High mobility group protein 14 and 17 can prevent the close packing of nucleosomes by increasing the strength of protein contacts in the linker DNA.
High mobility group (HMG) proteins 14 and 17 are abundant chromatin-associated proteins found in all higher eukaryotic nuclei. This observation demonstrates that HMGs 14 and 17 must have an important and universal function with regard to the structure and function of chromatin. What this function is, including how they interact with a nucleosomal array in vivo, is not known. Recently, we have demonstrated that HMGs 14 and 17 can organize nucleosomes into a regular array and increase the repeat length from 145 to about 160-165 base pairs in vitro. In addition, they can increase the apparent repeat length of chromatin deficient in histones H2A/H2B from 125 to approximately 145 base pairs. Importantly, this template was transcriptionally active. In this study, we report five new observations that begin to address the mechanism by which HMGs 14 and 17 space nucleosomal particles. First, we demonstrate that both human placenta HMG 14 and HMG 17 can space nucleosomes to produce a chromatin template with a repeat length around 160 base pairs. This result further highlights the similarity between these proteins in terms of protein structure and perhaps function. Second, we show that digestion of HMG containing chromatin with micrococcal nuclease produces DNA fragments that were approximately 10 and 20 base pairs longer than nucleosome core-particle DNA. This suggests that HMG 14 or HMG 17 can protect, directly or indirectly, at least an additional 10 base pairs of linker DNA from micrococcal digestion. However, this HMG-containing particle does not produce a strong kinetic block, and further digestion results in the eventual accumulation of DNA fragments 145 base pairs in length. Third, by comparing the full-length protein with different domains, we demonstrate that the acidic carboxyl-terminal domain is absolutely required for nucleosome spacing, neither the nucleosome binding domain of HMG 14 or HMG 17 nor the amino-terminal domain plus the nucleosome binding domain of HMG 14 could space nucleosomes. Fourth, we demonstrate that extensive micrococcal nuclease digestion of chromatin deficient in histones H2A/H2B led to the accumulation of DNA fragments about 110 base pairs in length, which is presumably the length of DNA associated with a nucleosomal particle deficient in one H2A/H2B dimer. Incorporation of either HMG 14 or HMG 17 into this chromatin results in the disappearance of this band and increase in the accumulation of fragments around 140-150 base pairs in length. Finally, in contrast to spacing of complete nucleosomes, we find that the nucleosome binding domain of HMG 17 (but not the nucleosome binding of HMG 14) is the only domain required for spacing of H2A/H2B-deficient chromatin.
Tremethick
D J
DJ
John Curtin School of Medical Research, Australian National University, Canberra, Australia.
Hyman
L
L
D000818:Animals; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D006609:High Mobility Group Proteins / Q000494:pharmacology*; D006657:Histones / Q000378:metabolism*; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D009707:Nucleosomes / Q000378:metabolism*; D014982:Xenopus laevis
D006609:High Mobility Group Proteins; D006657:Histones; D009707:Nucleosomes; D004247:DNA; D008274:Magnesium