High-Mobility Group Nucleosome-Binding Protein 1 Mediates Renal Fibrosis Correlating with Macrophages Accumulation and Epithelial-to-Mesenchymal Transition in Diabetic Nephropathy Mice Model.

BACKGROUND/AIM: Renal fibrosis is essential for the progression of diabetic nephropathy (DN). Macrophages accumulate in diabetic kidneys and are involved in epithelial-to-mesenchymal transition (EMT), a vital mechanism leading to renal fibrosis. Recently, high-mobility group nucleosome-binding protein 1(HMGN1) was documented in promoting the recruitment and activation of antigen-presenting cells. In this study, we first reported its roles in renal fibrosis and the underlying mechanism associated with macrophage filtration and EMT. METHODS: Twenty C57BL/6J mice were administered streptozotocin (STZ) to induce diabetes for 6 weeks and then divided into 4 groups: normal control group; DN group; benazepril-treated group, and insulin-treated group. Blood glucose, creatinine, and albumin in urine, hematoxylin and eosin, and Sirius red staining of kidney tissues were used to assess the renal pathology. ELISA, immunochemistry, and in situ hybridization were performed to determine the expression of HMGN1, CD68, F4/80, α-smooth muscle actin, and E-cadherin. RESULTS: The renal expression levels of HMGN1, macrophage markers, and EMT makers were increased in DN group, and insulin treatment could reduce the overexpression of these indicators with a better effect than benazepril treatment. Both treatments could not obviously ameliorate urine albumin-to-creatinine ratio, collagen expression, and renal histological changes in STZ-induced diabetic mice. Correlation analysis indicated that there was a relationship among HMGN1, macrophage markers, EMT markers, and collagen expression in DN mice. CONCLUSION: HMGN1 may promote macrophages accumulation and EMT, suggesting a potential therapeutic target for preventing renal fibrosis development in DN.

Interplay between H1 and HMGN epigenetically regulates OLIG1&2 expression and oligodendrocyte differentiation.

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.

HMGN proteins act in opposition to ATP-dependent chromatin remodeling factors to restrict nucleosome mobility.

The high-mobility group N (HMGN) proteins are abundant nonhistone chromosomal proteins that bind specifically to nucleosomes at two high-affinity sites. Here we report that purified recombinant human HMGN1 (HMG14) and HMGN2 (HMG17) potently repress ATP-dependent chromatin remodeling by four different molecular motor proteins. In contrast, mutant HMGN proteins with double Ser-to-Glu mutations in their nucleosome-binding domains are unable to inhibit chromatin remodeling. The HMGN-mediated repression of chromatin remodeling is reversible and dynamic. With the ACF chromatin remodeling factor, HMGN2 does not directly inhibit the ATPase activity but rather appears to reduce the affinity of the factor to chromatin. These findings suggest that HMGN proteins serve as a counterbalance to the action of the many ATP-dependent chromatin remodeling activities in the nucleus.

A role for chromosomal protein HMGN1 in corneal maturation.

Corneal differentiation and maturation are associated with major changes in the expression levels of numerous genes, including those coding for the chromatin-binding high-mobility group (HMG) proteins. Here we report that HMGN1, a nucleosome-binding protein that alters the structure and activity of chromatin, affects the development of the corneal epithelium in mice. The corneal epithelium of Hmgn1(-/-) mice is thin, has a reduced number of cells, is poorly stratified, is depleted of suprabasal wing cells, and its most superficial cell layer blisters. In mature Hmgn1(-/-)mice, the basal cells retain the ovoid shape of immature cells, and rest directly on the basal membrane which is disorganized. Gene expression was modified in Hmgn1(-/-) corneas: glutathione-S-transferase (GST)alpha 4 and GST omega 1, epithelial layer-specific markers, were selectively reduced while E-cadherin and alpha-, beta-, and gamma-catenin, components of adherens junctions, were increased. Immunofluorescence analysis reveals a complete co-localization of HMGN1 and p 63 in small clusters of basal corneal epithelial cells of wild-type mice, and an absence of p 63 expressing cells in the central region of the Hmgn1(-/-) cornea. We suggest that interaction of HMGN1 with chromatin modulates the fidelity of gene expression and affects corneal development and maturation.

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.

Heat shock, histone H3 phosphorylation and the cell cycle.

Genome-wide and gene-specific changes in histone H3 phosphorylation during heat shock have recently been described using two well-established experimental models, the "puffing" of heat shock loci in Drosophila polytene chromosomes and the induction of hsp70 mRNA transcripts in cultured mouse cells. Despite conservation of the molecular participants and overall stress response in these two organisms, some striking differences have emerged. Here, we summarize accounts of heat shock-modulated histone phosphorylation in Drosophila and mouse cells highlighting these differences. In addition, we describe a further complexity of this response in cultured mouse cells that becomes apparent when the nucleosomal response, referring to histone H3 and HMGN1 phosphorylation, is monitored through the cell cycle. This suggests that some heat shock-induced effects in mouse cells may be indirect and arise as a secondary consequence of the effect of heat shock on the cell cycle, complicating comparisons between the fly and mouse systems.

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.