Chromatin plays a key role in regulating gene expression programs necessary for the orderly progress of development and for preventing changes in cell identity that can lead to disease. The high mobility group N (HMGN) is a family of nucleosome binding proteins that preferentially binds to chromatin regulatory sites including enhancers and promoters. HMGN proteins are ubiquitously expressed in all vertebrate cells potentially affecting chromatin function and epigenetic regulation in multiple cell types. Here, we review studies aimed at elucidating the biological function of HMGN proteins, focusing on their possible role in vertebrate development and the etiology of disease. The data indicate that changes in HMGN levels lead to cell type-specific phenotypes, suggesting that HMGN optimize epigenetic processes necessary for maintaining cell identity and for proper execution of specific cellular functions. This manuscript contains tables that can be used as a comprehensive resource for all the English written manuscripts describing research aimed at elucidating the biological function of the HMGN protein family.

The dynamic nature of the chromatin epigenetic landscape plays a key role in the establishment and maintenance of cell identity, yet the factors that affect the dynamics of the epigenome are not fully known. Here we find that the ubiquitous nucleosome binding proteins HMGN1 and HMGN2 preferentially colocalize with epigenetic marks of active chromatin, and with cell-type specific enhancers. Loss of HMGNs enhances the rate of OSKM induced reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs), and the ASCL1 induced conversion of fibroblast into neurons. During transcription factor induced reprogramming to pluripotency, loss of HMGNs accelerates the erasure of the MEF-specific epigenetic landscape and the establishment of an iPSCs-specific chromatin landscape, without affecting the pluripotency potential and the differentiation potential of the reprogrammed cells. Thus, HMGN proteins modulate the plasticity of the chromatin epigenetic landscape thereby stabilizing, rather than determining cell identity.

The activation of naïve B lymphocyte involves rapid and major changes in chromatin organization and gene expression; however, the complete repertoire of nuclear factors affecting these genomic changes is not known. We report that HMGN proteins, which bind to nucleosomes and affect chromatin structure and function, co-localize with, and maintain the intensity of DNase I hypersensitive sites genome wide, in resting but not in activated B cells. Transcription analyses of resting and activated B cells from wild-type and Hmgn(-/-) mice, show that loss of HMGNs dampens the magnitude of the transcriptional response and alters the pattern of gene expression during the course of B-cell activation; defense response genes are most affected at the onset of activation. Our study provides insights into the biological function of the ubiquitous HMGN chromatin binding proteins and into epigenetic processes that affect the fidelity of the transcriptional response during the activation of B cell lymphocytes.

White adipose tissue browning is a key metabolic process controlled by epigenetic factors that facilitate changes in gene expression leading to altered cell identity. We find that male mice lacking the nucleosome binding proteins HMGN1 and HMGN2 (DKO mice), show decreased body weight and inguinal WAT mass, but elevated food intake, WAT browning and energy expenditure. DKO white preadipocytes show reduced chromatin accessibility and lower FRA2 and JUN binding at Pparγ and Pparα promoters. White preadipocytes and mouse embryonic fibroblasts from DKO mice show enhanced rate of differentiation into brown-like adipocytes. Differentiating DKO adipocytes show reduced H3K27ac levels at white adipocyte-specific enhancers but elevated H3K27ac levels at brown adipocyte-specific enhancers, suggesting a faster rate of change in cell identity, from white to brown-like adipocytes. Thus, HMGN proteins function as epigenetic factors that stabilize white adipocyte cell identity, thereby modulating the rate of white adipose tissue browning and affecting energy metabolism in mice.

High mobility group N (HMGN) is a family of intrinsically disordered nuclear proteins that bind to nucleosomes, alters the structure of chromatin and affects transcription. A major unresolved question is the extent of functional specificity, or redundancy, between the various members of the HMGN protein family. Here, we analyze the transcriptional profile of cells in which the expression of various HMGN proteins has been either deleted or doubled. We find that both up- and downregulation of HMGN expression altered the cellular transcription profile. Most, but not all of the changes were variant specific, suggesting limited redundancy in transcriptional regulation. Analysis of point and swap HMGN mutants revealed that the transcriptional specificity is determined by a unique combination of a functional nucleosome-binding domain and C-terminal domain. Doubling the amount of HMGN had a significantly larger effect on the transcription profile than total deletion, suggesting that the intrinsically disordered structure of HMGN proteins plays an important role in their function. The results reveal an HMGN-variant-specific effect on the fidelity of the cellular transcription profile, indicating that functionally the various HMGN subtypes are not fully redundant.

An interplay between the nucleosome binding proteins H1 and HMGN is known to affect chromatin dynamics, but the biological significance of this interplay is still not clear. We find that during embryonic stem cell differentiation loss of HMGNs leads to down regulation of genes involved in neural differentiation, and that the transcription factor OLIG2 is a central node in the affected pathway. Loss of HMGNs affects the expression of OLIG2 as well as that of OLIG1, two transcription factors that are crucial for oligodendrocyte lineage specification and nerve myelination. Loss of HMGNs increases the chromatin binding of histone H1, thereby recruiting the histone methyltransferase EZH2 and elevating H3K27me3 levels, thus conferring a repressive epigenetic signature at Olig1&2 sites. Embryonic stem cells lacking HMGNs show reduced ability to differentiate towards the oligodendrocyte lineage, and mice lacking HMGNs show reduced oligodendrocyte count and decreased spinal cord myelination, and display related neurological phenotypes. Thus, the presence of HMGN proteins is required for proper expression of neural differentiation genes during embryonic stem cell differentiation. Specifically, we demonstrate that the dynamic interplay between HMGNs and H1 in chromatin epigenetically regulates the expression of OLIG1&2, thereby affecting oligodendrocyte development and myelination, and mouse behavior.

DNase I hypersensitive sites (DHSs) are a hallmark of chromatin regions containing regulatory DNA such as enhancers and promoters; however, the factors affecting the establishment and maintenance of these sites are not fully understood. We now show that HMGN1 and HMGN2, nucleosome-binding proteins that are ubiquitously expressed in vertebrate cells, maintain the DHS landscape of mouse embryonic fibroblasts (MEFs) synergistically. Loss of one of these HMGN variants led to a compensatory increase of binding of the remaining variant. Genome-wide mapping of the DHSs in Hmgn1(-/-), Hmgn2(-/-), and Hmgn1(-/-)n2(-/-) MEFs reveals that loss of both, but not a single HMGN variant, leads to significant remodeling of the DHS landscape, especially at enhancer regions marked by H3K4me1 and H3K27ac. Loss of HMGN variants affects the induced expression of stress-responsive genes in MEFs, the transcription profiles of several mouse tissues, and leads to altered phenotypes that are not seen in mice lacking only one variant. We conclude that the compensatory binding of HMGN variants to chromatin maintains the DHS landscape, and the transcription fidelity and is necessary to retain wild-type phenotypes. Our study provides insight into mechanisms that maintain regulatory sites in chromatin and into functional compensation among nucleosome binding architectural proteins.

Nucleosomes containing acetylated H3K27 are a major epigenetic mark of active chromatin and identify cell-type specific chromatin regulatory regions which serve as binding sites for transcription factors. Here we show that the ubiquitous nucleosome binding proteins HMGN1 and HMGN2 bind preferentially to H3K27ac nucleosomes at cell-type specific chromatin regulatory regions. HMGNs bind directly to the acetylated nucleosome; the H3K27ac residue and linker DNA facilitate the preferential binding of HMGNs to the modified nucleosomes. Loss of HMGNs increases the levels of H3K27me3 and the histone H1 occupancy at enhancers and promoters and alters the interaction of transcription factors with chromatin. These experiments indicate that the H3K27ac epigenetic mark enhances the interaction of architectural protein with chromatin regulatory sites and identify determinants that facilitate the localization of HMGN proteins at regulatory sites to modulate cell-type specific gene expression.

DNA methylation and histone posttranslational modifications are epigenetics processes that contribute to neurophenotype of Down Syndrome (DS). Previous reports present strong evidence that nonhistone high-mobility-group N proteins (HMGN) are epigenetic regulators. They play important functions in various process to maintain homeostasis in the brain. We aimed to analyze the differential expression of five human HMGN genes in some brain structures and age ranks from DS postmortem brain samples. Methodology: We performed a computational analysis of the expression of human HMGN from the data of a DNA microarray experiment (GEO database ID GSE59630). Using the transformed log2 data, we analyzed the differential expression of five HMGN genes in several brain areas associated with cognition in patients with DS. Moreover, using information from different genome databases, we explored the co-expression and protein interactions of HMNGs with the histones of nucleosome core particle and linker H1 histone. Results: We registered that HMGN1 and HMGN5 were significantly overexpressed in the hippocampus and areas of prefrontal cortex including DFC, OFC, and VFC of DS patients. Age-rank comparisons between euploid control and DS individuals showed that HMGN2 and HMGN4 were overexpressed in the DS brain at 16 to 22 gestation weeks. From the BioGRID database, we registered high interaction scores of HMGN2 and HMGN4 with Hist1H1A and Hist1H3A. Conclusions: Overall, our results give strong evidence to propose that DS would be an epigenetics-based aneuploidy. Remodeling brain chromatin by HMGN1 and HMGN5 would be an essential pathway in the modification of brain homeostasis in DS.

Interactions between the nucleosome and the non-histone chromosomal proteins (HMGN1 and HMGN2) were studied by circular dichroism (CD) spectroscopy to elucidate structural changes in the nucleosome induced by HMGN binding. Unlike previous studies that used a nucleosome extracted from living cells, in this study we utilized a nucleosome reconstituted from unmodified recombinant histones synthesized in Escherichia coli and a 189-bp synthetic DNA fragment harboring a nucleosome positioning sequence. This DNA fragment consists of 5'-TATAAACGCC-3' repeats that has a high affinity to the histone octamer. A nucleosome containing a unique octamer-binding sequence at a specific location on the DNA was produced at sufficiently high yield for spectroscopic analysis. CD data have indicated that both HMGN1 and HMGN2 can increase the winding angle of the nucleosome DNA, but the extent of the structural changes induced by these proteins differs significantly. This suggests HMGN1 and HMGN2 would have different abilities to facilitate nucleosome remodeling.

Members of the HMGN protein family modulate chromatin structure and influence epigenetic modifications. HMGN1 and HMGN2 are highly expressed during early development and in the neural stem/progenitor cells of the developing and adult brain. Here, we investigate whether HMGN proteins contribute to the chromatin plasticity and epigenetic regulation that is essential for maintaining pluripotency in stem cells.

High-mobility group N (HMGN) proteins are the only non-histone proteins that specifically bind within the nucleosome between core histones and DNA. Among them, HMGN5 is one of the candidates that could participate in mouse endometrial decidualization; however, the specific role of HMGN5 remains to be clarified in human endometrial stromal cells (HESCs).

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.

Osteosarcoma is one of the most common malignant tumors in children and adolescents and is characterized by early metastasis. High-mobility group N (HMGN) domains are involved in the development of several tumors. Our previous study found that HMGN5 is highly expressed in osteosarcoma tissues and knockdown of HMGN5 inhibits migration and invasion of U-2 OS and Saos-2 cells. A hypoxic environment is commonly found in solid tumors such as osteosarcoma and is likely to be associated with tumor metastasis, so we further explored the relationship between HMGN5 and the hypoxic environment. Hypoxia-inducible factor 1A (HIF1A) is an adaptive factor in the hypoxic environment. We found that HIF1A and HMGN5 were upregulated in osteosarcoma (OS) cells cultured in the hypoxic environment, and the results of overexpression and knockdown experiments showed that HIF1A upregulated the transcription factor GATA1 and further promoted the expression of HMGN5. In addition, MMP2 and MMP9 were subsequently upregulated through the c-jun pathway, and finally, this promoted the migration and invasion of OS cells. It is suggested that HMGN5 may be an important downstream factor for HIF1A to promote osteosarcoma metastasis. It has an important clinical significance for the selection of therapeutic targets for osteosarcoma.

Tardigrades, also known as water bears, are animals that can survive extreme conditions. The tardigrade Ramazzottius varieornatus contains a unique nuclear protein termed Dsup, for damage suppressor, which can increase the resistance of human cells to DNA damage under conditions, such as ionizing radiation or hydrogen peroxide treatment, that generate hydroxyl radicals. Here we find that R. varieornatus Dsup is a nucleosome-binding protein that protects chromatin from hydroxyl radicals. Moreover, a Dsup ortholog from the tardigrade Hypsibius exemplaris similarly binds to nucleosomes and protects DNA from hydroxyl radicals. Strikingly, a conserved region in Dsup proteins exhibits sequence similarity to the nucleosome-binding domain of vertebrate HMGN proteins and is functionally important for nucleosome binding and hydroxyl radical protection. These findings suggest that Dsup promotes the survival of tardigrades under diverse conditions by a direct mechanism that involves binding to nucleosomes and protecting chromosomal DNA from hydroxyl radicals.

Nucleosome binding protein 1 (NSBP1) is identified as a new member of HMGN family and is abnormally overexpressed in a variety of tumors. However, it remains unclear whether NSBP1 is overexpressed and promotes gastric cancer. In this study we employed RNAi mediated knockdown of NSBP1 to investigate potential oncogenic role of NSBP1 in gastric cancer. In BGC823 and SGC7901 gastric cancer cell lines, we showed that NSBP1 knockdown decreased cell proliferation while increased apoptosis in vitro. Western blot analysis showed that NSBP1 knockdown decreased the levels of anti-apoptotic protein Bcl-2 while increased the levels of pro-apoptotic protein Bax. In addition, NSBP1 knockdown inhibited the growth and increased the apoptosis of SGC7901 cells xenografted in nude mice. In conclusion, this study provides the first evidence that NSBP1 enhances the proliferation while inhibits the apoptosis of gastric cancer cells, and this is related to the regulation of the expression of apoptosis related proteins by NSBP1. These data suggest that NSBP1 plays oncogenic role in gastric cancer.

Transcription-coupled repair (TCR) removes DNA lesions from the transcribed strand of active genes. Stalling of RNA polymerase II (RNAPII) at DNA lesions initiates TCR through the recruitment of the CSB and CSA proteins. The full repertoire of proteins required for human TCR - particularly in a chromatin context - remains to be determined. Studies in mice have revealed that the nucleosome-binding protein HMGN1 is required to enhance the repair of UV-induced lesions in transcribed genes. However, whether HMGN1 is required for human TCR remains unaddressed. Here, we show that knockout or knockdown of HMGN1, either alone or in combination with HMGN2, does not render human cells sensitive to UV light or Illudin S-induced transcription-blocking DNA lesions. Moreover, transcription restart after UV irradiation was not impaired in HMGN-deficient cells. In contrast, TCR-deficient cells were highly sensitive to DNA damage and failed to restart transcription. Furthermore, GFP-tagged HMGN1 was not recruited to sites of UV-induced DNA damage under conditions where GFP-CSB readily accumulated. In line with this, HMGN1 did not associate with the TCR complex, nor did TCR proteins require HMGN1 to associate with DNA damage-stalled RNAPII. Together, our findings suggest that HMGN1 and HMGN2 are not required for human TCR.

Chromatin structure plays a key role in regulating gene expression and embryonic differentiation; however, the factors that determine the organization of chromatin around regulatory sites are not fully known. Here we show that HMGN1, a nucleosome-binding protein ubiquitously expressed in vertebrate cells, preferentially binds to CpG island-containing promoters and affects the organization of nucleosomes, DNase I hypersensitivity, and the transcriptional profile of mouse embryonic stem cells and neural progenitors. Loss of HMGN1 alters the organization of an unstable nucleosome at transcription start sites, reduces the number of DNase I-hypersensitive sites genome wide, and decreases the number of nestin-positive neural progenitors in the subventricular zone (SVZ) region of mouse brain. Thus, architectural chromatin-binding proteins affect the transcription profile and chromatin structure during embryonic stem cell differentiation.

High-mobility group nucleosome-binding domain 5 (HMGN5) is the latest member of the HMGN family of proteins. Numerous studies have confirmed the carcinogenic role of HMGN5 in cancer, but its function in the regulation of chemosensitivity is largely unknown and controversial. A previous study by the authors of the present study demonstrated that HMGN5 contributes to the progression of urothelial bladder cancer (UBC) through regulating the expression of E-cadherin and vascular endothelial growth factor (VEGF)-C, which are associated with the sensitivity of tumor cells to cisplatin. Therefore, the present study aimed to elucidate the mechanisms underlying the regulation of HMGN5 and investigate the involvement of HMGN5 in cisplatin treatment. The results of the present study revealed that HMGN5 is able to positively regulate the expression of phosphorylated (p-)Akt in UBC cells. In addition, HMGN5 expression was negatively associated with the response of UBC cells to cisplatin. The findings indicated that HMGN5 may be a potential therapeutic target of cisplatin treatment, since cisplatin treatment reduced HMGN5 expression in a dose-dependent manner. It was also confirmed that the knockdown of HMGN5 decreased the viability, colony formation and invasion of 5637 cells but increased apoptosis under cisplatin treatment. The changes caused by HMGN5 knockdown in 5637 cells were able to be reversed by treatment with insulin-like growth factor-1 (IGF-1), which is a phosphoinositide 3-kinase (PI3K)/Akt signaling activator. Additionally, with the decreased expression of HMGN5, the expression of p-Akt, slug, E-cadherin and VEGF-C was subsequently inhibited. By contrast, the expression of cytochrome c, cleaved-caspase-3 and cleaved-poly ADP ribose polymerase was increased following HMGN5 knockdown. Consistently, these changes in protein expression were able to be reversed by IGF-1 treatment. In conclusion, findings from the in vitro experiments indicate that HMGN5 may a target of cisplatin treatment and that the inhibition of HMGN5 increases the chemosensitivity of UBC cells by inhibiting PI3K/Akt signaling.