Chromatin associated HMG proteins

Chromosomal proteins HMG-14/-17 are nucleosomal binding proteins, which alter the structure of the chromatin fiber and enhance transcription, but only from chromatin templates. In tissue culture cells, HMG-17 protein colocalizes with sites of active transcription. Incubation of permeabilized cells with a peptide corresponding to the nucleosomal binding domains of HMG-14/-17 specifically arrests polymerase II-dependent transcription. In these cells the peptide displaces HMG-17 from chromatin and reduces the cellular content of the protein. These results suggest that the presence of HMG-14/-17 in chromatin is required for efficient polymerase II transcription. In non-permeabilized, actively transcribing cells, the protein is dispersed in a punctate pattern, throughout the nucleus. Upon transcriptional inhibition by alpha-amanitin or actinomycin D, the protein gradually redistributes until it localizes fully to interchromatin granule clusters, together with the splicing factor SC35. The results suggest that the association of HMG-17 with chromatin is dynamic rather than static, and that in the absence of transcription, HMG-17 is released from chromatin and accumulates in interchromatin granule clusters. Thus, the intranuclear distribution of chromosomal proteins that act as architectural elements of chromatin structure may be dynamic and functionally related to the transcriptional activity of the cell (Hock, 1998a).

The high mobility group 14/17 (HMG-14/-17) proteins form specific complexes with nucleosome core particles and produce distinct footprints on nucleosomal DNA. Therefore, they could be an integral part of the chromatin fiber. During the cell cycle these proteins are transiently dissociated from chromatin. They colocalize with the nuclear DNA in interphase and prophase but not in metaphase and anaphase. They relocate into the nucleus and colocalize again with the DNA in late telophase, concomitantly with the appearance of the nuclear envelope. Thus, these nucleosomal binding proteins are not always associated with chromatin. Using reconstituted nuclei and permeabilized cells, it has been demonstrated that these two small proteins, with a molecular mass <10 kD, are actively imported into the nucleus. The major elements involved in the nuclear import of these chromosomal proteins are identified: HMG-14/-17 proteins contain an intrinsic bipartite nuclear localization signal, and their entry into the nucleus through nuclear pores requires energy and the participation of importin alpha. These findings suggest that the cell cycle-related association of HMG-14/-17 with chromatin is dependent on, and perhaps regulated by, nuclear import processes (Hock, 1998b).

The effect of HMG17 on transcription by RNA polymerase II has been examined by the assembly and analysis of HMG17-containing chromatin templates consisting of regularly spaced nucleosomal arrays. Structural analysis of the chromatin indicates that HMG17 is incorporated into chromatin in a physiological manner with the full complement of core histones. The transcriptional studies reveal that HMG17 stimulates transcription in conjunction with the sequence-specific activator GAL4-VP16. This effect is observed with chromatin, but not with non-nucleosomal templates, and requires the presence of HMG17 during chromatin assembly. The incorporation of HMG17 into chromatin results in a 7- to 40-fold stimulation of GAL4-VP16-activated transcription to levels that are comparable to those observed with histone-free DNA templates. In contrast, transcription from HMG17-containing chromatin is not detectable in the absence of GAL4-VP16 or with a GAL4 derivative [GAL4(1-147)] lacking the VP16 activation domain. Finally, the incorporation of HMG17 into chromatin is found to increase the efficiency of transcription initiation, but not the extent of transcriptional elongation. Thus, HMG17 is a chromatin-specific transcriptional coactivator that increases the efficiency of initiation of transcription by RNA polymerase II (Paranjape, 1995).

Chromosomal proteins HMG-14 and HMG-17 enhance the transcriptional potential of chromatin when incorporated into nucleosomes during (but not after) chromatin assembly on replicating DNA. Two molecules of either HMG-14 or HMG-17 can bind to nucleosome cores, independently of the underlying DNA sequence, in a cooperative fashion to limit nucleosome mobility and stabilize the structure of the nucleosome core without stabilizing the higher order chromatin structure. By modifying the structure of nucleosomes, the proteins affect the local structure of the chromatin fiber leading to an increase in the rate of transcriptional elongation but not initiation. It is thought that HMG-14/-17 are architectural elements that assist in the assembly of an unfolded chromatin fiber, thereby decreasing the repressive activity of histones and facilitating transcriptional processes (Bustin, 1995).

Chromosomal proteins HMG-14/HMG-17 enhanced transcription from a chromatin template carrying a 5S rRNA gene, but not from a DNA template. 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. It is thought 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 (Trieschmann, 1995).

Nucleosome core particles interact with an equimolar mixture of the chromosomal proteins HMG-14 and HMG-17 to form, exclusively, complexes containing two molecules of either HMG-14 or HMG-17 (homodimers). Analysis of the binding of various mixtures of wild-type proteins and their deletion mutants indicates that homodimer formation is not dependent on contacts between the nucleosome-bound HMG-14/-17 proteins themselves. It is thought that HMG-14/-17 proteins in nucleosomes cross-talk by inducing specific allosteric transitions in the chromatin subunit (Postnikov, 1995).

Histone H1 promotes the generation of a condensed, transcriptionally inactive, higher-order chromatin structure. Consequently, histone H1 activity must be antagonized in order to convert chromatin to a transcriptionally competent, more extended structure. Using simian virus 40 minichromosomes as a model system, it has been demonstrated that the nonhistone chromosomal protein HMG-14, which is known to preferentially associate with active chromatin, completely alleviates histone H1-mediated inhibition of transcription by RNA polymerase II. HMG-14 also partially disrupts histone H1-dependent compaction of chromatin. Both the transcriptional enhancement and chromatin-unfolding activities of HMG-14 are mediated through its acidic, C-terminal region. Strikingly, transcriptional and structural activities of HMG-14 are maintained upon replacement of the C-terminal fragment by acidic regions from either GAL4 or HMG-2. These data support the model that the acidic C terminus of HMG-14 is involved in unfolding higher-order chromatin structure to facilitate transcriptional activation of mammalian genes (Ding, 1997).

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. PCAF, a transcription coactivator with intrinsic histone acetyltransferase activity, specifically acetylates HMG-17 but not HMG-14. Using mass spectrum sequence analysis, the lysine at position 2 has been identified 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 it has been demonstrated that acetylation reduces the affinity of HMG-17 to nucleosome cores. In addition, 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 (Herrera, 1999).

HMG-14 and HMG-17 form specific complexes with and stabilize the structure of the nucleosome cores. In these complexes, the C-terminal region of HMG-14/-17 proteins is in close proximity to the N-terminal region of histone H3, which is the main nucleosomal target of PCAF. The presence of both HMG-14 and HMG-17 inhibits histone H3 acetylation. The C-terminally deleted form of HMG-17, which binds to nucleosomes with the same affinity as does the intact protein, inhibits the acetylation of H3 to a significantly lower degree than does intact HMG-17. Therefore, it is suggested that in the HMG-nucleosome complex the C terminus of HMG-17 (and HMG-14) sterically hinders the interaction between PCAF and H3. The reversible acetylation of the N termini of the histones is a key step in the reorganization of the chromatin structure leading to transcriptional activation. The finding that the presence of HMG-14/-17 affects the acetylation of H3 points to an additional mechanism whereby these proteins may affect DNA-dependent processes occurring in chromatin (Herrera, 1999 and references).

Results from several laboratories indicate that HMG-14/-17 proteins enhance transcription and replication, but only from chromatin and not from DNA templates. The enhancement of these DNA-dependent activities is associated with the unfolding of the higher-order chromatin structure and is due to the interaction of the proteins with the N termini of the core histones and with histone H1. By unfolding the higher-order chromatin structure, these HMG proteins facilitate the access of various chromatin-modifying factors to the underlying oligonucleosomal chain, i.e., to the primary level of DNA packing in chromatin. However, it is well documented that the binding of HMG-14/-17 stabilizes the structure of the nucleosomes, a situation that seems inconsistent with transcriptional activation. It is generally accepted that nucleosomes repress transcription and that destabilizing this structure is a prerequisite for transcriptional activation. The finding that PCAF acetylates a known in vivo acetylation site and that this modification reduces the affinity of HMG-17 to nucleosomes suggests a possible mechanism to resolve this HMG conflict. Conceivably, the binding of HMG-17 to nucleosomes unfolds the higher-order chromatin fiber and enhances the accessibility of various factors, including HATs, to the primary level of chromatin organization, i.e., the nucleosomes in the 10-nm chromatin fiber. The temporary stabilization of the nucleosome in this first stage of HMG-17 action may promote transcription from chromatin, especially if certain components of the transcriptional machinery preferentially recognize the nucleosome core particle. In a later stage, a HAT-containing complex acetylates the protein, thereby reducing its affinity to nucleosomes and alleviating its stabilizing affects on the nucleosome structure. This model provides a unifying concept for the effect of HATs on the interaction of nuclear proteins with their targets. Specific acetylation of the structural nonhistone protein HMG-17 reduces its binding affinity to nucleosome cores much as the specific acetylation of the N termini of histones reduces their affinity for the nucleosomal DNA (Herrera, 1999 and references).

The nucleosomal response refers to the rapid phosphorylation of histone H3 on serine 10 and HMG-14 on serine 6 that occurs concomitantly with immediate-early (IE) gene induction in response to a wide variety of stimuli. Using antibodies against the phosphorylated residues, it has been shown that H3 and HMG-14 phosphorylation is mediated via different MAP kinase (MAPK) cascades, depending on the stimulus. The nucleosomal response elicited by TPA is ERK-dependent, whereas that elicited by anisomycin is p38 MAPK-dependent. In intact cells, the nucleosomal response can be selectively inhibited using the protein kinase inhibitor H89. MAPK activation and phosphorylation of transcription factors are largely unaffected by H89, whereas induction of IE genes is inhibited and its characteristics markedly altered. MSK1 is considered the most likely kinase to mediate this response because (1) it is activated by both ERK and p38 MAPKs; (2) it is an extremely efficient kinase for HMG-14 and H3, utilizing the physiologically relevant sites; and (3) its activity towards H3/HMG-14 is uniquely sensitive to H89 inhibition. Thus, the nucleosomal response is an invariable consequence of ERK and p38 but not JNK/SAPK activation, and MSK1 potentially provides a link to complete the circuit between cell surface and nucleosome (Thomson, 1999).