HMGN1 and 2 remodel core and linker histone tail domains within chromatin.

The structure of the nucleosome, the basic building block of the chromatin fiber, plays a key role in epigenetic regulatory processes that affect DNA-dependent processes in the context of chromatin. Members of the HMGN family of proteins bind specifically to nucleosomes and affect chromatin structure and function, including transcription and DNA repair. To better understand the mechanisms by which HMGN 1 and 2 alter chromatin, we analyzed their effect on the organization of histone tails and linker histone H1 in nucleosomes. We find that HMGNs counteract linker histone (H1)-dependent stabilization of higher order 'tertiary' chromatin structures but do not alter the intrinsic ability of nucleosome arrays to undergo salt-induced compaction and self-association. Surprisingly, HMGNs do not displace H1s from nucleosomes; rather these proteins bind nucleosomes simultaneously with H1s without disturbing specific contacts between the H1 globular domain and nucleosomal DNA. However, HMGNs do alter the nucleosome-dependent condensation of the linker histone C-terminal domain, which is critical for stabilizing higher-order chromatin structures. Moreover, HMGNs affect the interactions of the core histone tail domains with nucleosomal DNA, redirecting the tails to more interior positions within the nucleosome. Our studies provide new insights into the molecular mechanisms whereby HMGNs affect chromatin structure.

Elevated HMGN4 expression potentiates thyroid tumorigenesis.

Thyroid cancer originates from genetic and epigenetic changes that alter gene expression and cellular signaling pathways. Here, we report that altered expression of the nucleosome-binding protein HMGN4 potentiates thyroid tumorigenesis. Bioinformatics analyses reveal increased HMGN4 expression in thyroid cancer. We find that upregulation of HMGN4 expression in mouse and human cells, and in the thyroid of transgenic mice, alters the cellular transcription profile, downregulates the expression of the tumor suppressors Atm, Atrx and Brca2, and elevates the levels of the DNA damage marker γH2AX. Mouse and human cells overexpressing HMGN4 show increased tumorigenicity as measured by colony formation, by tumor generation in nude mice, and by the formation of preneoplastic lesions in the thyroid of transgenic mice. Our study identifies a novel epigenetic factor that potentiates thyroid oncogenesis and raises the possibility that HMGN4 may serve as an additional diagnostic marker, or therapeutic target in certain thyroid cancers.

Functional interplay between histone H1 and HMG proteins in chromatin.

The dynamic interaction of nucleosome binding proteins with their chromatin targets is an important element in regulating the structure and function of chromatin. Histone H1 variants and High Mobility Group (HMG) proteins are ubiquitously expressed in all vertebrate cells, bind dynamically to chromatin, and are known to affect chromatin condensation and the ability of regulatory factors to access their genomic binding sites. Here, we review the studies that focus on the interactions between H1 and HMGs and highlight the functional consequences of the interplay between these architectural chromatin binding proteins. H1 and HMG proteins are mobile molecules that bind to nucleosomes as members of a dynamic protein network. All HMGs compete with H1 for chromatin binding sites, in a dose dependent fashion, but each HMG family has specific effects on the interaction of H1 with chromatin. The interplay between H1 and HMGs affects chromatin organization and plays a role in epigenetic regulation.

Loss of the nucleosome-binding protein HMGN1 affects the rate of N-nitrosodiethylamine-induced hepatocarcinogenesis in mice.

UNLABELLED: We report that HMGN1, a nucleosome-binding protein that affects chromatin structure and function, affects the growth of N-nitrosodiethylamine (DEN)-induced liver tumors. Following a single DEN injection at 2 weeks of age, Hmgn1(tm1/tm1) mice, lacking the nucleosome-binding domain of HMGN1, had earlier signs of liver tumorigenesis than their Hmgn1(+/+) littermates. Detailed gene expression profiling revealed significant differences between DEN-injected and control saline-injected mice, but only minor differences between the injected Hmgn1(tm1/tm1) mice and their Hmgn1(+/+) littermates. Pathway analysis revealed that the most significant process affected by loss of HMGN1 involves the lipid/sterol metabolic pathway. Our study indicates that in mice, loss of HMGN1 leads to transcription changes that accelerate the progression of DEN-induced hepatocarcinogenesis, without affecting the type of tumors or the final total tumor burden of these mice. IMPLICATIONS: Loss of HMGN1 leads to accelerated progression of DEN-induced hepatocarcinogenesis in mice.

The nucleosome binding protein HMGN1 interacts with PCNA and facilitates its binding to chromatin.

Proliferating cell nuclear antigen (PCNA) is a ubiquitous protein that interacts with multiple partners and regulates nuclear activities, including chromatin assembly, histone modifications, replication, and DNA damage repair. The role of specific partners in regulating PCNA activities is not fully understood. Here we identify the nucleosome binding protein HMGN1 as a new PCNA-interacting protein that enhances the binding of PCNA to chromatin but not to purified DNA. Two tetrapeptides in the conservative domain of HMGN1 contain amino acids necessary for the binding of HMGN1 to PCNA. Deletion of both tetrapeptides abolishes the HMGN1-PCNA interaction. PCNA preferentially binds to the linker DNA adjacent to an HMGN-containing nucleosome. In living cells, loss of HMGN1 decreases the rate of PCNA recruitment to damaged DNA sites. Our study identifies a new factor that facilitates the interaction of PCNA with chromatin and provides insights into mechanisms whereby nucleosome binding architectural proteins affect the cellular phenotype.

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

Alarmins are endogenous mediators capable of promoting the recruitment and activation of antigen-presenting cells (APCs), including dendritic cells (DCs), that can potentially alert host defense against danger signals. However, the relevance of alarmins to the induction of adaptive immune responses remains to be demonstrated. In this study, we report the identification of HMGN1 (high-mobility group nucleosome-binding protein 1) as a novel alarmin and demonstrate that it contributes to the induction of antigen-specific immune responses. HMGN1 induced DC maturation via TLR4 (Toll-like receptor 4), recruitment of APCs at sites of injection, and activation of NF-κB and multiple mitogen-activated protein kinases in DCs. HMGN1 promoted antigen-specific immune response upon co-administration with antigens, and Hmgn1(-/-) mice developed greatly reduced antigen-specific antibody and T cell responses when immunized with antigens in the presence of lipopolysaccharide (LPS). The impaired ability of Hmgn1(-/-) mice to mount antigen-specific immune responses was accompanied by both deficient DC recruitment at sites of immunization and reduced production of inflammatory cytokines. Bone marrow chimera experiments revealed that HMGN1 derived from nonleukocytes was critical for the induction of antigen-specific antibody and T cell responses. Thus, extracellular HMGN1 acts as a novel alarmin critical for LPS-induced development of innate and adaptive immune responses.

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 phosphorylation of serine 1 in histone H2A.

Here we demonstrate that HMGN1, a nuclear protein that binds specifically to nucleosomes, modulates the level of histone H2A phosphorylation. In Hmgn1-/- cells, loss of HMGN1 elevates the steady-state levels of H2AS1ph throughout the cell cycle. In vitro, HMGN1 reduces the rate of Rsk2- and Msk1-mediated phosphorylation of nucleosomal, but not free, histone H2A. HMGN1 inhibits H2A phosphorylation by binding to nucleosomes since an HMGN mutant, which cannot bind to chromatin, does not inhibit the Rsk2- mediated H2A phosphorylation. HMGN2 also inhibits H2A phosphorylation, suggesting that the inhibition of H2A phosphorylation is not specific to only one member of this protein family. Thus, the present data add modifications of histone H2A to the list of histone modifications affected by HMGN proteins. It supports the suggestion that structural chromatin binding proteins can modify the whole profile of post-translational modifications of core histones.

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.

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.

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.

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.

Chromosomal protein HMGN1 modulates histone H3 phosphorylation.

Here we demonstrate that HMGN1, a nuclear protein that binds to nucleosomes and reduces the compaction of the chromatin fiber, modulates histone posttranslational modifications. In Hmgn1-/- cells, loss of HMGN1 elevates the steady-state levels of phospho-S10-H3 and enhances the rate of stress-induced phosphorylation of S10-H3. In vitro, HMGN1 reduces the rate of phospho-S10-H3 by hindering the ability of kinases to modify nucleosomal, but not free, H3. During anisomycin treatment, the phosphorylation of HMGN1 precedes that of H3 and leads to a transient weakening of the binding of HMGN1 to chromatin. We propose that the reduced binding of HMGN1 to nucleosomes, or the absence of the protein, improves access of anisomysin-induced kinases to H3. Thus, the levels of posttranslational modifications in chromatin are modulated by nucleosome binding proteins that alter the ability of enzymatic complexes to access and modify their nucleosomal targets.

HMGN dynamics and chromatin function.

Recent studies indicate that most nuclear proteins, including histone H1 and HMG are highly mobile and their interaction with chromatin is transient. These findings suggest that the structure of chromatin is dynamic and the protein composition at any particular chromatin site is not fixed. Here we discuss how the dynamic behavior of the nucleosome binding HMGN proteins affects the structure and function of chromatin. The high intranuclear mobility of HMGN insures adequate supply of protein throughout the nucleus and serves to target these proteins to their binding sites. Transient interactions of the proteins with nucleosomes destabilize the higher order chromatin, enhance the access to nucleosomal DNA, and impart flexibility to the chromatin fiber. While roaming the nucleus, the HMGN proteins encounter binding partners and form metastable multiprotein complexes, which modulate their chromatin interactions. Studies with HMGN proteins underscore the important role of protein dynamics in chromatin function.

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.

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 approximately 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.