The chromatin unfolding domain of chromosomal protein HMG-14 targets the N-terminal tail of histone H3 in nucleosomes

Abstract

Nonhistone chromosomal protein HMG-14 is a nucleosomal binding protein that unfolds the higher-order chromatin structure and enhances the transcriptional potential of chromatin, but not that of DNA. Both the transcriptional enhancement and the chromatin unfolding activities of HMG-14 are mediated through the C-terminal region of the protein. Here we study the molecular interactions of both this region and the N-terminal region of HMG-14 with nucleosome cores. By protein photocrosslinking we demonstrate that the N-terminal domain of HMG-14 targets a restricted region in histone H2B, whereas the C-terminal chromatin unfolding domain of HMG-14 targets a restricted region in the N terminus of histone H3. The N-terminal regions of the core histones are involved in the folding of oligonucleosomes and are the target of various activities associated with chromatin unfolding and transcriptional activation. We suggest that specific interactions between the C-terminal domain of HMG-14 and the N-terminal tail of histone H3 reduce the compaction of chromatin. These findings provide insights into the molecular mechanism whereby HMG-14/-17 proteins reduce the repressive effect of chromatin, and they also broaden the scope of the molecular interactions involving the N termini of the core histones in nucleosomes.

Figure 1

Procedure for mapping nucleosome core–HMG-14 interactions. Radioactively labeled S-[N-(4-azidosalicyl)cysteaminyl]-2-thiopyridyl is attached to either 7C or 88C HMG-14 point mutants. The labeled point mutants are complexed with nucleosome cores and the phenylazide moiety is activated by exposure to UV. The bond between the HMG protein and the crosslinking agent is disrupted by 2-mercaptoethanol, and the target of crosslinking is identified by polyacrylamide gel electrophoresis and autoradiography. The ¹²⁵I-labeled histones are purified by HPLC and proteolyzed, and the resulting peptides are fractionated by HPLC and polyacrylamide gel electrophoresis. The targeted peptides are detected by autoradiography and identified by their amino acid sequence. See text for further details.

Figure 2

Mobility-shift assays indicate that modified HMG-14 point mutants bind to nucleosome cores. Nucleosome cores containing either nonacetylated (n) or acetylated (ac) histones were incubated either without (lanes 1, 4, and 7) or with a 4-fold molar excess (lanes 2, 5, and 8) or an 8-fold molar excess (lanes 3, 6, and 9) of the modified HMG-14 point mutant indicated on the top of the lanes. C, position of free core particle; C+2, position of core containing two molecules of HMG-14 protein (see also Fig. 3).

Figure 3

Identification of the targets of the HMG-14 point mutants. Acetylated (ac) or nonacetylated (n) cores containing the ¹²⁵I-labeled HMG-14 point mutants indicated at the top of the lanes were exposed to UV, treated with 2-mercaptoethanol, and analyzed by SDS/PAGE. (A) Autoradiograph. (B) Coomassie blue stain. Lanes 5–8 contained only the labeled HMG-14 mutants. Lanes 7 and 8 were not exposed to UV and therefore the radioactive label runs with the ion front. (C) The degree of acetylation of the histones in the nucleosome cores was assessed by Coomassie blue staining of Triton/acid/urea polyacrylamide gels (33).

Figure 4

Identification of the region in histone H3 targeted by the HMG-14 88C point mutant. A proteolytic digest of the ¹²⁵I-labeled histone was fractionated by HPLC (A) and the peptides present in the fractions were analyzed by PAGE. The targeted peptides were identified by autoradiography (B) of Coomassie blue-stained gels (C).

Figure 5

Identification of the region in histone H2B targeted by the HMG-14 7C point mutant. A proteolytic digest of the ¹²⁵I-labeled histone was fractionated by HPLC (A) and the peptides present in the fractions were analyzed by PAGE. The targeted peptides were identified by autoradiography (B) of Coomassie blue-stained gels (C).

Figure 6

Organization of HMG-14 in nucleosome cores. (A) Specific crosslinks between HMG-14 and histones in nucleosome cores. The α-helical regions in the histones are depicted as boxes. The evolutionarily conserved domains in HMG-14 are depicted as cylinders. The amino acid positions at which the proteases cleaved the histones are indicated above their sequence. The radioactive counts present in each of the peptides is indicated, expressed as percent of total. The regions targeted in H2B and H3 by amino acid residues 7 and 88 of HMG-14, respectively, are indicated by the striped boxes. (B) Model of the HMG-14 binding sites. The ribbon traces for the DNA and histones in the core particles were reproduced by permission from the recent article in Nature by Luger et al. (12), copyright 1997, Macmillan Magazines Ltd. The solid white symbols, in the two major grooves flanking the dyad axis and approximately 25 bp from the end of the DNA, indicate the regions where HMG-14/-17 protect the DNA from hydroxyl radical cleavage. The open white circles represent the approximate location of the crosslinks identified in the present study. The red arrow points to the N-terminal region of histone H3. Histones H2B and H3 are represented by red and blue ribbons, respectively.