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Dental enamel formation is coordinated by ameloblast differentiation, production of enamel matrix proteins, and crystal growth. The factors regulating ameloblast differentiation are not fully understood. Here we show that the high mobility group N (HMGN) nucleosomal binding proteins modulate the rate of ameloblast differentiation and enamel formation. We found that HMGN1 and HMGN2 proteins are downregulated during mouse ameloblast differentiation. Genetically altered mice lacking HMGN1 and HMGN2 proteins show faster ameloblast differentiation and a higher rate of enamel deposition in mice molars and incisors. In vitro differentiation of induced pluripotent stem cells to dental epithelium cells showed that HMGN proteins modulate the expression and chromatin accessibility of ameloblast-specific genes and affect the binding of transcription factors epiprofin and PITX2 to ameloblast-specific genes. Our results suggest that HMGN proteins regulate ameloblast differentiation and enamel mineralization by modulating lineage-specific chromatin accessibility and transcription factor binding to ameloblast regulatory sites., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The chromatin-associated high mobility group protein N2 (HMGN2) cofactor regulates transcription factor activity through both chromatin and protein interactions. Hmgn2 expression is known to be developmentally regulated, but the post-transcriptional mechanisms that regulate Hmgn2 expression and its precise roles in tooth development remain unclear. Here, we demonstrate that HMGN2 inhibits the activity of multiple transcription factors as a general mechanism to regulate early development. Bimolecular fluorescence complementation, pull-down, and coimmunoprecipitation assays show that HMGN2 interacts with the transcription factor Lef-1 through its HMG-box domain as well as with other early development transcription factors, Dlx2, FoxJ1, and Pitx2. Furthermore, EMSAs demonstrate that HMGN2 binding to Lef-1 inhibits its DNA-binding activity. We found that Pitx2 and Hmgn2 associate with H4K5ac and H3K4me2 chromatin marks in the proximal Dlx2 promoter, demonstrating Hmgn2 association with open chromatin. In addition, we demonstrate that microRNAs (miRs) mir-23a and miR-23b directly target Hmgn2, promoting transcriptional activation at several gene promoters, including the amelogenin promoter. In vivo, we found that decreased Hmgn2 expression correlates with increased miR-23 expression in craniofacial tissues as the murine embryo develops. Finally, we show that ablation of Hmgn2 in mice results in increased amelogenin expression because of increased Pitx2, Dlx2, Lef-1, and FoxJ1 transcriptional activity. Taken together, our results demonstrate both post-transcriptional regulation of Hmgn2 by miR-23a/b and post-translational regulation of gene expression by Hmgn2-transcription factor interactions. We conclude that HMGN2 regulates tooth development through its interaction with multiple transcription factors., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

High Mobility Group Chromosomal Protein N2 (HMGN2) can recognize tumor cells and enhance the anti-tumor effect of immune cells. This study aimed to establish a lentiviral vector of recombinant HMGN2 gene, establish recombinant T cells (HMGN2-T cells), and observe their anti-tumor effects. Total RNA was isolated from peripheral blood mononuclear cells. HMGN2, cluster of differentiation (CD) 8 A, CD28, CD137, and CD3ζ genes were amplified and connected. Jurkat cells were transfected with the recombinant lentivirus vector. The viability, apoptosis, and cell cycle of HMGN2-T cells were detected using Cell Counting Kit-8 assay and flow cytometry. The co-culture was performed by adding HMGN2-T cells to tumor cells with different effect-to-target (E:T) ratios. The cytotoxic activity was measured by lactate dehydrogenase (LDH) releasing assay. The sequences of HMGN2, CD8A, CD28, CD137, and CD3ζ gene plasmids were confirmed using gene sequencing. After the lentiviral transfection for 72 h, green fluorescence cells (HMGN2-T cells) could be seen. Cell viability and apoptosis were increased in HMGN2-T cells. The cytokine levels of interleukin 2 (IL-2) and tumor necrosis factor α (TNF-α) increased in cell supernatants of HMGN2-T cells. The percentage of G0/G1 phase cells was lower, the rate of S phase cells was higher in HMGN2-T cells than control cells. The co-culture of HMGN2-T cells and tumor cells could promote the cytokines' release. The LDH level was increased with the elevation of E:T ratios. In conclusion, the HMGN2-T cells were well-established and have the effect of secreting cytokines and killing tumor cells., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

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.

The hormone prolactin has been implicated in breast cancer pathogenesis and regulates chromatin engagement by the transcription factor, STAT5A. STAT5A is known to inducibly bind promoters and cis-regulatory elements genome-wide, though the mechanisms by which it exerts specificity and regulation of target gene expression remain enigmatic. We previously identified HDAC6 and HMGN2 as cofactors that facilitate prolactin-induced, STAT5A-mediated gene expression. Here, multicondition STAT5A, HDAC6, and HMGN2 chromatin immunoprecipitation and sequencing with parallel condition RNA-seq are utilized to reveal the cis-regulatory landscape and cofactor dynamics underlying prolactin-stimulated gene expression in breast cancer. We find that prolactin-regulated genes are significantly enriched for cis-regulatory elements bound by HDAC6 and HMGN2, and that inducible STAT5A binding at enhancers, rather than promoters, conveys specificity for prolactin-regulated genes. The selective HDAC6 inhibitor, ACY-241, blocks prolactin-induced STAT5A chromatin engagement at cis-regulatory elements as well as a significant proportion of prolactin-stimulated gene expression. We identify functional pathways known to contribute to the development and/or progression of breast cancer that are activated by prolactin and inhibited by ACY-241. Additionally, we find that the DNA sequences underlying shared STAT5A and HDAC6 binding sites at enhancers are differentially enriched for estrogen response elements (ESR1 and ESR2 motifs) relative to enhancers bound by STAT5A alone. Gene set enrichment analysis identifies significant overlap of ERα-regulated genes with genes regulated by prolactin, particularly prolactin-regulated genes with promoters or enhancers co-occupied by both STAT5A and HDAC6. Lastly, the therapeutic efficacy of ACY-241 is demonstrated in in vitro and in vivo breast cancer models, where we identify synergistic ACY-241 drug combinations and observe differential sensitivity of ER+ models relative to ER- models., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aim: The objective of this work was to investigate the prognostic role of the HMGN family in acute myeloid leukemia (AML). Methods: A total of 155 AML patients with HMGN1-5 expression data from the Cancer Genome Atlas database were enrolled in this study. Results: In the chemotherapy-only group, patients with high HMGN2 expression had significantly longer event-free survival (EFS) and overall survival (OS) than those with low expression (all p < 0.05), whereas high HMGN5 expressers had shorter EFS and OS than the low expressers (all p < 0.05). Multivariate analysis identified that high HMGN2 expression was an independent favorable prognostic factor for patients who only received chemotherapy (all p < 0.05). HMGN  family expression had no impact on EFS and OS in AML patients receiving allogeneic hematopoietic stem cell transplantation. Conclusion: High HMGN2/5 expression is a potential prognostic indicator for AML.

It has been reported that high mobility group nucleosomal binding domain 2 (HMGN2) is a nucleus-related protein that regulates gene transcription and plays a critical role in bacterial clearance. An elevated level of HMGN2 reduced integrin α5/β1 expression of human pulmonary epithelial A549 cells was demonstrated during Klebsiella pneumoniae infection, thus weakening bacterial adhesion and invasion. However, the mechanism by which HMGN2 regulates integrin expression remains unclear. This study found that a transcription factor-nuclear factor I (NFI), which serves as the potential target of HMGN2 regulated integrin expression. The results showed that HMGN2 was able to promote NFIA and NFIB expression by increasing H3K27 acetylation of NFIA/B promoter regions. The integrin α5/β1 expression was significantly enhanced by knockdown of NFIA/B via a siRNA approach. Meanwhile, NFIA/B silence could also compromise the inhibition effect of HMGN2 on the integrin α5/β1 expression. Mechanistically, it was demonstrated that HMGN2 facilitated the recruitment of NFI on the promoter regions of integrin α5/β1 according to the chromatin immunoprecipitation assay. In addition, it was further demonstrated that the knockdown of NFIA/B induced more adhesion of Klebsiella pneumoniae on pulmonary epithelial A549 cells, which could be reversed by the application of an integrin inhibitor RGD. The results revealed a regulatory role of HMGN2 on the transcription level of integrin α5/β1, indicating a potential treatment strategy against Klebsiella pneumoniae-induced infectious lung diseases., (© 2020 The Societies and John Wiley & Sons Australia, Ltd.)

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.

The surface area of the human cerebral cortex undergoes dramatic expansion during late fetal development, leading to cortical folding, an evolutionary feature not present in rodents. Microcephaly is a neurodevelopmental disorder defined by an abnormally small brain, and many gene mutations have been found to be associated with primary microcephaly. However, mouse models generated by ablating primary microcephaly-associated genes often fail to recapitulate the severe loss of cortical surface area observed in individuals with this pathology. Here, we show that a mouse model with deficient expression of high-mobility group nucleosomal binding domain 2 (HMGN2) manifests microcephaly with reduced cortical surface area and almost normal radial corticogenesis, with a pattern of incomplete penetrance. We revealed that altered cleavage plane and mitotic delay of ventricular radial glia may explain the rising ratio of intermediate progenitor cells to radial glia and the displacement of neural progenitor cells in microcephalic mutant mice. These led to decreased self-renewal of the radial glia and reduction in lateral expansion. Furthermore, we found that HMGN2 protected corticogenesis by maintaining global chromatin accessibility mainly at promoter regions, thereby ensuring the correct regulation of the transcriptome. Our findings underscore the importance of the regulation of chromatin structure in cortical development and highlight a mouse model with critical insights into the etiology of microcephaly., (© 2020 Gao et al.)

Background: 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., Results: We show that loss of Hmgn1 or Hmgn2 in pluripotent embryonal carcinoma cells leads to increased levels of spontaneous neuronal differentiation. This is accompanied by the loss of pluripotency markers Nanog and Ssea1, and increased expression of the pro-neural transcription factors Neurog1 and Ascl1. Neural stem cells derived from these Hmgn-knockout lines also show increased spontaneous neuronal differentiation and Neurog1 expression. The loss of HMGN2 leads to a global reduction in H3K9 acetylation, and disrupts the profile of H3K4me3, H3K9ac, H3K27ac and H3K122ac at the Nanog and Oct4 loci. At endodermal/mesodermal genes, Hmgn2-knockout cells show a switch from a bivalent to a repressive chromatin configuration. However, at neuronal lineage genes whose expression is increased, no epigenetic changes are observed and their bivalent states are retained following the loss of HMGN2., Conclusions: We conclude that HMGN1 and HMGN2 maintain the identity of pluripotent embryonal carcinoma cells by optimising the pluripotency transcription factor network and protecting the cells from precocious differentiation. Our evidence suggests that HMGN2 regulates active and bivalent genes by promoting an epigenetic landscape of active histone modifications at promoters and enhancers.

Non-tuberculous mycobacteria (NTM), also known as an environmental and atypical mycobacteria, can cause the chronic pulmonary infectious diseases. Macrophages have been suggested as the main host cell to initiate the innate immune responses to NTM infection. However, the molecular mechanism to regulate the antimicrobial immune responses to NTM is still largely unknown. Current study showed that the NTM clinical groups, Mycobacterium abscessus and Mycobacterium smegmatis, significantly induced the M1 macrophage polarization with the characteristic production of nitric oxide (NO) and marker gene expression of iNOS, IFNγ, TNF-α, IL1-β and IL-6. Interestingly, a non-histone nuclear protein, HMGN2 (high-mobility group N2), was found to be spontaneously induced during NTM-activated M1 macrophage polarization. Functional studies revealed that HMGN2 deficiency in NTM-infected macrophage promotes the expression of M1 markers and the production of NO via the enhanced activation of NF-κB and MAPK signalling. Further studies exhibited that HMGN2 knock-down also enhanced IFNγ-induced M1 macrophage polarization. Finally, we observed that silencing HMGN2 affected the survival of NTM in macrophage, which might largely relevant to enhanced macrophage polarization into M1 phenotype under the NTM infection. Collectively, current studies thus suggested a novel function of HMGN2 in regulating the anti-non-tuberculous mycobacteria innate immunity of macrophage., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

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 urinary tract is vulnerable to frequent challenges from environmental microflora. Uropathogenic Escherichia coli (UPEC) makes a major contribution to urinary tract infection (UTI). Previous studies have characterized positive roles of non-histone nuclear protein HMGN2 in lung epithelial innate immune response. In the study presented here, we found HMGN2 expression was up-regulated in UPEC J96-infected urothelium. Surprisingly, over-expression of HMGN2 promoted disruption of BECs 5637 cells' intercellular junctions by down-regulating tight junction (TJs) components' expression and physical structure under J96 infection. Further investigation showed that BECs 5637 monolayer, in which HMGN2 was over-expressed, had significantly increased permeability to J96. Our study systemically explored the regulatory roles of HMGN2 in BECs barrier function during UPEC infection and suggested different modulations of intracellular and paracellular routes through which UPEC invades the bladder epithelium.

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., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objective: To investigate the antibacterial mechanism of high-mobility group nucleosomal-binding domain 2 (HMGN2) on Escherichia coli K12, focusing on the antibacterial and antibiofilm formation effects. Its chemotactic activity on human neutrophils was also investigated., Methods: Human tissue-derived HMGN2 (tHMGN2) was extracted from fresh uterus fiber cystadenoma and purified by HP1100 reversed-phase high-performance liquid chromatography (RP-HPLC). Recombinant human HMGN2 (rHMGN2) was generated in E. coli DE3 carrying PET-32a-c(+)-HMGN2. Antibacterial activity of HMGN2 was determined using an agarose diffusion assay and minimum inhibitory concentration (MIC) of HMGN2 was determined by the microdilution broth method. Bacterial membrane permeability assay and DNA binding assay were performed. The antibiofilm effect of HMGN2 was investigated using a crystal violet assay and electron microscopy scanning. The activating effect and chemotactic activity of HMGN2 on neutrophils were determined using a nitroblue tetrazolium (NBT) reduction assay and Transwell chamber cell migration assay, respectively., Results: HMGN2 showed a relatively high potency against Gram-negative bacteria E. coli and the MIC of HMGN2 was 16.25 μg/ml. Elevated bacterial membrane permeability was observed in HMGN2-treated E. coli K12. HMGN2 could also bind the bacterial plasmid and genomic DNA in a dose-dependent manner. The antibiofilm effect of HMGN2 on E. coli K12 was confirmed by crystal violet staining and scanning electron microscopy. However, the activating effects and chemotactic effects of HMGN2 on human neutrophils were not observed., Conclusions: As an antimicrobial peptide (AMP), HMGN2 possessed a good capacity for antibacterial and antibiofilm activities on E. coli K12. This capacity might be associated with disruption of the bacterial membrane and combination of DNA, which might affect the growth and viability of E. coli.

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., (Published by Oxford University Press on behalf of Nucleic Acids Research 2016.)

Background/aims: Hmgn2 is involved in regulating embryonic development, but its physiological function during embryo implantation and decidualization remains unknown., Methods: In situ hybridization, real-time PCR, RNA interference, gene overexpression and MTS assay were used to examine the expression of Hmgn2 in mouse uterus during the pre-implantation period and explore its function and regulatory mechanisms in epithelial adhesion junction and stromal cell proliferation and differentiation., Results: Hmgn2 was primarily accumulated in uterine luminal epithelia on day 4 of pregnancy and subluminal stromal cells around the implanting blastocyst at implantation sites on day 5. Similar results were observed during delayed implantation and activation. Meanwhile, Hmgn2 expression was visualized in the decidua. In uterine epithelial cells, silencing of Hmgn2 by specific siRNA reduced the expression of adhesion molecules Cdh1, Cdh2 and Ctnnb1 and enhanced the expression of Muc1, whereas constitutive activation of Hmgn2 exhibited the opposite effects, suggesting a role for Hmgn2 in attachment reaction during embryo implantation. Estrogen stimulated the expression of Hmgn2 in uterine epithelia, but the stimulation was abrogated by ER antagonist ICI 182,780. Further analysis evidenced that attenuation of Hmgn2 might eliminate the regulation of estrogen on the expression of Cdh1, Cdh2 and Ctnnb1. In uterine stromal cells, progesterone induced the accumulation of Hmgn2 which advanced the expression of Prl8a2 and Prl3c1, two well-known differentiation markers for decidualization, but did not affect the proliferation of stromal cells. Knockdown of Hmgn2 blocked the progesterone-induced differentiation of uterine stromal cells. Moreover, Hmgn2 might serve as an intermediate to mediate the regulation of progesterone on Hand2., Conclusion: Hmgn2 may play an important role during embryo implantation and decidualization., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

Integrin receptors, a large family of adhesion receptors, are involved in the attachment of Klebsiella pneumoniae to respiratory epithelial cells, and subsequently cause the internalization of K. pneumoniae by host cells. Although a number of molecules have been reported to regulate the expression and activity of integrin receptors in respiratory epithelial cells, the specific underlying molecular mechanisms remain largely unknown. High mobility group nucleosomal binding domain 2 (HMGN2), a non-histone nuclear protein, is present in eukaryotic cells as a ubiquitous nuclear protein. Our previous studies have demonstrated that HMGN2 affects chromatin function and modulates the expression of antibacterial peptide in A549 cells exposed to lipopolysaccharide, which indicates the critical role of HMGN2 in innate immune responses. In addition, our cDNA microarray analysis suggested that HMGN2 knockdown induced the enhanced expression of α5β1 integrin in A549 cells. Therefore, we hypothesized that intercellular HMGN2 may mediate the internalization of K. pneumoniae by altering the expression of α5β1 integrin. Using the A549 cell line, we demonstrated that HMGN2 knockdown induced the increased expression of α5β1 integrin on cell membranes, which resulted in a significant increase in K. pneumoniae internalization. Further results revealed that HMGN2 silencing induced the expression of talin and the activation of α5β1 integrin, which led to actin polymerization following the phosphorylation of FAK and Src. This study suggests a possible therapeutic application for bacterial internalization by targeting HMGN2 in order to treat K. pneumoniae infection.

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., (© 2015 Deng et al.; Published by Cold Spring Harbor Laboratory Press.)

High mobility group N (HMGNs) are members of the high mobility group protein family, and are involved in the development and progression of several tumors. HMGN1 and HMGN5 were previously shown to be associated with the bioactivities of osteosarcoma. However, the effects and molecular mechanisms of HMGN2 on osteosarcoma progression remain to be determined. In order to characterize the endogenous expression of HMGN2 in osteosarcoma cell lines, RT-PCR and western blot analysis were performed. Recombinant HMGN2 lentivirus was used to infect the osteosarcoma cell lines with relatively low HMGN2 expression to determine the functional relevance of HMGN2 overexpression in osteosarcoma cell growth and migration in vitro and in vivo, and to investigate the expression levels of Ki-67, PCNA, cyclin D1 and cyclin E. The results showed that osteosarcoma cell proliferation and migration were significantly reduced by HMGN2, as indicated by cell count and wound-healing assays. Cell apoptosis was markedly induced and HMGN2 increased the sensitivity to chemotherapy. When HMGN2 expression was enhanced, the expression of cyclin D1 and PCNA was downregulated in osteosarcoma cells. In addition, the tumor volumes in SaO2 and U2-OS subcutaneous nude mouse models treated with HMGN2 lentivirus were significantly decreased as compared to those of the GFP group. These results suggested that the enhanced expression of HMGN2 in osteosarcoma cells by HMGN2 lentivirus, exerts inhibitory effects on growth and migration of osteosarcoma cells.

High mobility group nucleosomal binding domain 2 (HMGN2) is a small and unique non-histone protein that has many functions in a variety of cellular processes, including regulation of chromatin structure, transcription, and DNA repair. In addition, it may have other roles in antimicrobial activity, cell homing, and regulating cytokine release. Although the biochemical properties of HMGN2 protein are regulated by acetylation and phosphorylation, it is not yet known whether HMGN2 activity can also be regulated by SUMOylation. In this study, we demonstrated for the first time that HMGN2 is modified by covalent attachment of small ubiquitin-related modifier 1 (SUMO1) by pro-inflammatory signal and identified the major SUMOylated lysine residues that localize to the HMGN2 nucleosome-binding domain at Lys-17 and Lys-35. SENP1 can deSUMOylate SUMOylated HMGN2, and PIAS1 is the E3 ligase responsible for SUMOylation of HMGN2. Finally, using SUMO1-conjugated HMGN2 purified from a basal SUMOylation system in Escherichia coli, we demonstrated that SUMOylated HMGN2 has decreased the binding affinity to nucleosome core particles in comparison to unSUMOylated HMGN2. These observations potentially provide new perspectives for understanding the functions of HMGN2 in inflammatory reaction., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Patients with Axenfeld-Rieger Syndrome (ARS) present various dental abnormalities, including hypodontia, and enamel hypoplasia. ARS is genetically associated with mutations in the PITX2 gene, which encodes one of the earliest transcription factors to initiate tooth development. Thus, Pitx2 has long been considered as an upstream regulator of the transcriptional hierarchy in early tooth development. However, because Pitx2 is also a major regulator of later stages of tooth development, especially during amelogenesis, it is unclear how mutant forms cause ARS dental anomalies. In this report, we outline the transcriptional mechanism that is defective in ARS. We demonstrate that during normal tooth development Pitx2 activates Amelogenin (Amel) expression, whose product is required for enamel formation, and that this regulation is perturbed by missense PITX2 mutations found in ARS patients. We further show that Pitx2-mediated Amel activation is controlled by chromatin-associated factor Hmgn2, and that Hmgn2 prevents Pitx2 from efficiently binding to and activating the Amel promoter. Consistent with a physiological significance to this interaction, we show that K14-Hmgn2 transgenic mice display a severe loss of Amel expression on the labial side of the lower incisors, as well as enamel hypoplasia-consistent with the human ARS phenotype. Collectively, these findings define transcriptional mechanisms involved in normal tooth development and shed light on the molecular underpinnings of the enamel defect observed in ARS patients who carry PITX2 mutations. Moreover, our findings validate the etiology of the enamel defect in a novel mouse model of ARS.

EMT (epithelial-mesenchymal transition) is crucial for cancer cells to acquire invasive phenotypes. In A549 lung adenocarcinoma cells, TGF-β elicited EMT in Smad-dependent manner and TNF-α accelerated this process, as confirmed by cell morphology, expression of EMT markers, capacity of gelatin lysis and cell invasion. TNF-α stimulated the phosphorylation of Smad2 linker region, and this effect was attenuated by inhibiting MEK or JNK pathway. Comprehensive expression analysis unraveled genes differentially regulated by TGF-β and TNF-α, such as cytokines, chemokines, growth factors and ECM (extracellular matrices), suggesting the drastic change in autocrine/paracrine signals as well as cell-to-ECM interactions. Integrated analysis of microRNA signature enabled us to identify a subset of genes, potentially regulated by microRNAs. Among them, we confirmed TGF-β-mediated induction of miR-23a in lung epithelial cell lines, target genes of which were further identified by gene expression profiling. Combined with in silico approaches, we determined HMGN2 as a downstream target of miR-23a. These findings provide a line of evidence that the effects of TGF-β and TNF-α were partially mediated by microRNAs, and shed light on the complexity of molecular events elicited by TGF-β and TNF-α.

Hmgn2 (high mobility group nucleosomal 2), a ubiquitous nucleosome-binding protein that unfolds chromatin fibres and enhances DNA replication, reportedly regulates differentiation of epithelial and mesenchymal cells. To investigate how Hmgn2 regulates HC (haemopoietic cell) differentiation, we quantified Hmgn2 expression in HCs of mouse FL (fetal liver) during erythroid differentiation. Hmgn2 expression levels were >10-fold higher in immature erythroid progenitors than in mature erythroid cells, suggesting that Hmgn2 antagonizes erythroid differentiation. To address this issue, Hmgn2 were transfected into both Friend erythroleukaemia cells and FL HCs. There was a 3.3-fold decrease in relatively mature c-Kit(+)/CD71(+) erythroid cells, a 2.9-fold increase in immature c-Kit(+)/CD71(-) erythroid cells in transfected Friend cells, a 1.1-fold decrease in relatively mature CD71(+)/Ter119(+) erythroid cells, and a 1.7-fold increase in relatively immature c-Kit(+)/CD71(+) erythroid cells in FL HCs accompanied by down-regulation of genes encoding the erythroid transcription factors, Gata1 and Klf1. Two days after Hmgn2 transfection of Friend erythroleukaemia cells, the number of S-phase cells increased, whereas the number of cells in G(1) decreased, while that of mitotic cells remained unchanged. We conclude that ectopic expression of Hmgn2 antagonizes mouse erythroid differentiation in vitro, which may be due to enhancement of DNA replication and/or blocking entry of mitosis at S-phase.

Study Aim: Immunohistochemical screening of hMLH1 and hMSH2 gene mutations in patients diagnosed with colorectal cancers, suspected of having microsatellite instability, as diagnosed between January 2002 and December 2009 in the Surgery Department of the CF Clinical Hospital Cluj-Napoca (prospective non-randomised study)., Methods: Inclusion criteria were adenocarcinoma pathology finding and also minimum one of the revised Bethesda criteria for genetic testing of microsatellite instability in colorectal cancers. 110 eligible patients were divided in 2 study groups according to the number of Bethesda criteria met (group A - 1 criteria; group B - 2 or more criteria). Both groups were statistically compared considering the clinical and pathological parameters specific to the Lynch syndrome. We performed immunohistochemical staining to determine the expression of hMLH1 and hMSH2 genes in the tumors of all the patients., Results: We found the differences in age, colorectal family history and right colon tumor site between the two groups to be statistically significant. Immunohistochemical stainings showed lack of hMLH1 gene expresion in 9 patients and of hMSH2 gene in 4 patients respectively., Conclusions: Immunohistochemical staining can identify patients who need to be genetically tested for mutations of the DNA mismatch repair genes, in order to establish the correct diagnostic of Lynch syndrome.

Since bacterial invasion into host cells is an important step in the infection process, using the agents to interfere with bacterial internalization is an attractive approach to block the infection process. In this work, we describe a new, previously unrecognized role of the human cationic host defense peptide HMGN2 during Klebsiella pneumoniae infections. Our results revealed that the internalization of K. pneumoniae strain 03183 into cultured bladder epithelial cells (T24) was significantly reduced at HMGN2 concentrations that were unable to produce any bacteriostatic or bactericidal effect. Using microarrays and follow-up studies, we demonstrated that HMGN2 affected the internalization of K. pneumoniae strain 03183 by inhibiting the attachment of bacteria, and then decreasing bacteria-induced ERK1/2 activation and actin polymerization, which might contribute to bacterial internalization into T24 cells. This disruption of bacterial internalization implied that HMGN2 could provide protection against K. pneumoniae infections.

Chromatin structure and function are regulated by numerous proteins through specific binding to nucleosomes. The structural basis of many of these interactions is unknown, as in the case of the high mobility group nucleosomal (HMGN) protein family that regulates various chromatin functions, including transcription. Here, we report the architecture of the HMGN2-nucleosome complex determined by a combination of methyl-transverse relaxation optimized nuclear magnetic resonance spectroscopy (methyl-TROSY) and mutational analysis. We found that HMGN2 binds to both the acidic patch in the H2A-H2B dimer and to nucleosomal DNA near the entry/exit point, "stapling" the histone core and the DNA. These results provide insight into how HMGNs regulate chromatin structure through interfering with the binding of linker histone H1 to the nucleosome as well as a structural basis of how phosphorylation induces dissociation of HMGNs from chromatin during mitosis. Importantly, our approach is generally applicable to the study of nucleosome-binding interactions in chromatin.

Objective: To construct a prokaryotic expression recombinant for the expression of HMGN2 and to evaluate its antiviral activity against human hepatitis virus., Methods: The extracellular region cDNA of HMGN2 was isolated and amplified by RT-PCR, and introduced to the prokaryotic expression vector pGEX-4T-1. HMGN2 protein was expressed under IPTG induction and purified by GST protein purification system, then identified by SDS-PAGE and Western blot. The cytotoxicity of fusion HMGN2 to HBV-transfected HepG2. 2.15 cell was evaluated with MTT assay. Different concentration of fusion HMGN2 was applied on the HepG2. 2.15 cell and the cell culture supernatants were harvested after 3 and 6 days treatment. The HBsAg and HBeAg in the supernatants were detected by ELISA and the HBV DNA was detected by RT-PCR., Results: In the range of tested 1-100 microg/mL of HMGN2, no cytotoxicity to HepG2. 2.15 cells was detected by MTT assay. When incubated with HMGN2 at 15 microg/mL for 72 h or 144 h, there was a significant reduction in HBeAg and HBsAg expression as well as the HBV DNA copies., Conclusion: pGEX-4T-1/HMGN2 vector was success constructed, and the recombinant HMGN2 protein could inhibit HBV expression and replication in vitro remarkably.

Human β-defensin-2 (HBD-2) is an antimicrobial peptide produced by the epithelial cells that plays an important role in innate and adaptive immunity. Here we report that high mobility group protein N2 (HMGN2), a member of the high mobility group superfamily that affects chromatin function, modulates the expression of HBD-2 in A549 cells treated by lipopolysaccharide. Mechanistically, HMGN2 prolongs the retention time and enhances the accumulation of nuclear factor κB p65 in the nucleus, and promotes the acetylation of p65 through increasing histone acetyltransferase activity and enhancing p65-Ser536 phosphorylation. Additionally, chromatin immunoprecipitation reveals that HMGN2 and p65 synergistically promote their specific binding to HBD-2 promoter, thereby affecting the downstream transcription. Taken together, these results suggest that HMGN2 acts as a positive modulator of nuclear factor κB signalling to promote lipopolysaccharide-induced β-defensin expression., (© 2011 The Authors Journal compilation © 2011 FEBS.)

The HMGN family comprises nuclear proteins that bind to nucleosomes and alter the structure of chromatin. Here, we report that DT40 chicken cells lacking either HMGN2 or HMGN1a, or lacking both HMGN1a and HMGN2, are hypersensitive to killing by UV irradiation. Loss of both HMGN1a and HMGN2 or only HMGN2 increases the extent of UV-induced G(2)-M checkpoint arrest and the rate of apoptosis. HMGN null mutant cells showed slower removal of UV-induced DNA lesions from native chromatin, but the nucleotide excision repair remained intact, as measured by host cell reactivation assays. These results identify HMGN2 as a component of the global genome repair subpathway of the nucleotide excision repair pathway, and may indicate that HMGN2 facilitates the ability of the DNA repair proteins to access and repair UV-induced DNA lesions in chromatin. Our finding that HMGNs play a role in global DNA repair expands the role of these proteins in the maintenance of genome integrity.

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.

The HMGN proteins are a group of non-histone nuclear proteins that associate with the core nucleosome and alter the structure of the chromatin fiber. We investigated the distribution of the three best characterized HMGN family members, HMGN1, HMGN2 and HMGN3 during mouse eye development. HMGN1 protein is evenly distributed in all ocular structures of 10.5 days post-coitum (dpc) mouse embryos however, by 13.5dpc, relatively less HMGN1 is detected in the newly formed lens fiber cells compared to other cell types. In the adult, HMGN1 is detected throughout the retina and lens, although in the cornea, HMGN1 protein is predominately located in the epithelium. HMGN2 is also abundant in all ocular structures of mouse embryos, however, unlike HMGN1, intense immunolabeling is maintained in the lens fiber cells at 13.5dpc. In the adult eye, HMGN2 protein is still found in all lens nuclei while in the cornea, HMGN2 protein is mostly restricted to the epithelium. In contrast, the first detection of HMGN3 in the eye is in the presumptive corneal epithelium and lens fiber cells at 13.5dpc. In the lens, HMGN3 remained lens fiber cell preferred into adulthood. In the cornea, HMGN3 is transiently upregulated in the stroma and endothelium at birth while its expression is restricted to the corneal epithelium in adulthood. In the retina, HMGN3 upregulates around 2 weeks of age and is found at relatively high levels in the inner nuclear and ganglion cell layers of the adult retina. RT-PCR analysis determined that the predominant HMGN3 splice form found in ocular tissues is HMGN3b which lacks the chromatin unfolding domain although HMGN3a mRNA is also detected. These results demonstrate that the HMGN class of chromatin proteins has a dynamic expression pattern in the developing eye.

Numerous nuclear proteins bind to chromatin by targeting unique DNA sequences or specific histone modifications. In contrast, HMGN proteins recognize the generic structure of the 147-bp nucleosome core particle. HMGNs alter the structure and activity of chromatin by binding to nucleosomes; however, the determinants of the specific interaction of HMGNs with chromatin are not known. Here we use systematic mutagenesis, quantitative fluorescence recovery after photobleaching, fluorescence imaging, and mobility shift assays to identify the determinants important for the specific binding of these proteins to both the chromatin of living cells and to purified nucleosomes. We find that several regions of the protein affect the affinity of HMGNs to chromatin; however, the conserved sequence RRSARLSA, is the sole determinant of the specific interaction of HMGNs with nucleosomes. Within this sequence, each of the 4 amino acids in the R-S-RL motif are the only residues absolutely essential for anchoring HMGN protein to nucleosomes, both in vivo and in vitro. Our studies identify a new chromatin-binding module that specifically recognizes nucleosome cores independently of DNA sequence or histone tail modifications.

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.

Aim: To examine the antimicrobial spectrum and functional structure of high mobility group nucleosomal binding domain 2 (HMGN2)., Methods: OMIGA protein structure software was used to analyze the two-dimensional structure of HMGN2. Synthetic short peptides were generated for studying the relationship between function and structure. Prokaryotic expression vectors were constructed for the holo-HMGN2 and its helical domain. Their E coli-based products were also prepared for antimicrobial testing. The antimicrobial assay included minimal effective concentration, minimal inhibitory concentration, and minimal bactericidal concentration., Results: OMIGA protein structure software analysis revealed a transmembrane alpha-helical structure (the putative antimicrobial domain) located from position 18 to 48 of the HMGN2 protein sequence. The antimicrobial assay showed that the MIC of the recombinant holo-HMGN2 against E coli ML-35p (an ampicillin-resistance strain), Pseudomonas aeruginosa ATCC 27853 and Candida albicans ATCC 10231 were 12.5, 25, and 100 mg/L, respectively. Against the same microorganisms, the MIC of the synthetic HMGN2 alpha-helical domain were 12.5, 25, and 100 mg/L, respectively, that is, the same as with the recombinant form of HMGN2. In contrast, recombinant holo-HMGN2 was inactive against Staphylococcus aureus ATCC 25923. The synthetic N-terminal and C-terminal fragments of HMGN2 had no antimicrobial activity against E coli ML-35p, P aeruginosa ATCC 27853 or C albicans ATCC 10231., Conclusion: HMGN2 showed potent antimicrobial activity against E coli ML-35p, P aeruginosa ATCC 27853 and, to some extent, against C albicans ATCC 10231, but was inactive against S aureus ATCC 25923 in these assay systems. Itos alpha-helical structure may be essential for the antimicrobial activity of HMGN2.

Total RNA was extracted from human LAK cell, and a cDNA encoding mature peptide HMG-17 and its alpha helix domain was amplified by RT-PCR. The recombinant prokaryotic expression vector pGEX-1lambdaT-HMG-17 and pGEX-1lambdaT HMG-17alpha helix was constructed. Using affinity chromatography, thrombin cleaving and AU-PAGE elution, we obtained the purified HMG-17. Analyses of MIC, MEC and MBC indicated that HMG-17 and HMG-17alpha had strong antibacterial activity. MIC of the alpha-helic domain was almost the same as that of HMG17, suggesting that the alpha-helic structure would be essential for the antibacterial activity of HMG-17.

HMG-17 proteins are nucleosomal proteins, implicated in control of chromatin structure and transcription, in a way that has not yet been fully understood. In this report, quantification of HMG-17 in porcine tissues was performed, by ELISA, using previously produced and characterized specific rabbit anti-HMG-17 antibodies. Our results showed high levels of HMG-17 compared to the DNA and H1 content of the tissues (on the contrary to previous reports), and more specifically that there were 1.7 molecules of HMG-17 per molecule of histone H1.

Identification of genes selectively expressed in tumors or individual tissues is a crucial prerequisite for molecular diagnosis and treatment of cancer by addressing molecular targets. By screening an expression database, we identified the novel gene D-PCa-2 (Dresden prostate carcinoma 2), which is highly overexpressed in normal prostate tissue and prostate carcinoma (PCa). The corresponding transcript contained an open reading frame of 453 nucleotides encoding a putative protein of 150 amino acids. A large part of exon 8 of the D-PCa-2 gene shows strong similarity to the high-mobility-group nucleosomal binding protein 2 (HMGN2) cDNA. The highly specific transcription of the D-PCa-2 gene in normal and malignant prostate tissues and in a few additional tumors was demonstrated by using multiple tissue dot blot, cancer profiling dot blot and real-time PCR analyses. Examination of 18 pairs of tumorous and nontumorous prostate tissues from PCa patients by quantitative RT-PCR revealed D-PCa-2 transcripts in all specimens. The potential usefulness of D-PCa-2 as a sensitive marker for metastatic prostate carcinoma cells in lymph nodes was demonstrated by the detection of one LNCaP cell in 1 x 10(5) normal lymph node cells using real-time RT-PCR. Examination of 22 lymph nodes from PCa patients either containing metastatic prostate cancer cells or diagnosed as cancer-free was in full concordance with histopathologic diagnoses. These results validate D-PCa-2 as a transcript with high tissue specificity and with a potential application in the diagnosis of PCa., (Copyright 2004 Wiley-Liss, Inc.)

HMG-17 is a nucleosomal protein which is an immune target of autoantibodies in systemic lupus erythematosus (SLE) and other autoimmune diseases. Autoantibody production in SLE is believed to result from autoantigen specific immune stimulation and subsequently, it is expected that antigenic determinants recognized by SLE autoantibodies and induced antibodies by immunization are quite similar. To examine this issue, rabbits were immunized with purified HMG-17. The produced antiserum showed cross reactivity on blots and in inhibition ELISA with histone H1, even after its affinity purification with immobilized HMG-17. Finally, purification of the antiserum over H1 absorbed on nitrocellulose membrane produced specific anti-HMG-17 antibodies in the supernatant and anti-HMG-17/H1 antibodies that were bound to H1. SLE sera positive for HMG-17 had also cross reactivity with H1, and following the same procedure as before we received HMG-17 specific SLE autoantibodies and anti-HMG-17/H1 autoantibodies. Using the multipin epitope mapping technology, 19 overlapping 15-mer HMG-17 peptides and six 15-peptides, corresponding to known epitopes of histone H1, were synthesized. Four major epitopes were identified on the HMG-17 molecule, reactive with induced anti-HMG-17 antibodies, and these were the same as major autoepitopes In SLE. The sequence 25-51 of HMG-17, part of its DNA-binding domain, was recognized by the anti-HMG-17/H1 antibodies that were bound to H1. These antibodies recognized also defined epitopes of H1. Our results show that SLE autoantibodies can be directed against the same or similar epitopes as do IgGs evoked during the active immunization of animals, and provide additional evidence that autosensitization with an autoantigen might be operative. The possibility that the same or similar epitopes are found on different molecules (in this study HMG-17 and H1) supports the fact that there are rules by which nature selects the most dominant immunodeterminant to a given protein, which often represents functional or structural sites in the autoantigen.

Butyrate, a short chain fatty acid (SCFA), is generated by anaerobic fermentation of undigested carbohydrates within the colon. Butyrate enhances acetylation of core histones, a process directly linked to the formation of active chromatin and gene expression. However, additional chromatin components also contribute to the formation of transcriptionally active chromatin. The high mobility group protein N2 (HMG-N2), a nonhistone protein, is involved in chromatin structure modulation. We examined the effects of butyrate on HMG-N2 expression, hyperacetylation and chromatin binding. HT29 human adenocarcinoma cells were incubated with butyrate. Levels of HMG-N2 mRNA and of total or acetylated HMG-N2 protein were analyzed. Protein dynamics were investigated with transfected cells expressing HMG-N2-EGFP fusion proteins. Treatment of HT29 cells with butyrate led to significant hyperacetylation of HMG-N2. Levels of HMG-N2 protein remained unchanged. Northern blot analysis revealed a significant reduction in HMG-N2 mRNA levels after treatment with butyrate. Analysis of HMG-N2-EGFP transfected HT29 cells demonstrated that butyrate treatment changes the binding properties of HMG-N2-EGFP to chromatin. In addition, butyrate treatment resulted in solubilization of endogenous acetylated HMG-N2 into the supernatant of permeabilized cells. We demonstrate that butyrate treatment is associated with hyperacetylation of HMG-N2 protein in HT29 cells. The modulation of this nonhistone chromatin protein resulted in altered binding properties to chromatin. This may represent an additional step in changing chromatin structure and composition with subsequent consequences for transcription and gene expression. Modulation of nonhistone chromatin proteins, like the ubiquitous HMG-N2 proteins, may be partly responsible for the wide range of butyrate-associated effects., (Copyright 2001 Wiley-Liss, Inc.)

We studied the relationship between proliferation, differentiation, and the expression of high-mobility-group protein 17 (HMG-17) during metanephric kidney development. Proliferating cells were found homogenously throughout the early kidney rudiment. The expression pattern of HMG-17 mRNA, on the other hand, was distinctly uneven: In the undifferentiated mesenchyme, the cells in the cranial "tail" part of the mesenchyme showed only a weak signal, whereas a group of cells lying close to the tip of the ureteric bud showed strong HMG-17 expression. The region distinctly positive for HMG-17 is known to contain the first cells to undergo mesenchyme-to-epithelium transition. Using the transfilter model system, strong expression of HMG-17 mRNA, followed by mesenchyme-to-epithelium transition, could be induced also in the "tail" part of the mesenchyme. The upregulation of HMG-17 in the metanephrogenic mesenchyme thus results from interaction with an inductor tissue. Throughout the renal development, the HMG-17 mRNA was also abundant in those epithelial and mesenchymal cells that were undergoing active cell differentiation, and the transcript was downregulated in mature cells. HMG-17 expression thus correlated with the processes of induction and differentiation rather than with proliferation. The present results suggest that HMG-17 could have a role in the activation of the genes regulating kidney organogenesis.