Interaction of HMG 14 and 17 with actively transcribed genes

The interaction of HMG 14 and 17 with actively transcribed genes was studied by monitoring the sensitivity of specific genes to DNAase I after reconstitution of HMG-depleted chromatin with HMG 14 and 17. Our experiments lead to the following conclusions: most actively transcribed genes become sensitized to DNAase I by HMG 14 and 17; either HMG 14 or HMG 17 can sensitize most genes to DNAase I; genes transcribed at different rates have about the same affinity for HMG 14 and 17; HMG 14 and 17 bind stoichiometrically to actively transcribed nucleosomes; and HMG 14 and 17 can restore DNAase I sensitivity to purified nucleosome core particles depleted of HMGs. This last observation suggests that during reconstitution, low levels of HMG 14 and 17 can associate with the active nucleosomes in the presence of a 10–20 fold excess of inactive nucleosomes. Consequently, we conclude that besides their association with HMGs, active nucleosomes also have at least one other unique feature that distinguishes them from bulk nucleosomes and insures proper HMG binding during reconstitution.     

The distribution of nuclear proteins and transcriptionally-active sequences in rat liver chromatin fractions

Chromatin fractions from rat liver nuclei digested by nucleases were separated by differential solubility into several fractions. Material solubilized during digestion (predominantly monomer nucleosomes and polynucleosomes) had the highest HMG14 + 17/DNA ratios but were not enriched in active gene sequences (albumin and c-Ha-ras1 genes). Material soluble in a low ionic strength buffer containing 0.2 mM MgCl2 (monomer nucleosomes and polynucleosomes) contained in addition to the histones, HMG14 and 17 plus a 41K non-histone protein. This fraction was depleted in active gene sequences and enriched in inactive sequences. The insoluble material was highly enriched in active sequences and had the lowest HMG14 + 17/DNA ratio. This fraction could be further fractionated into a histone-containing 2 M NaCl-soluble fraction and a 2 M NaCl-insoluble matrix-bound fraction, both of which were enriched in active sequences. The results show that the HMG proteins do not partition with active sequences during fractionation of chromatin. The 41K protein may be associated with inactive chromatin fraction.     

Interactions between HMG proteins and the core sequence of DNaseI hypersensitive site 2 in the locus control region (LCR) of the human β-like globin gene cluster

HMG proteins are abundant chromosomal non-histone proteins. It has been suggested that the HMG proteins may play an important role in the structure and function of chromatin. In the present study, the binding of HMG proteins (HMG1/2 and HMG14/17) to the core DNA sequence of DNaseI hypersensitive site 2 (HS2core DNA sequence, -10681--10970 bp) in the locus control region (LCR) of the human b-like globin gene cluster has been examined by using both the in vitro nucleosome reconstitution and the gel mobility shift assays. Here we show that HMG1/2 can bind to the naked HS2core DNA sequence, however, HMG14/17 cannot. Using the in vitro nucleosome reconstitution we demonstrate that HMG14/17 can bind to the HS2core DNA sequence which is assembled into nucleosomes with the core histone octamer transferred from chicken erythrocytes. In contrast, HMG1/2 cannot bind to the nucleosomes reconstituted in vitro with the HS2core DNA sequence. These results indicate that the binding patterns between HMG proteins and the HS2core DNA sequence which exists in different states (the naked DNA or the in vitro reconstituted nucleosomal DNA) are quite different. We speculate that HMG proteins might play a critical role in the regulation of the human β-like globin gene's expression.     

Effect of histone acetylation on structure and in vitro transcription of chromatin.

n-Butyrate treatment of growing Hela cells produces a dramatic increase in the levels of histone acetylation. We have exploited this system to study the effect of histone acetylation on chromatin structure. Chromatin containing highly acetylated histones is more rapidly digested to acid-soluble material by DNase I, but not by micrococcal nuclease. The same pattern of nuclease sensitivity was exhibited by in vitro-assembled chromatin consisting of SV40 DNA Form I and the 2 M salt-extracted core histones from butyrate-treated cells. Using this very defined system, it was possible to demonstrate that acetylated nucleosomes do not have a greatly diminished stability. Stability was measured in terms of exhange of histone cores onto competing naked DNA or sliding of histone cores along ligated naked DNA. Finally, it was shown that acetylated nucleosomes are efficient inhibitors of in vitro RNA synthesis by the E. coli holoenzyme as well as by the mammalian polymerases A and B.     

Evidence for a shared structural role for HMG1 and linker histones B4 and H1 in organizing chromatin.

The high mobility group proteins 1 and 2 (HMG1/2) and histone B4 are major components of chromatin within the nuclei assembled during the incubation of Xenopus sperm chromatin in Xenopus egg extract. To investigate their potential structural and functional roles, we have cloned and expressed Xenopus HMG1 and histone B4. Purified histone B4 and HMG1 form stable complexes with nucleosomes including Xenopus 5S DNA. Both proteins associate with linker DNA and stabilize it against digestion with micrococcal nuclease, in a similar manner to histone H1. However, neither histone B4 nor HMG1 influence the DNase I or hydroxyl radical digestion of DNA within the nucleosome core. We suggest that HMG1/2 and histone B4 have a shared structural role in organizing linker DNA in the nucleosome.     

Interactions between HMG proteins and the core sequence of DNaseI hypersensitive site 2 in the locus control region (LCR) of the human β-like globin gene cluster

HMG proteins are abundant chromosomal non-histone proteins. It has been suggested that the HMG proteins may play an important role in the structure and function of chromatin. In the present study, the binding of HMG proteins (HMG1/2 and HMG14/17) to the core DNA sequence of DNaseI hypersensitive site 2 (HS2core DNA sequence, -10681-10970 bp) in the locus control region (LCR) of the human β-like globin gene cluster has been examined by using both thein vitro nucleosome reconstitution and the gel mobility shift assays. Here we show that HMG1/2 can bind to the naked HS2core DNA sequence, however, HMG14/17 cannot. Using thein vitro nucleosome reconstitution we demonstrate that HMG14/17 can bind to the HS2core DNA sequence which is assembled into nucleosomes with the core histone octamer transferred from chicken erythrocytes. In contrast, HMG1/2 cannot bind to the nucleosomes reconstitutedin vitro with the HS2core DNA sequence. These results indicate that the binding patterns between HMG proteins and the HS2core DNA sequence which exists in different states (the naked DNA or thein vitro reconstituted nucleosomal DNA) are quite different. We speculate that HMG proteins might play a critical role in the regulation of the human β-like globin gene’s expression.     

Interactions between HMG proteins and the core sequence of DNaseI hypersensitive site 2 in the locus control region (LCR) of the human β-like globin gene cluster

HMG proteins are abundant chromosomal non-histone proteins. It has been suggested that the HMG proteins may play an important role in the structure and function of chromatin. In the present study, the binding of HMG proteins (HMG1/2 and HMG14/17) to the core DNA sequence of DNaseI hypersensitive site 2 (HS2core DNA sequence, -10681-10970 bp) in the locus control region (LCR) of the human β-like globin gene cluster has been examined by using both thein vitro nucleosome reconstitution and the gel mobility shift assays. Here we show that HMG1/2 can bind to the naked HS2core DNA sequence, however, HMG14/17 cannot. Using thein vitro nucleosome reconstitution we demonstrate that HMG14/17 can bind to the HS2core DNA sequence which is assembled into nucleosomes with the core histone octamer transferred from chicken erythrocytes. In contrast, HMG1/2 cannot bind to the nucleosomes reconstitutedin vitro with the HS2core DNA sequence. These results indicate that the binding patterns between HMG proteins and the HS2core DNA sequence which exists in different states (the naked DNA or thein vitro reconstituted nucleosomal DNA) are quite different. We speculate that HMG proteins might play a critical role in the regulation of the human β-like globin gene’s expression.     

My favourite molecule: Polyamines,chromatin structure and transcription

Nucleosomes are the basic elements of chromatin structure. Polyamines, such as spermine and spermidine, are small ubiquitous molecules absolutely required for cell growth. Photoaffinity polyamines bind to specific locations in nucleosomes and can change the helical twist of DNA in nucleosomes. Acetylation of polyamines reduces their affinity for DNA and nucleosomes, thus the helical twist of DNA in nucleosomes could be regulated by cells through acetylation. I suggest that histone and polyamine acetylation act synergistically to modulate chromatin structure. On naked DNA, the photoaffinity spermine bound preferentially to a specific ‘TATA’ sequence element, suggesting that polyamines may be involved in the unusual chromatin structure in this region. Further work is needed to test whether the specificities shown by photoaffinity polyamines are also shown by cellular polyamines; such experiments are now feasible.     

Effect of HMG protein 17 on the thermal stability of control and acetylated HeLa oligonucleosomes.

Many studies have implicated histone acetylation and HMG proteins 14 and 17 in the structure of active chromatin. Studies of the binding of HMG 14 and 17 to chromatin core particles have shown that there are two binding sites for HMG 14 or 17 located within 20-25 bp of the DNA ends of the core particles [13-15]. Such binding sites may result from the free DNA ends in the core particle being available for the binding of HMG 14 and 17. We have studied the effects of the binding of HMG 17 on the thermal denaturation of DNA in mono, di and trinucleosomes. In each case the binding of 1 HMG 17 molecule per nucleosome reduces the DNA premelt region by 50%, while the binding of 2 HMG 17 molecules per nucleosome abolishes the premelt region. From this it is concluded that there are two HMG 17 binding sites per nucleosome which are located between the entry and exit points to the nucleosome and the strongly complexed central DNA region. Highly acetylated mono, di and trinucleosomes have been isolated from butyrate treated HeLa S3 cells. For this series of acetylated oligonucleosomes, it has been found that there are also two HMG 17 binding sites per acetylated nucleosome.     

Binding of wheat and chicken high mobility group chromosomal proteins to DNA and to wheat and chicken mononucleosomes

We have used an electrophoretic retardation assay to investigate the interactions of wheat high mobility group (HMG) proteins with DNA and with isolated trimmed mononucleosomes (complexes which contain a histone octamer and approximately 146 base pairs of DNA). In order to characterize these interactions, we have compared the binding of each of the wheat HMG proteins, HMGa, b, c, and d, with those of the low molecular weight chicken HMG proteins HMG14 and 17. These vertebrate animal HMG proteins have previously been shown to occupy two specific binding sites on animal nucleosomes and to have a greater affinity for nucleosomes than for naked DNA (Mardian, J. K. W., Paton, A. E., Bunick, G. J., and Olins, D. E. (1980) Science 209, 1534-1536; Sandeen, G., Wood, W. I., and Felsenfeld, G. (1980) Nucleic Acids Res. 8, 3757-3778). As a criterion for "specific binding," we have used the property of HMG14 and 17 binding of causing a discontinuous shift of nucleosomes to a distinct band of lower electrophoretic mobility. According to this criterion, wheat HMGb, c, and d do not bind nucleosomes specifically. These HMG proteins have approximately the same affinity for nucleosomes and naked DNA. Wheat HMGa does bind nucleosomes specifically by this criterion, but other aspects of the binding are reminiscent of histone H1-nucleosome binding. We present evidence that trimmed mononucleosomes of wheat are conformationally distinct from their animal counterparts. Despite the conformational differences, competition studies indicate that chicken and wheat mononucleosomes have essentially identical affinity for the low molecular weight animal HMG proteins.     

The nuclease sensitivity of active genes.

Brief micrococcal nuclease digestion of chick embryonic red blood cells results in preferential excision and solubilization of monomer nucleosomes associated with beta-globin sequences and also 5'-sequences flanking the beta-globin gene. Both regions are DNAse-I sensitive in nuclei. Such salt-soluble nucleosomes are enriched in all four major HMG proteins but HMG1 and 2 are only weakly associated. These nucleosomes appear to have lost much of the DNAse-I sensitivity of active genes. The HMG14 and 17-containing salt-soluble nucleosomes separated by electrophoresis are not DNAse-I sensitive and contain inactive gene sequences as well as active sequences. Reconstitution of HMG proteins onto bulk nucleosomes or chromatin failed to reveal an HMG-dependent sensitivity of active genes as assayed by dot-blot hybridization and it was found that the DNAse-I sensitivity of ASV proviral sequences as assayed by dot-blot hybridization was not HMG-dependent. These results indicate that higher order chromatin structures might be responsible for nuclease sensitivity of active genes.