Takashi Furusawa

Chromosomal protein HMGN1 enhances the rate of DNA repair in chromatin

We report that HMGN1, a nucleosome binding protein that destabilizes the higher‐order chromatin structure, modulates the repair rate of ultraviolet light (UV)‐induced DNA lesions in chromatin. Hmgn1−/− mouse embryonic fibroblasts (MEFs) are hypersensitive to UV, and the removal rate of photoproducts from the chromatin of Hmgn1−/− MEFs is decreased as compared with the chromatin of Hmgn1+/+ MEFs; yet, host cell reactivation assays and DNA array analysis indicate that the nucleotide excision repair (NER) pathway in the Hmgn1−/− MEFs remains intact. The UV hypersensitivity of Hmgn1−/− MEFs could be rescued by transfection with plasmids expressing wild‐type HMGN1 protein, but not with plasmids expressing HMGN1 mutants that do not bind to nucleosomes or do not unfold chromatin. Transcriptionally active genes, the main target of the NER pathways in mice, contain HMGN1 protein, and loss of HMGN1 protein reduces the accessibility of transcribed genes to nucleases. By reducing the compaction of the higher‐order chromatin structure, HMGN1 facilitates access to UV‐damaged DNA sites and enhances the rate of DNA repair in chromatin.

Activation of ATM depends on chromatin interactions occurring before induction of DNA damage

Efficient and correct responses to double-stranded breaks (DSB) in chromosomal DNA are crucial for maintaining genomic stability and preventing chromosomal alterations that lead to cancer. The generation of DSB is associated with structural changes in chromatin and the activation of the protein kinase ataxia-telangiectasia mutated (ATM), a key regulator of the signalling network of the cellular response to DSB. The interrelationship between DSB-induced changes in chromatin architecture and the activation of ATM is unclear. Here we show that the nucleosome-binding protein HMGN1 modulates the interaction of ATM with chromatin both before and after DSB formation, thereby optimizing its activation. Loss of HMGN1 or ablation of its ability to bind to chromatin reduces the levels of ionizing radiation (IR)-induced ATM autophosphorylation and the activation of several ATM targets. IR treatments lead to a global increase in the acetylation of Lys 14 of histone H3 (H3K14) in an HMGN1-dependent manner and treatment of cells with histone deacetylase inhibitors bypasses the HMGN1 requirement for efficient ATM activation. Thus, by regulating the levels of histone modifications, HMGN1 affects ATM activation. Our studies identify a new mediator of ATM activation and demonstrate a direct link between the steady-state intranuclear organization of ATM and the kinetics of its activation after DNA damage.

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.

Increased Tumorigenicity and Sensitivity to Ionizing Radiation upon Loss of Chromosomal Protein HMGN1

We report that loss of HMGN1, a nucleosome-binding protein that alters the compaction of the chromatin fiber, increases the cellular sensitivity to ionizing radiation and the tumor burden of mice. The mortality and tumor burden of ionizing radiation-treated Hmgn1-/- mice is higher than that of their Hmgn1+/+ littermates. Hmgn1-/- fibroblasts have an altered G2-M checkpoint activation and are hypersensitive to ionizing radiation. The ionizing radiation hypersensitivity and the aberrant G2-M checkpoint activation of Hmgn1-/- fibroblasts can be reverted by transfections with plasmids expressing wild-type HMGN1, but not with plasmids expressing mutant HMGN proteins that do not bind to chromatin. Transformed Hmgn1-/- fibroblasts grow in soft agar and produce tumors in nude mice with a significantly higher efficiency than Hmgn1+/+ fibroblasts, suggesting that loss of HMGN1 protein disrupts cellular events controlling proliferation and growth. Hmgn1-/- mice have a higher incidence of multiple malignant tumors and metastases than their Hmgn1+/+ littermates. We suggest that HMGN1 optimizes the cellular response to ionizing radiation and to other tumorigenic events; therefore, loss of this protein increases the tumor burden in mice.

An orphan nuclear receptor lacking a zinc-finger DNA-binding domain: interaction with several nuclear receptors

The yeast two-hybrid screening was applied to cloning cDNAs of proteins that interact with peroxisome proliferator-activated receptor alpha (PPAR alpha). We obtained from a rat liver cDNA library a clone encoding a protein related to the ligand-binding domain of the members of nuclear hormone receptor superfamily, whereas apparently lacking the zinc-finger DNA-binding domain. This protein interacted with the activated forms of several nuclear receptors, and thus is a novel type of heterodimer-forming nuclear receptor.

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.

The Nucleosome Binding Protein HMGN3 Modulates the Transcription Profile of Pancreatic β Cells and Affects Insulin Secretion

ABSTRACT Improper glucose-stimulated insulin secretion from pancreatic β cells is a major factor in the onset of type 2 diabetes. We now report that HMGN3, a nuclear protein that binds to nucleosomes and affects chromatin function, is highly expressed in β cells and that in mice, loss of HMGN3 impairs glucose-stimulated insulin secretion and leads to a diabetic phenotype. In pancreatic β cells, loss of HMGN3 affects the transcription of several genes involved in glucose-stimulated insulin secretion, including that of the Glut2 glucose transporter. Chromatin immunoprecipitation reveals that HMGN3 and the transcription factor PDX1 mutually reinforce their specific binding to the chromatin in the promoter of the Glut2 gene, thereby regulating GLUT2 protein levels in pancreatic islets and in β cells. Our results identify a new regulator of glucose homeostasis and demonstrate a link between the activity of a nucleosome binding structural protein and the regulation of insulin secretion.

Nuclear receptor binding factor-2 (NRBF-2), a possible gene activator protein interacting with nuclear hormone receptors.

A protein named nuclear receptor binding factor-2 (NRBF-2) was identified by yeast two-hybrid screening, as an interaction partner of peroxisome proliferator-activated receptor alpha as well as several other nuclear receptors. NRBF-2 exhibited a gene activation function, when tethered to a heterologous DNA binding domain, in both mammalian cells and yeast.

Developmental function of HMGN proteins

High mobility group N (HMGN) proteins are the only nuclear proteins known to specifically recognize the generic structure of the 147-bp nucleosome core particle. Both in vitro and in vivo experiments demonstrate that HMGN proteins are involved in epigenetic regulation by modulating chromatin structure and levels of posttranslational modifications of nucleosomal histones. Expression of HMGN proteins is developmentally regulated, and the loss or overexpression of these proteins can lead to developmental abnormalities. This review will focus on the role and on the possible molecular mechanism whereby HMGN proteins affect cellular differentiation and development.

Chromatin decompaction by the nucleosomal binding protein HMGN5 impairs nuclear sturdiness

In most metazoan nuclei, heterochromatin is located at the nuclear periphery in contact with the nuclear lamina, which provides mechanical stability to the nucleus. We show that in cultured cells, chromatin de-compaction by the nucleosome binding protein HMGN5 decreases the sturdiness, elasticity, and rigidity of the nucleus. Mice overexpressing HMGN5, either globally or only in the heart, are normal at birth but develop hypertrophic heart with large cardiomyoctyes, deformed nuclei and disrupted lamina, and die of cardiac malfunction. Chromatin de-compaction is seen in cardiomyocytes of newborn mice but misshaped nuclei with disrupted lamina are seen only in adult cardiomyocytes, suggesting that loss of heterochromatin diminishes the ability of the nucleus to withstand the mechanical forces of the contracting heart. Thus, heterochromatin enhances the ability of the nuclear lamina to maintain the sturdiness and shape of the eukaryotic nucleus; a structural role for chromatin that is distinct from its genetic functions.