Michael Bustin

Structural features of the HMG chromosomal proteins and their genes.

I Laboratory of Molecular Carcinogenesis, National Cancer Institute and 2 National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD (U.S.A.) (Received 30 March 1990) Key words: Chromosomal protein; Chromatin structure; Gene structure; DNA-protein interaction; High mobility group protein

High mobility group box-1 protein induces the migration and activation of human dendritic cells and acts as an alarmin

High mobility group box‐1 (HMGB1) protein is a nonhistone, DNA‐binding protein that plays a critical role in regulating gene transcription. Recently, HMGB1 has also been shown to act as a late mediator of endotoxic shock and to exert a variety of proinflammatory, extracellular activities. Here, we report that HMGB1 simultaneously acts as a chemoattractant and activator of dendritic cells (DCs). HMGB1 induced the migration of monocyte‐derived, immature DCs (Mo‐iDCs) but not mature DCs. The chemotactic effect of HMGB1 on iDCs was pertussis toxin‐inhibitable and also inhibited by antibody against the receptor of advanced glycation end products (RAGE), suggesting that HMGB1 chemoattraction of iDCs is mediated by RAGE in a Gi protein‐dependent manner. In addition, HMGB1 treatment of Mo‐iDCs up‐regulated DC surface markers (CD80, CD83, CD86, and HLA‐A, B,C), enhanced DC production of cytokines (IL‐6, CXCL8, IL‐12p70, and TNF‐α), switched DC chemokine responsiveness from CCL5‐sensitive to CCL21‐sensitive, and acquired the capacity to stimulate allogeneic T cell proliferation. Based on its dual DC‐attracting and ‐activating activities as well as its reported capacity to promote an antigen‐specific immune response, we consider HMGB1 to have the properties of an immune alarmin.

Network of Dynamic Interactions between Histone H1 and High-Mobility-Group Proteins in Chromatin

ABSTRACT Histone H1 and the high-mobility group (HMG) proteins are chromatin binding proteins that regulate gene expression by modulating the compactness of the chromatin fiber and affecting the ability of regulatory factors to access their nucleosomal targets. Histone H1 stabilizes the higher-order chromatin structure and decreases nucleosomal access, while the HMG proteins decrease the compactness of the chromatin fiber and enhance the accessibility of chromatin targets to regulatory factors. Here we show that in living cells, each of the three families of HMG proteins weakens the binding of H1 to nucleosomes by dynamically competing for chromatin binding sites. The HMG families weaken H1 binding synergistically and do not compete among each other, suggesting that they affect distinct H1 binding sites. We suggest that a network of dynamic and competitive interactions involving HMG proteins and H1, and perhaps other structural proteins, constantly modulates nucleosome accessibility and the local structure of the chromatin fiber.

Antibodies against chromosomal HMG proteins stain the cytoplasm of mammalian cells

Antibodies specific to protein HMG-1 were purified by affinity chromatography on Sepharose columns to which HMG-1 was covalently bound. Immunofluorescence studies with these antibodies reveal that HMG-1 or components which immunologically cross-react with HMG-1 are present in the cytoplasm of Chinese hamster V-79, rat liver TR-12 and bovine trachea EBTr-NBL-4 cells. At selected antibody concentrations, the fluorescence present in the cytoplasm is more intense than that observed in the nucleus. The presence of HMG-1 protein in the cytoplasm of rat liver cells was verified by direct examination of the protein content of selected cytoplasmic fractions. A protein with electrophoretic mobility identical to HMG-1 was detected by electrophoresis on polyacrylamide gels containing either sodium dodecylsulfate or urea. Furthermore, the cytoplasmic extracts yielded a positive complement fixation with anti-HMG-1, while no reaction was obtained with control anti-H1 sera. We suggest that HMG protins, rather than functioning in the nucleus alone, are important structural elements of the entire cell.

Dynamic interaction of HMGA1a proteins with chromatin

High-mobility-group proteins A1 (HMGA1; previously named HMGI/Y) function as architectural chromatin-binding proteins and are involved in the transcriptional regulation of several genes. We have used cells expressing proteins fused to green fluorescent protein (GFP) and fluorescence recovery after photobleaching (FRAP) to analyze the distribution and dynamics of HMGA1a in vivo. HMGA1-GFP proteins localize preferentially to heterochromatin and remain bound to chromosomes during mitosis. FRAP experiments showed that they are highly mobile components of euchromatin, heterochromatin and of mitotic chromosomes, although with different resident times. For a more-detailed investigation on the interaction of HMGA1a with chromatin, the contribution of the AT-hook DNA-binding motifs was analyzed using point-mutated HMGA1a-GFP proteins. Furthermore, by inhibiting kinase or histone deacetylase activities, and with the help of fusion proteins lacking specific phosphorylation sites, we analyzed the effect of reversible modifications of HMGA1a on chromatin binding. Collectively our data show that the kinetic properties of HMGA1a proteins are governed by the number of functional AT-hooks and are regulated by specific phosphorylation patterns. The higher residence time in heterochromatin and chromosomes, compared with euchromatic regions, correlates with an increased phosphorylation level of HMGA1a. The regulated dynamic properties of HMGA1a fusion proteins indicate that HMGA1 proteins are mechanistically involved in local and global changes in chromatin structure.

High-mobility group nucleosome-binding protein 1 acts as an alarmin and is critical for lipopolysaccharide-induced immune responses

HMGN1 is a novel alarmin that signals through TLR4 and is required for LPS-induced immune responses in vivo.

Dynamic relocation of chromosomal protein HMG‐17 in the nucleus is dependent on transcriptional activity

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. Here we show that 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 arrested 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 α‐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 which act as architectural elements of chromatin structure may be dynamic and functionally related to the transcriptional activity of the cell.

Down-Regulation of Nucleosomal Binding Protein HMGN1 Expression during Embryogenesis Modulates Sox9 Expression in Chondrocytes

ABSTRACT We find that during embryogenesis the expression of HMGN1, a nuclear protein that binds to nucleosomes and reduces the compaction of the chromatin fiber, is progressively down-regulated throughout the entire embryo, except in committed but continuously renewing cell types, such as the basal layer of the epithelium. In the developing limb bud, the expression of HMGN1 is complementary to Sox9, a master regulator of the chondrocyte lineage. In limb bud micromass cultures, which faithfully mimic in vivo chondrogenic differentiation, loss of HMGN1 accelerates differentiation. Expression of wild-type HMGN1, but not of a mutant HMGN1 that does not bind to chromatin, in Hmgn1 −/− micromass cultures inhibits Sox9 expression and retards differentiation. Chromatin immunoprecipitation analysis reveals that HMGN1 binds to Sox9 chromatin in cells that are poised to express Sox9. Loss of HMGN1 elevates the amount of HMGN2 bound to Sox9, suggesting functional redundancy among these proteins. These findings suggest a role for HMGN1 in chromatin remodeling during embryogenesis and in the activation of Sox9 during chondrogenesis.

Chromatin structure visualization by immunoelectron microscopy

Antibodies elicited in rabbits by chromatin and by purified histone H2B have been used to study the structure of chromatin by immunoelectron microscopy. Chromatin spread on grids reveals a structure of closely packed spherical particles with an average diameter of 104 A, arranged either in clusters or in linear arrays of beads, some of which have a supercoil-like arrangement. No DNA strings connecting the beads could be observed. Upon antibody binding, the diameter of the particles increases up to 300 A. This size is compatible with a model where one layer of gamma globulin molecules 110 A long encircles a sphere of chromatin 100 A in diameter. The presence of rabbit gamma globulins on the enlarged beads has been verified by the addition of ferritin-labeled goat anti-rabbit gamma globulins. Anti-chromatin sera which react with nonhistone proteins but not with free histones or DNA react with more than 95% of the beads; this suggests that most of the beads contain nonhistone proteins. Since the number of nonhistone proteins is large, it is improbable that each sphere contains a full complement of these proteins. We therefore suggest that the various chromatin spheres contain different types of nonhistone proteins. About 90% of the chromatin spheres reacted with antibodies to histone H2B, suggesting the most of the chromatin beads contain this type of histone.

Distinct Domains in High Mobility Group N Variants Modulate Specific Chromatin Modifications

We have demonstrated that levels of specific modification in histone H3 are modulated by members of the nucleosome-binding high mobility group N (HMGN) protein family in a variant-specific manner. HMGN1 (but not HMGN2) inhibits the phosphorylation of both H3S10 and H3S28, whereas HMGN2 enhances H3K14 acetylation more robustly than HMGN1. Two HMGN domains are necessary for modulating chromatin modifications, a non-modification-specific domain necessary for chromatin binding and a modification-specific domain localized in the C terminus of the HMGNs. Thus, chromatin-binding structural proteins such as HMGNs affect the levels of specific chromatin modifications and therefore may play a role in epigenetic regulation.