The role of chromosomal proteins HMG‐14 and HMG‐17 in the generation of transcriptionally active chromatin was studied in a Xenopus laevis egg extract which supports complementary DNA strand synthesis and chromatin assembly. Chromosomal proteins HMG‐14/HMG‐17 enhanced transcription from a chromatin template carrying a 5S rRNA gene, but not from a DNA template. The transcriptional potential of chromatin was enhanced only when these proteins were incorporated into the template during, but not after, chromatin assembly. HMG‐14 and HMG‐17 stimulate transcription by increasing the activity, and not the number, of transcribed templates. They unfold the chromatin template without affecting the nucleosomal repeat or decreasing the content of histone B4. We suggest that HMG‐14/HMG‐17 enhance transcription by inducing an extended conformation in the chromatin fiber, perhaps due to interactions with histone tails in nucleosomes. By disrupting the higher order chromatin structure HMG‐14/HMG‐17 increase the accessibility of target sequences to components of the transcriptional apparatus.

Nonhistone chromosomal proteins HMG-14 and HMG-17 are closely related nucleosomal binding proteins that unfold the higher-order chromatin structure, thereby enhancing the transcription and replication potential of chromatin. Here we report that PCAF, a transcription coactivator with intrinsic histone acetyltransferase activity, specifically acetylates HMG-17 but not HMG-14. Using mass spectrum sequence analysis, we identified the lysine at position 2 as the predominant site acetylated by PCAF. Lysine 2 is a prominent acetylation site in vivo, suggesting that this PCAF-mediated acetylation is physiologically relevant. Experiments with HMG-17 deletion mutants and competition studies with various protein fragments indicate that the specific acetylation of HMG-17 is not determined solely by the primary sequence near the acetylation site. By equilibrium dialysis we demonstrated that acetylation reduces the affinity of HMG-17 to nucleosome cores. In addition, we found that the binding of HMG-14 and HMG-17 to nucleosome cores inhibits the PCAF-mediated acetylation of histone H3. Thus, the presence of HMG-14 and HMG-17 affects the ability of PCAF to acetylate chromatin, while the acetylation of HMG-17 reduces its binding affinity to chromatin. Conceivably, in HMG-17-containing chromatin, acetylation of HMG-17 precedes the acetylation of histones.Reversible acetylation of the N-terminal tails of histones plays a key role in the regulation of various nuclear activities such as chromatin assembly, replication, and transcription (2,19,29,39,49,51,52). The acetylation of lysine residues within nucleosomes weakens the interaction of the histone tails with the DNA and leads to chromatin decompaction (16,17). These structural transitions enhance the accessibility of the underlying DNA sequence to various factors, thereby reducing the repressive effect of the nucleosome on transcription and replication. The relationship between transcriptional regulation and histone acetylation has been strengthened considerably by the discovery that certain factors associated with transcriptional activation have intrinsic histone acetylase activity (7,20,30,31,44,53), while factors associated with transcriptional repression contain histone deacetylase activity (26,44). It is significant that in some cases this reversible acetylation is targeted and specific. For example, Tetrahymena GCN5 preferentially acetylates residues K8 and K16 of histone H4 and K14 of histone H3 (13, 24). In contrast, in Saccharomyces cerevisiae, transcriptional repression by UME6 involves the specific deacetylation of K5 in histone H4 by the deacetylase RPD3 (40). Furthermore, the pattern of H4 acetylation in heterochromatin is unique, suggesting that specific acetylation marks discrete functional states of chromatin structure (5, 32). Taken together with other findings, these results suggest that the reversible acetylation of histones is not merely a mechanism for indiscriminately unfolding chromatin but is a key step in the selective regulation of the expression of spe...

A cell‐free system from Xenopus eggs was used to study the role of chromosomal protein HMG‐17 in the generation of the chromatin structure of transcriptionally active genes. Addition of HMG‐17 protein to the extracts, which do not contain structural homologs of the HMG‐14/−17 protein family, indicates the protein is incorporated into the nascent template during replication, prior to completion of chromatin assembly. The protein binds to and stabilizes the structure of the nucleosomal core thereby improving the apparent periodicity of the nucleosomal spacing of nascent chromatin. Assembly of HMG‐17 into the nascent chromatin structure significantly increased the transcription potential of the 5S RNA gene and satellite I chromatin. Kinetic studies indicate that the increase in transcriptional potential is observed only when HMG‐17 is incorporated into nucleosomes during chromatin assembly.

Chromosomal proteins HMG-14 and HMG-17 are the only known nuclear proteins which specifically bind to the nucleosome core particle and are implicated in the generation and/or maintenance of structural features specific to active chromatin. The two proteins facilitate polymerase II and III transcription from in vitro- and in vivo-assembled circular chromatin templates. Here we used deletion mutants and specific peptides to identify the transcriptional enhancement domain and delineate the nucleosomal binding domain of the HMG-14 and -17 proteins. Deletion of the 22 C-terminal amino acids of HMG-17 or 26 C-terminal amino acids of HMG-14 reduces significantly the ability of the proteins to enhance transcription from chromatin templates. In contrast, N-terminal truncation mutants had the same transcriptional enhancement activity as the full-length proteins. We conclude that the negatively charged C-terminal region of the proteins is required for transcriptional enhancement. Chromatin transcription enhancement assays, which involve binding competition between the full-length proteins and peptides derived from their nucleosomal binding regions, indicate that the minimal nucleosomal binding domain of human HMG-17 is 24 amino acids long and spans residues 17 to 40. The results suggest that HMG-14 and -17 proteins have a modular structure and contain distinct functional domains.

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-14͞-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.