HIGH-MOBILITY GROUP PROTEIN-14 AND PROTEIN-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-as sociated proteins found in all higher eukaryotic nuclei. This observat ion demonstrates that HMGs 14 and 17 must have an important and univer sal function with regard to the structure and function of chromatin. W hat this function is, including how they interact with a nucleosomal a rray in vivo, is not known. Recently, we have demonstrated that HMGs 1 4 and 17 can organize nucleosomes into a regular array and increase th e repeat length from 145 to about 160-165 base pairs in vitro. In addi tion, they can increase the apparent repeat length of chromatin defici ent in histones H2A/H2B from 125 to approximately 145 base pairs. Impo rtantly, this template was transcriptionally active. In this study, we report five new observations that begin to address the mechanism by w hich HMGs 14 and 17 space nucleosomal particles. First, we demonstrate that both human placenta HMG 14 and HMG 17 can space nucleosomes to p roduce 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 t hat digestion of HMG containing chromatin with micrococcal nuclease pr oduces DNA fragments that were approximately 10 and 20 base pairs long er than nucleosome core-particle DNA. This suggests that HMG 14 or HMG 17 can protect, directly or indirectly, at least an additional 10 bas e pairs of linker DNA from micrococcal digestion. However, this HMG-co ntaining particle does not produce a strong kinetic block, and further digestion results in the eventual accumulation of DNA fragments 145 b ase pairs in length. Third, by comparing the full-length protein with different domains, we demonstrate that the acidic carboxyl-terminal do main is absolutely required for nucleosome spacing; neither the nucleo some 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 o f chromatin deficient in histones H2A/H2B led to the accumulation of D NA fragments about 110 base pairs in length, which is presumably the l ength of DNA associated with a nucleosomal particle deficient in one H 2A/H2B dimer. Incorporation of either HMG 14 or HMG 17 into this chrom atin results in the disappearance of this band and increase in the acc umulation of fragments around 140-150 base pairs in length. Finally, i n contrast to spacing of complete nucleosomes, we find that the nucleo some binding domain of HMG 17 (but not the nucleosome binding of HMG 1 4) is the only domain required for spacing of H2A/H2B-deficient chroma tin.