The High Mobility Group (HMG) Proteins

The High Mobility Group (HMG) Proteins

Other than spesific enzymes (to be discussed in the next section), the HMG proteins are the most extensively characterized group of non-histone proteins. They are readly identified on the basis of their high solubility in the mineral acids, their lack of tissue or specificity, and their high concentration in the nucleus^{28, 73, 80, 95}. Four main HMG proteins have been have been characterized in calf thymus, designated HMG1, HMG2, HMG14, HMG17 in order of increasing electrophoretic mobility in acid urea; smiliar proteins have been identified in most other tissue and species examined. One possible exception is trout testes, which has only two major HMG proteins; the H6 protein which has 50% sequence homology with calf HMG17, and HMG-T, which has been composed with calf HMG1 and HMG2^{56, 95}. The HMG proteins are all presents in nucleosomes isolated from chromatin by limited digestion with micrococcal nuclease, but can be completely dissociated from chromatin by the treatment with 0.35 mol/ℓ NaCl. Isolated HMG proteins will be rebind to DNA at low ionic strength and although there is no apperent  sequence specificity in this binding, HMG1 and HMG2 have a preferential affinity for single stranded DNA and the potential to unwind double stranded DNA^{1, 38, 41, 82, 103}.

            Experiments in which transcriptionally active chromatin has been selectively solubilized by various types of nuclease tratment have led to the general genes. This conclusion is not without controversy and apparent contradictions, however probably because HMG protein relase has not always been adequately quantified and it is sometimes difficult to compare HMG proteins from different organisms. Thus preferential relase of HMG1 and HMG2 by selective digestion of active chromatin with Dnase I has been reported for calf thymus, pig thymus, mouse brain, and duck erythrocytes^{53, 72, 93}, but such release has not been confirmed in studies on rabbit thymus and liver²⁹. Likewise HMG-T, a trout testis HMG protein which is supposedly similar to HMG1 and HMG2, is not preferentially released by DNase I⁵⁷. On the other hand pereferentially release of H6 has been reported in trout testis digests^{55, 56}, while HMG17, which is sometimes compared to H6, is not released in some laboratories^{29, 52, 72}, but is released in others⁹⁸. All the HMG proteins have been found associated with 0.1 mol/ℓ NaCl soluble mononucleosomes^{30, 53, 54}, which are reportedly enriched in transcribed DNA sequences.

            The sub-nucleosomal localization of the HMG proteins is difficult to define with precision at present. In trout testis, H6 has been found on nucleosomal core particles in transcribed chromatin, whereas HMG-T appears to be associated with the internucleosomal linker regions immediately adjacent to those core nucleosomes containing proteins H6⁵⁴. Similarly, in other organisms HMG14 and HMG17 appear to be closely associated with core particles^30.62, while HMG1 and HMG2 may be present on linker DNA. The polar nature of HMG14 and HMG17 is believed to permit their binding to both the histone octamer and the nucleosomal DNA in core particles, where  as HMG1 and HMG2 bind DNA, are apparently found as homopolymers in vivo³⁷ and can partake in hydrophobic interactions¹⁷, all of which may point to their presence on linker DNA  where they may either crosslink the chromatin filament by head-to-tail interactions with other HMG proteins or with Histone H1. Alternatively, it hasbeenproposed that they replace histone H1 on the linker DNA, and are thereby involved in the decondensation of transcribed chromatin.

            The role of HMG14 and HMG17 in the structure of transcribed chromatin is less controversial. It has been observed that transcribed genomic sequencesin chromatin lose their preferential sensitivity to DNase I in the absence of HMG 14 and HMG17, but this sensitivity can be restored by reconstitution of HMG14 and HMG17 back to HMG-depleted chromatin^{25, 31, 84, 98, 99 100}. This observation is also true for crude mononucleosomes preparations. Indicating that the HMG proteins sensitive the nucleosomal DNA to DNase I in a linker DNA-independent fashion. Such a conclusion is further supported by the observation that in whole chromatin HMG14 and HMG17 sensitize transcribed sequences in the absence of histone H1 (or H5). The reconstitution studies and the use of affinity chromatography with immobilized HMG14 and HMG17have estabilished that HMG14 and HMG17 selectively bind to nuclosomes containing actively transcribed sequences conferring upon them sensitivy to DNase I digestion. Thus, HMG14 and HMG17 appear to bind to nucleosomes in a sequence-spesific fashion, in spite of the fact that they bind naked DNA with no apparent sequences specificity.

            The biochemical basis for the specific binding of HMG14 and HMG17 to active nucleosomes is not clear at present. Attemps to identify the factors involved have included the use of nucleosomes washed with 0.6 mol/ ℓ NaCl , as well as nucleosomes dissociated  in high salt urea and then reconstituted, but in both cases the spectificity of HMG binding is retained⁹⁹. In addition, no individual type of non-histone protein is present on nucleosomes in sufficient amounts to imply a role in HMG-nucleosomes recognitionsin. In fact, the 0.6 mol/ ℓ NaCl washed nucleosomes have no detectablenon-histone proteins at all. Nucleosomes to which HMG14 and HMG17 bind selectively have been reported to be distinguished by an enrichment in acetylated histones and undermethylated DNA sequences^{97, 99}. While the role of the undermethylane DNA sequences in HMG recognitionin uncertain, the levels of histone acetylation appear to be insufficient in themselves to explain the molar specifity of HMG binding to active nucleosomes. Competition studies have shown that the histone H2A-H2B dimer, but not the H3-H4 dimer, wil, compete with HMG14 and HMG17 for binding sites on active nucleosomes , suggesting that the HMG proteins bind to a site which normally interacts with Histone H2A and H2B, perhaps the H3- H4; DNA complex HMG14 and HMG17 thus appear to be able to recognize a specific nucleosomal conformation which may be induced by a combination of histone and DNA post-synthetic modifications, implying that enzimatyc non-histone proteins present in substoichiometric amounts may be ultimately responsible for determining the structureof chromosomal sequences destined to be transcribed or repressed.