Yuri V. Postnikov

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.

Chromosomal protein HMGN1 enhances the acetylation of lysine 14 in histone H3

The acetylation levels of lysine residues in nucleosomes, which are determined by the opposing activities of histone acetyltransferases (HATs) and deacetylases, play an important role in regulating chromatin‐related processes, including transcription. We report that HMGN1, a nucleosomal binding protein that reduces the compaction of the chromatin fiber, increases the levels of acetylation of K14 in H3. The levels of H3K14ac in Hmgn1−/− cells are lower than in Hmgn1+/+ cells. Induced expression of wild‐type HMGN1, but not of a mutant that does not bind to chromatin, in Hmgn1−/− cells elevates the levels of H3K14ac. In vivo, HMGN1 elevates the levels of H3K14ac by enhancing the action of HAT. In vitro, HMGN1 enhances the ability of PCAF to acetylate nucleosomal, but not free, H3. Thus, HMGN1 modulates the levels of H3K14ac by binding to chromatin. We suggest that HMGN1, and perhaps similar architectural proteins, modulates the levels of acetylation in chromatin by altering the equilibrium generated by the opposing enzymatic activities that continuously modify and de‐modify the histone tails in nucleosomes.

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.

The interaction of NSBP1/HMGN5 with nucleosomes in euchromatin counteracts linker histone-mediated chromatin compaction and modulates transcription.

Structural changes in specific chromatin domains are essential to the orderly progression of numerous nuclear processes, including transcription. We report that the nuclear protein NSBP1 (HMGN5), a recently discovered member of the HMGN nucleosome-binding protein family, is specifically targeted by its C-terminal domain to nucleosomes in euchromatin. We find that the interaction of NSBP1 with nucleosomes alters the compaction of cellular chromatin and that in living cells, NSBP1 interacts with linker histones. We demonstrate that the negatively charged C-terminal domain of NSBP1 interacts with the positively charged C-terminal domain of H5 and that NSBP1 counteracts the linker histone-mediated compaction of a nucleosomal array. Dysregulation of the cellular levels of NSBP1 alters the transcription level of numerous genes. We suggest that mouse NSBP1 is an architectural protein that binds preferentially to euchromatin and modulates the fidelity of the cellular transcription profile by counteracting the chromatin-condensing activity of linker histones.

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.

Regulation of chromatin structure and function by HMGN proteins.

High mobility group nucleosome-binding (HMGN) proteins are architectural non-histone chromosomal proteins that bind to nucleosomes and modulate the structure and function of chromatin. The interaction of HMGN proteins with nucleosomes is dynamic and the proteins compete with the linker histone H1 chromatin-binding sites. HMGNs reduce the H1-mediated compaction of the chromatin fiber and facilitate the targeting of regulatory factors to chromatin. They modulate the cellular epigenetic profile, affect gene expression and impact the biological processes such as development and the cellular response to environmental and hormonal signals. Here we review the role of HMGN in chromatin structure, the link between HMGN proteins and histone modifications, and discuss the consequence of this link on nuclear processes and cellular phenotype.

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.

Metastable Macromolecular Complexes Containing High Mobility Group Nucleosome-binding Chromosomal Proteins in HeLa Nuclei

High mobility group nucleosome-binding (HMGN) proteins belong to a family of nuclear proteins that bind to nucleosomes and enhance transcription from chromatin templates by altering the structure of the chromatin fiber. The intranuclear organization of these proteins is dynamic and related to the metabolic state of the cell. Here we report that ∼50% of the HMGN proteins are organized into macromolecular complexes in a fashion that is similar to that of other nuclear activities that modify the structure of the chromatin fiber. We identify several distinct HMGN-containing complexes that are relatively unstable and find that the inclusion of HMGN in the complexes varies according to the metabolic state of the cell. The nucleosome binding ability of HMGN in the complex is stronger than that of the free HMGN. We suggest that the inclusion of HMGN proteins into metastable multiprotein complexes serves to target the HMGN proteins to specific sites in chromatin and enhances their interaction with nucleosomes.

Chromosomal Protein HMGN1 Enhances the Heat Shock-induced Remodeling of Hsp70 Chromatin

The nucleosome-binding protein HMGN1 affects the structure and function of chromatin; however, its role in regulating specific gene expression in living cells is not fully understood. Here we use embryonic fibroblasts from Hmgn1+/+ and Hmgn1–/– mice to examine the effect of HMGN1 on the heat shock-induced transcriptional activation of Hsp70, a well characterized gene known to undergo a rapid chromatin re-structuring during transcriptional activation. We find that loss of HMGN1 decreases the levels of Hsp70 transcripts at the early stages of heat shock. HMGN1 enhances the rate of heat shockinduced changes in the Hsp70 chromatin but does not affect the chromatin structure before induction, an indication that it does not predispose the gene to rapid activation. Heat shock elevates the levels of H3K14 acetylation in the Hsp70 chromatin of wild type cells more efficiently than in the chromatin of Hmgn1–/– cells, whereas treatment with histone deacetylase inhibitors abrogates the effects of HMGN1 on the heat shock response. We suggest that HMGN1 enhances the rate of heat shock-induced H3K14 acetylation in the Hsp70 promoter, thereby enhancing the rate of chromatin remodeling and the subsequent transcription during the early rounds of Hsp70 activation when the gene is still associated with histones in a nucleosomal conformation.

Chromosomal protein HMGN1 modulates the expression of N-cadherin.

HMGN1 is a nuclear protein that binds to nucleosomes and alters the accessibility of regulatory factors to their chromatin targets. To elucidate its biological function and identify specific HMGN1 target genes, we generated Hmgn1–/– mice. DNA microarray analysis of Hmgn1+/+ and Hmgn1–/– embryonic fibroblasts identified N‐cadherin as a potential HMGN1 gene target. RT‐PCR and western blot analysis confirmed a linkage between HMGN1 expression and N‐cadherin levels. In both transformed and primary mouse embryonic fibroblasts (MEFs), HMGN1 acted as negative regulator of N‐cadherin expression. Likewise, the N‐cadherin levels in early embryos of Hmgn1–/– mice were higher than those of their Hmgn1+/+ littermates. Loss of HMGN1 increased the adhesiveness, motility and aggregation potential of Hmgn1–/– MEFs, a phenotype consistent with increased levels of N‐cadherin protein. Re‐expression of wild‐type HMGN1, but not of the mutant HMGN1 protein that does not bind to chromatin, in Hmgn1–/– MEFs, decreased the levels of N‐cadherin and restored the Hmgn1+/+ phenotype. These studies demonstrate a role for HMGN1 in the regulation of specific gene expression. We suggest that in MEFs, and during early mouse development, the interaction of HMGN1 with chromatin down‐regulates the expression of N‐cadherin.