-histone proteins called the High Mobility Group (HMG) proteins [l] . There are four main HMG proteins, HMG 1,2, 14 and 17, in thymus which we have shown to be present in isolated nucleosomes [2]. Three of these proteins, HMG 1,2 and 17, and an HMG protein from trout testis, HMG T, have been isolated in a pure form [3,4,5] . The amino acid sequence of HMG 17 has recently been determined [6]. The fourth thymus HMG protein, HMG 14, is present in chromatin in much smaller quantities than the other three and has been more difficult to isolate in a pure form. In this paper we report the large scale isolation of this protein from pig thymus.

Molecular cloning of polybromo, a nuclear protein containing multiple domains including five bromodomains, a truncated HMG-box, and two repeats of a novel domain

A number of transcription factors that act as adaptor proteins have been found to contain an 87 amino acid domain called the bromodomain. In a study to identify and characterise bromodomain proteins expressed in chicken cells, a novel gene has been isolated which encodes five repeats of the bromodomain. In addition, the encoded protein, termed polybromo, contains four other domains: an unusual truncated HMG box, two repeats of a novel domain which we term the BAH domain and a sequence related to a region within the regulatory domain of the DNA cytosine-5 methyltransferase enzyme. Polybromo was found to be related to a yeast protein U19102 which has two bromo domains, a BAH domain and the DNA methyltransferase-related sequence. Antibodies that were raised against polybromo were used in confocal microscopy analysis to show that the 180-kDa polybromo protein is located within the nucleus but excluded from the nucleolus. Gel filtration analysis of nuclear extracts demonstrate that polybromo is part of a large complex with a mass of approximately 2 million dalton.

Microheterogeneity in a non-histone chromosomal protein.

The non-histone proteins that are extracted from chromatin with 0.35 M NaCl contain a group of proteins which are characterised by their solubility in trichloroacetic acid and by their relatively low molecular weights (< 30 000) [l] . These proteins, termed the high mobility group (HMG) proteins, have been fractionated and the main components isolated in a pure form [2]. A recent detailed comparison of two of the HMG proteins which have high contents of basic and acidic amino acids (proteins HMG 1 and HMG 2) has indicated that their amino acid sequences are closely related [3]. However, the situation has been somewhat complicated by the recent finding that both proteins appear to exhibit multiple forms when analysed by isoelectric focusing. Protein HMG 1 displays a rather complex banding pattern in the pH 6-8 range which could be due to aggregation, but protein HMG 2 clearly has four main subfractions. In view of the possible importance of microheterogeneity in these non-h&one proteins, reminiscent of that found with histone Fl [4], we are currently developing procedures for isolating the subfractions. In this paper a method for isolating protein HMG 2 subfractions is presented.