HMGN1 loss sensitizes lung cancer cells to chemotherapy.

The high mobility group nucleosome binding (HMGN) family, constitutes a large family of non-histone protein family known to bind the acidic patch of the nucleosomes with various key cellular functions. Several studies have highlighted the pivotal roles of HMGNs in the pathogenic process of various cancer types. However, the roles of HMGN family in lung adenocarcinoma (LUAD) have not been fully elucidated. Herein, integrative analyses of multiple-omics data revealed that HMGNs frequently exhibit dysregulation in LUAD. Subsequent analysis of the clinical relevance of HMGN1 demonstrated its association with poor prognosis in LUAD and its potential as a diagnostic marker to differentiate LUAD from healthy controls. Additionally, functional enrichment analysis suggested that HMGN1 was mainly involved in DNA repair. To corroborate these findings, cellular experiments were conducted, confirming HMGN1's crucial involvement in homologous recombination repair and its potential to enhance the sensitivity of LUAD cells to standard chemotherapeutic drugs. This study proposes HMGN1 as a novel prognostic biomarker and a promising target for chemotherapy in lung adenocarcinoma.

HMGN1 down-regulation in the diabetic kidney attenuates tubular cells injury and protects against renal inflammation via suppressing MCP-1 and KIM-1 expression through TLR4.

BACKGROUND: Renal tubular injury, accompanied by damaging inflammation, has been identified to drive diabetic kidney disease (DKD) toward end-stage renal disease. However, it is unclear how damage-associated molecular patterns (DAMPs) activate innate immunity to mediate tubular epithelial cell (TEC) injury, which in turn causes with subsequent sterile inflammation in diabetic kidneys. High mobility group nucleosome-binding protein 1 (HMGN1) is a novel DAMP that contributes to generating the innate immune response. In this study, we focused on determining whether HMGN1 is involved in DKD progression. METHODS: Streptozotocin (STZ)-induced diabetic mice model was established. Then we downrergulated HMGN1 expression in kidney with or without HMGN1 administration. The renal dysfunction and morphological lesions in the kidneys were evaluated. The expressions of KIM-1, MCP-1, F4/80, CD68, and HMGN1/TLR4 signaling were examined in the renal tissue. In vitro, HK2 cells were exposed in the high glucose with or without HMGN1, and further pre-incubated with TAK242 was applied to elucidate the underlying mechanism. RESULTS: We demonstrated that HMGN1 was upregulated in the tubular epithelial cells of streptozotocin (STZ)-induced type 1 and type 2 diabetic mouse kidneys compared to controls, while being positively correlated with increased TLR4, KIM-1, and MCP-1. Down-regulation of renal HMGN1 attenuated diabetic kidney injury, decreased the TLR4, KIM-1, and MCP-1 expression levels, and reduced interstitial infiltrating macrophages. However, these phenotypes were reversed after administration of HMGN1. In HK-2 cells, HMGN1 promoted the expression of KIM-1 and MCP-1 via regulating MyD88/NF-κB pathway; inhibition of TLR4 effectively diminished the in vitro response to HMGN1. CONCLUSIONS: Our study provides novel insight into HMGN1 signaling mechanisms that contribute to tubular sterile injury and low-grade inflammation in DKD. The study findings may help to develop new HMGN1-targeted approaches as therapy for immune-mediated kidney damage rather than as an anti-infection treatments.

[High mobility group nucleosome binding protein 1 (HMGN1) induces activation of mouse BV2 microglia and upregulates their pro-inflammatory mediator expression by activating TLR4/MyD88/NF-κB p65/IKK-ß signal pathway].

Objective To explore the effects and mechanism of high-mobility group nucleosome-binding protein 1 (HMGN1) on the inflammatory response of mouse BV2 microglia. Methods BV2 cells were incubated with recombinant HMGN1 at different concentrations (0, 100, 200, 500, 1000, 2000 ng/mL) for 6 hours, and the morphological changes were observed under a microscope. The mRNA levels of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), interleukin-1ß (IL-1ß) and monocyte chemotactic protein 1 (MCP-1) were detected by real time quantitative PCR. Microglial cells were then randomly divided into a control group, model group, inhibitor group and antagonist group. The cells in the model group were treated with 500 ng/mL HMGN1, while the antagonist group was treated with 500 ng/mL TAK-242 (resatorvid), a Toll-like receptor 4 (TLR4) antagonist, in addition to HMGN1. Real time quantitative PCR and immunofluorescence were used to detect the expression of M1/M2 markers in the four groups, and Western blot analysis was used to measure the protein expression levels of inducible nitric-oxide synthase (iNOS), TLR4, myeloid differentiation factor88 (MyD88), nuclear factor κB p65 (NF-κB p65) and inhibitor of NF-κB(IκB)kinase ß(IKK-ß). Results After the treatment of HMGN1, the morphology of BV2 cells changed significantly, showing an amoeba-like appearance. The mRNA levels of TNF-α, IL-6, IL-1ß and MCP-1 increased with the HMGN1 concentration, with a statistically significant difference compared to the 0 ng/mL HMGN1 group. At the same time, the mRNA level of iNOS, a M1 phenotype marker, increased with the HMGN1 concentration, while the level of CD206, a M2 phenotype marker, decreased with HMGN1 concentration, showing a statistically significant difference compared to the 0 ng/mL HMGN1 group. Compared with the model group, the mRNA level of M1 phenotypic marker iNOS in the antagonist group was significantly lower, and the level of M2 phenotypic marker CD206 was significantly higher. The results of immunofluorescence cytochemistry also showed that the expression of M1 phenotypic marker iNOS in the antagonist group was lower. The results of Western blot suggested that the protein expression levels of iNOS, TLR4, MyD88, NF-κB p65 and IKK-ß decreased significantly in the antagonist group. Conclusion HMGN1 may induce the activation of BV2 microglial cells by upregulating pro-inflammatory mediators through activating the TLR4/MyD88/NF-κB p65/IKK-ß signaling pathway.

Chromatin binding protein HMGN1 promotes HBV cccDNA transcription and replication by regulating the phosphorylation of histone 3.

BACKGROUND AND AIMS: Direct elimination of cccDNA remains a formidable obstacle due to the persistent and stable presence of cccDNA in hepatocyte nuclei. The silencing of cccDNA transcription enduringly is one of alternative strategies in the treatment of hepatitis B. Protein binding to cccDNA plays an important role in its transcriptional regulation; thus, the identification of key factors involved in this process is of great importance. APPROACHES AND RESULTS: In the present study, high mobility group nucleosome binding domain 1 (HMGN1) was screened out based on our biotin-avidin enrichment system. First, chromatin immunoprecipitation and fluorescent in situ hybridization assays confirmed the binding of HMGN1 with cccDNA in the nucleus. Second, functional experiments in HBV-infected cells showed that the promoting effect of HMGN1 on HBV transcription and replication depended on the functional region of the nucleosomal binding domain, while transfection of the HMGN1 mutant showed no influence on HBV compared with the vector. Third, further mechanistic exploration revealed that the silencing of HMGN1 increased the level of phosphorylase CLK2 and promoted H3 phosphorylation causing the reduced accessibility of cccDNA. Moreover, silenced HMGN1 was mimicked in HBV (r) cccDNA mouse model of HBV infection in vivo. The results showed that silencing HMGN1 inhibited HBV replication in vivo. CONCLUSIONS: In summary, our study identified that a host protein can bind to cccDNA and promote its transcription, providing a candidate strategy for anti-HBV targeting to interfere with the transcriptional activity of cccDNA microchromosomes.

Epigenetic Regulation of Ameloblast Differentiation by HMGN Proteins.

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.

HMGN1 enhances CRISPR-directed dual-function A-to-G and C-to-G base editing.

C-to-G base editors have been successfully constructed recently, but limited work has been done on concurrent C-to-G and A-to-G base editing. In addition, there is also limited data on how chromatin-associated factors affect the base editing. Here, we test a series of chromatin-associated factors, and chromosomal protein HMGN1 was found to enhance the efficiency of both C-to-G and A-to-G base editing. By fusing HMGN1, GBE and ABE to Cas9, we develop a CRISPR-based dual-function A-to-G and C-to-G base editor (GGBE) which is capable of converting simultaneous A and C to G conversion with substantial editing efficiency. Accordingly, the HMGN1 role shown in this work and the resulting GGBE tool further broaden the genome manipulation capacity of CRISPR-directed base editors.

Overexpression screen of chromosome 21 genes reveals modulators of Sonic hedgehog signaling relevant to Down syndrome.

Trisomy 21 and mutations in the Sonic hedgehog (SHH) signaling pathway cause overlapping and pleiotropic phenotypes including cerebellar hypoplasia, craniofacial abnormalities, congenital heart defects and Hirschsprung disease. Trisomic cells derived from individuals with Down syndrome possess deficits in SHH signaling, suggesting that overexpression of human chromosome 21 genes may contribute to SHH-associated phenotypes by disrupting normal SHH signaling during development. However, chromosome 21 does not encode any known components of the canonical SHH pathway. Here, we sought to identify chromosome 21 genes that modulate SHH signaling by overexpressing 163 chromosome 21 cDNAs in a series of SHH-responsive mouse cell lines. We confirmed overexpression of trisomic candidate genes using RNA sequencing in the cerebella of Ts65Dn and TcMAC21 mice, model systems for Down syndrome. Our findings indicate that some human chromosome 21 genes, including DYRK1A, upregulate SHH signaling, whereas others, such as HMGN1, inhibit SHH signaling. Individual overexpression of four genes (B3GALT5, ETS2, HMGN1 and MIS18A) inhibits the SHH-dependent proliferation of primary granule cell precursors. Our study prioritizes dosage-sensitive chromosome 21 genes for future mechanistic studies. Identification of the genes that modulate SHH signaling may suggest new therapeutic avenues for ameliorating Down syndrome phenotypes.

Shaking up the silence: consequences of HMGN1 antagonizing PRC2 in the Down syndrome brain.

Intellectual disability is a well-known hallmark of Down Syndrome (DS) that results from the triplication of the critical region of human chromosome 21 (HSA21). Major studies were conducted in recent years to gain an understanding about the contribution of individual triplicated genes to DS-related brain pathology. Global transcriptomic alterations and widespread changes in the establishment of neural lineages, as well as their differentiation and functional maturity, suggest genome-wide chromatin organization alterations in trisomy. High Mobility Group Nucleosome Binding Domain 1 (HMGN1), expressed from HSA21, is a chromatin remodeling protein that facilitates chromatin decompaction and is associated with acetylated lysine 27 on histone H3 (H3K27ac), a mark correlated with active transcription. Recent studies causatively linked overexpression of HMGN1 in trisomy and the development of DS-associated B cell acute lymphoblastic leukemia (B-ALL). HMGN1 has been shown to antagonize the activity of the Polycomb Repressive Complex 2 (PRC2) and prevent the deposition of histone H3 lysine 27 trimethylation mark (H3K27me3), which is associated with transcriptional repression and gene silencing. However, the possible ramifications of the increased levels of HMGN1 through the derepression of PRC2 target genes on brain cell pathology have not gained attention. In this review, we discuss the functional significance of HMGN1 in brain development and summarize accumulating reports about the essential role of PRC2 in the development of the neural system. Mechanistic understanding of how overexpression of HMGN1 may contribute to aberrant brain cell phenotypes in DS, such as altered proliferation of neural progenitors, abnormal cortical architecture, diminished myelination, neurodegeneration, and Alzheimer's disease-related pathology in trisomy 21, will facilitate the development of DS therapeutic approaches targeting chromatin.

Spatial and Temporal Expression of High-Mobility-Group Nucleosome-Binding (HMGN) Genes in Brain Areas Associated with Cognition in Individuals with Down Syndrome.

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.

Function of chromatin modifier Hmgn1 during neural crest and craniofacial development.

The neural crest is a dynamic embryonic structure that plays a major role in the formation of the vertebrate craniofacial skeleton. Neural crest formation is regulated by a complex sequence of events directed by a network of transcription factors working in concert with chromatin modifiers. The high mobility group nucleosome binding protein 1 (Hmgn1) is a nonhistone chromatin architectural protein, associated with transcriptionally active chromatin. Here we report the expression and function of Hmgn1 during Xenopus neural crest and craniofacial development. Hmgn1 is broadly expressed at the gastrula and neurula stages, and is enriched in the head region at the tailbud stage, especially in the eyes and the pharyngeal arches. Hmgn1 knockdown affected the expression of several neural crest specifiers, including sox8, sox10, foxd3, and twist1, while other genes (sox9 and snai2) were only marginally affected. The specificity of this phenotype was confirmed by rescue, where injection of Hmgn1 mRNA was able to restore sox10 expression in morphant embryos. The reduction in neural crest gene expression at the neurula stage in Hmgn1 morphant embryos correlated with a decreased number of sox10- and twist1-positive cells in the pharyngeal arches at the tailbud stage, and hypoplastic craniofacial cartilages at the tadpole stage. These results point to a novel role for Hmgn1 in the control of gene expression essential for neural crest and craniofacial development. Future work will investigate the precise mode of action of Hmgn1 in this context.

Combining an Alarmin HMGN1 Peptide with PD-L1 Blockade Results in Robust Antitumor Effects with a Concomitant Increase of Stem-Like/Progenitor Exhausted CD8+ T Cells.

The expansion of intratumoral stem-like/progenitor exhausted CD8+ T (Tstem/Tpex) cells provides a potential approach to improve the therapeutic efficacy of immune checkpoint blockade (ICB). Thus, here we demonstrate a strategy to facilitate Tstem/Tpex cell expansion by combining an alarmin high-mobility group nucleosome binding domain 1 (HMGN1) peptide with programmed death-ligand 1 (PD-L1) blockade. The antitumor effects of HMGN1, anti-PD-L1, and their combined treatment were monitored in the B16F10, LLC, Colon26, or EO771 tumor-bearing mice. The comprehensive immunologic analyses, such as high-dimensional flow cytometry, transcriptome analysis, and single-cell RNA-sequencing (scRNA-seq), were used to investigate the cellular and molecular mechanisms of antitumor immune responses after treatments. We identified the immunostimulatory domain (EPKRR SARLS AKPPA KVEAK PKK) on HMGN1 and synthesized this domain as a therapeutic peptide (minP1). Combined treatment with minP1 and PD-L1 blockade induced durable tumor regression in tumor-bearing mice. minP1 increased the number of intratumoral mature DCs enriched in immunoregulatory molecules (mregDC) and enhanced their MHC class I antigen-presenting program. minP1 also synergized with PD-L1 blockade in augmenting intratumoral Tstem/Tpex cell number. Analysis of our scRNA-seq dataset by CellPhonDB suggested potential interactions between mregDCs and Tstem/Tpex cells in tumors. Our results indicate that HMGN1 peptide (minP1) serves as an immunoadjuvant to promote effective anti-PD-L1 immunotherapy with increased Tstem/Tpex cells in tumors.

Alarmin-painted exosomes elicit persistent antitumor immunity in large established tumors in mice.

Treating large established tumors is challenging for dendritic cell (DC)-based immunotherapy. DC activation with tumor cell-derived exosomes (TEXs) carrying multiple tumor-associated antigen can enhance tumor recognition. Adding a potent adjuvant, high mobility group nucleosome-binding protein 1 (HMGN1), boosts DCs' ability to activate T cells and improves vaccine efficiency. Here, we demonstrate that TEXs painted with the functional domain of HMGN1 (TEX-N1ND) via an exosomal anchor peptide potentiates DC immunogenicity. TEX-N1ND pulsed DCs (DCTEX-N1ND) elicit long-lasting antitumor immunity and tumor suppression in different syngeneic mouse models with large tumor burdens, most notably large, poorly immunogenic orthotopic hepatocellular carcinoma (HCC). DCTEX-N1ND show increased homing to lymphoid tissues and contribute to augmented memory T cells. Importantly, N1ND-painted serum exosomes from cancer patients also promote DC activation. Our study demonstrates the potency of TEX-N1ND to strengthen DC immunogenicity and to suppress large established tumors, and thus provides an avenue to improve DC-based immunotherapy.

Chromatin accessibility promotes hematopoietic and leukemia stem cell activity.

Chromatin organization is a highly orchestrated process that influences gene expression, in part by modulating access of regulatory factors to DNA and nucleosomes. Here, we report that the chromatin accessibility regulator HMGN1, a target of recurrent DNA copy gains in leukemia, controls myeloid differentiation. HMGN1 amplification is associated with increased accessibility, expression, and histone H3K27 acetylation of loci important for hematopoietic stem cells (HSCs) and leukemia, such as HoxA cluster genes. In vivo, HMGN1 overexpression is linked to decreased quiescence and increased HSC activity in bone marrow transplantation. HMGN1 overexpression also cooperates with the AML-ETO9a fusion oncoprotein to impair myeloid differentiation and enhance leukemia stem cell (LSC) activity. Inhibition of histone acetyltransferases CBP/p300 relieves the HMGN1-associated differentiation block. These data nominate factors that modulate chromatin accessibility as regulators of HSCs and LSCs, and suggest that targeting HMGN1 or its downstream effects on histone acetylation could be therapeutically active in AML.

Human HMGN1 and HMGN2 are not required for transcription-coupled DNA repair.

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.

Maintenance of active chromatin states by HMGN2 is required for stem cell identity in a pluripotent stem cell model.

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.

Improving CRISPR-Cas9 Genome Editing Efficiency by Fusion with Chromatin-Modulating Peptides.

Bacterial-derived CRISPR-Cas9 nucleases have become a common tool in genome engineering. However, the editing efficiency by even the best-crafted Cas9 nucleases varies considerably with different genomic sites, and efforts to explore the vast natural Cas9 diversity have often met with mixed or little success. Here, we show that modification of the widely used Streptococcus pyogenes Cas9 by fusion with chromatin-modulating peptides (CMPs), derived from high mobility group proteins HMGN1 and HMGB1, histone H1, and chromatin remodeling complexes, improves its activity by up to several fold, particularly on refractory target sites. We further show that this CMP fusion strategy (termed CRISPR-chrom) is also effective in improving the activities of smaller Cas9 nucleases from Streptococcus pasteurianus and Campylobacter jejuni, as well as four newly characterized Cas9 orthologs from Bacillus smithii, Lactobacillus rhamnosus, Mycoplasma canis, and Parasutterella excrementihominis. Our findings suggest that this CRISPR-chrom strategy can be used to improve established Cas9 nucleases and facilitate exploration of novel Cas9 orthologs for genome modification.

Combining Rapid Data Independent Acquisition and CRISPR Gene Deletion for Studying Potential Protein Functions: A Case of HMGN1.

CRISPR-Cas gene editing holds substantial promise in many biomedical disciplines and basic research. Due to the important functional implications of non-histone chromosomal protein HMG-14 (HMGN1) in regulating chromatin structure and tumor immunity, gene knockout of HMGN1 is performed by CRISPR in cancer cells and the following proteomic regulation events are studied. In particular, DIA mass spectrometry (DIA-MS) is utilized, and more than 6200 proteins (protein- FDR 1%) and more than 82 000 peptide precursors are reproducibly measured in the single MS shots of 2 h. HMGN1 protein deletion is confidently verified by DIA-MS in all of the clone- and dish- replicates following CRISPR. Statistical analysis reveals 147 proteins change their expressions significantly after HMGN1 knockout. Functional annotation and enrichment analysis indicate the deletion of HMGN1 induces histone inactivation, various stress pathways, remodeling of extracellular proteomes, cell proliferation, as well as immune regulation processes such as complement and coagulation cascade and interferon alpha/ gamma response in cancer cells. These results shed new lights on the cellular functions of HMGN1. It is suggested that DIA-MS can be reliably used as a rapid, robust, and cost-effective proteomic-screening tool to assess the outcome of the CRISPR experiments.

Combined treatment with HMGN1 and anti-CD4 depleting antibody reverses T cell exhaustion and exerts robust anti-tumor effects in mice.

BACKGROUND: Transient depletion of CD4+ T cells results in tumor suppression and survival benefit in murine models; however, the tumor progression and recurrence still occur over more long-term monitoring of mice. Thus, we explored an additional strategy to enhance endogenous immune responses by an alarmin, high mobility group nucleosome binding protein 1 (HMGN1). METHODS: The anti-tumor effects of HMGN1, anti-CD4 depleting antibody, and their combined treatment were monitored in the Colon26 or the B16F10 subcutaneous murine models. The tumor-infiltrating CD8+ T cell proliferation, differentiation, exhaustion, and its gene expression were determined by flow cytometry, transcriptome analysis, and quantitative real-time PCR. RESULTS: Our results show that a systemic administration of low doses of HMGN1 with an anti-CD4 depleting antibody (HMGN1/αCD4) promoted expansion of CD8+ T cell populations (e.g. CD137+ PD-1+ and CD44hi PD-1+), recruited CCR7+ migratory dendritic cells to the tumor, and reduced co-inhibitory molecules (e.g. PD-1, LAG-3, and TIM-3) to counteract CD8+ T cell exhaustion. CONCLUSION: The HMGN1/αCD4 treatment expanded effector CD8+ T cells and prolonged their anti-tumor activities by rescuing them from exhaustion, thus resulting in tumor regression and even rejection in long-term monitored mice.

Binding of HMGN proteins to cell specific enhancers stabilizes cell identity.

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.

Trisomy of a Down Syndrome Critical Region Globally Amplifies Transcription via HMGN1 Overexpression.

Down syndrome (DS, trisomy 21) is associated with developmental abnormalities and increased leukemia risk. To reconcile chromatin alterations with transcriptome changes, we performed paired exogenous spike-in normalized RNA and chromatin immunoprecipitation sequencing in DS models. Absolute normalization unmasks global amplification of gene expression associated with trisomy 21. Overexpression of the nucleosome binding protein HMGN1 (encoded on chr21q22) recapitulates transcriptional changes seen with triplication of a Down syndrome critical region on distal chromosome 21, and HMGN1 is necessary for B cell phenotypes in DS models. Absolute exogenous-normalized chromatin immunoprecipitation sequencing (ChIP-Rx) also reveals a global increase in histone H3K27 acetylation caused by HMGN1. Transcriptional amplification downstream of HMGN1 is enriched for stage-specific programs of B cells and B cell acute lymphoblastic leukemia, dependent on the developmental cellular context. These data offer a mechanistic explanation for DS transcriptional patterns and suggest that further study of HMGN1 and RNA amplification in diverse DS phenotypes is warranted.