Nucleosome-binding Protein HMGN1 Research Articles

Site-specific modification and segmental isotope labelling of HMGN1 reveals long-range conformational perturbations caused by posttranslational modifications.

Interactions between histones, which package DNA in eukaryotes, and nuclear proteins such as the high mobility group nucleosome-binding protein HMGN1 are important for regulating access to DNA. HMGN1 is a highly charged and intrinsically disordered protein (IDP) that is modified at several sites by posttranslational modifications (PTMs) – acetylation, phosphorylation and ADP-ribosylation. These PTMs are thought to affect cellular localisation of HMGN1 and its ability to bind nucleosomes; however, little is known about how these PTMs regulate the structure and function of HMGN1 at a molecular level. Here, we combine the chemical biology tools of protein semi-synthesis and site-specific modification to generate a series of unique HMGN1 variants bearing precise PTMs at their N- or C-termini with segmental isotope labelling for NMR spectroscopy. With access to these precisely-defined variants, we show that PTMs in both the N- and C-termini cause changes in the chemical shifts and conformational populations in regions distant from the PTM sites; up to 50–60 residues upstream of the PTM site. The PTMs investigated had only minor effects on binding of HMGN1 to nucleosome core particles, suggesting that they have other regulatory roles. This study demonstrates the power of combining protein semi-synthesis for introduction of site-specific PTMs with segmental isotope labelling for structural biology, allowing us to understand the role of PTMs with atomic precision, from both structural and functional perspectives.

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.

Nucleosome Binding Protein HMGN1 Promotes HSPC Competitive Advantage with Decreased Quiescence and Cooperates with AML Oncogenes to Block Differentiation and Increase Clonogenicity

Acute myeloid leukemia (AML) is characterized by rapid growth and differentiation arrest of immature myeloblasts. The AML blast is defined by having “open” chromatin, thus, we hypothesized that alterations of chromatin compaction may promote AML. The nucleosome binding protein HMGN1 is known to decompact chromatin and is located on chr21q22 in one of the most recurrently amplified regions in AML. We previously presented in vitro data showing that HMGN1 overexpression delayed myeloid differentiation in association with an increase in global levels of Histone H3K27 acetylation downstream of the histone acetyltransferases (HATs) CBP and p300. The goal of this study was to define the effect of HMGN1 overexpression on hematopoiesis in vivo and to identify leukemia oncogenes that cooperate with HMGN1 in primary hematopoietic cells.We analyzed native WT and OE-HMGN1 (transgenic overexpressing HMGN1) hematopoiesis at steady state in young and older mice. OE-HMGN1 transgenic mice had a significant increase in lineage-negative cells with age (Fig A, *p=0.02). We next performed competitive bone marrow transplantation with CD45.1 (WT) vs CD45.2 (WT or OE-HMGN1) donors measuring the relative contribution of CD45.1 and 45.2 cells to hematopoiesis over time after 3 consecutive serial transplants, each at 16 weeks. OE-HMGN1 stem and progenitor cells (HSPCs) had a progressive clonal advantage during serial transplantation (Fig B, *p<0.0002). This clonal advantage was observed across multiple HSPC populations and in all mature lineages. Lin-Sca1+Kit+ (LSK) and Lin-Kit+ (LK) HSPCs were sorted 4 months after transplantation for cell cycle analysis, ex vivo differentiation assays, and RNA-sequencing. OE-HMGN1 HSPCs were less quiescent in vivo, with fewer cells in G0 (Fig C, LK p= 0.0007, LSK p=0.01), and had a relative defect in differentiation capacity in vitro compared to WT. Gene set enrichment analysis was consistent with these phenotypes, showing enrichment of senescence and cell cycle regulatory gene expression programs in WT compared to OE-HMGN1 HSPCs.HMGN1 overexpression alone did not cause leukemia in mice. To screen for synergy between HMGN1 and AML oncogenes, we immortalized myeloid progenitors from WT or OE-HMGN1 mice with a HoxB8 transgene and measured growth properties after transduction with retroviruses expressing one of several AML-associated fusion proteins (BCR-ABL, MLL-AF9, PML-RAR, AML-ETO9a, MOZ-TIF2) or mutated oncogenes (FLT3, NPM1, NRAS, GATA2) with GFP. AML-ETO9a (AE9a) and MOZ-TIF2 had the most significant cooperation with HMGN1, most notably by enhancing their ability to block myeloid differentiation in vitro. This is of interest because, like we found for HMGN1, AE9a and MT2 are known promote leukemia via chromatin effects dependent on CBP/p300 and/or acetyltransferase activity. In confirmatory experiments, combination of AE9a and HMGN1 exacerbated the delay in myeloid differentiation induced by either gene alone (p<0.05), and HMGN1 enhanced serial colony formation capacity of AE9a in semi-solid media (p<0.05).In vivo transplantation of WT or OE-HMGN1 c-Kit+ HSPCs transduced with AE9a led to development of AML ~110 days after injection in both groups. However, subpopulation analysis of AE9a-expressing leukemias showed that LSK and MPP-like populations were expanded in the context of OE-HMGN1 compared to WT (p<0.05). Remarkably, global H3K27ac levels were significantly higher in LT-HSC-like HMGN1-OE AE9a leukemia cells using single cell subpopulation analysis by intracellular flow cytometry compared to WT (Fig D p=0.008), similar to what we observed in HSPCs after competitive transplant. Ex vivo clonogenic capacity was increased in AE9a leukemias that overexpressed HMGN1 in an LTC-IC assay (Fig E *p<0.05). Limiting dilution secondary transplants are ongoing to test if these differences correlate with increased leukemia-initiating capacity per cell in vivo in leukemias that overexpress HMGN1.Our study suggests that overexpression of HMGN1 provides a clonal advantage to hematopoietic populations including long-term stem cells. HMGN1 also cooperates with specific AML oncogenes, possibly those associated with H3K27 acetylation/HAT activity, to enhance leukemic phenotypes. Further study is needed to determine if HMGN1 overexpression confers specific therapeutic vulnerabilities, such as to HAT inhibitors or other chromatin-targeting agents. [Display omitted] DisclosuresLane:Stemline Therapeutics: Research Funding; N-of-one: Consultancy.

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

SUMMARYDown 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.

The Alarmin HMGN1 contributes to antitumor immunity and is a potent immunoadjuvant.

Alarmins are endogenous mediators that are elicited rapidly in response to danger signals, enhancing innate and adaptive immune responses by promoting the recruitment and maturation of antigen-presenting cells (APC). The nucleosome-binding protein HMGN1 is a potent alarmin that binds TLR4 and induces antigen-specific Th1 immune responses, but its contributions to antitumor immunity have not been explored. We found that ovalbumin (OVA)-expressing EG7 mouse thymoma cells grew much faster in Hmgn1-deficient mice than littermate-matched controls. Tumor-bearing Hmgn1(-/-) mice generated fewer OVA-specific CD8 cells in the spleen than EG7-bearing Hmgn1(+/+) mice, suggesting that HMGN1 supported T cell-mediated antitumor immunity. In addition, EG7 tumors expressing HMGN1 grew more slowly than control EG7 tumors, suggesting greater resistance to HMGN1-expressing tumors. This resistance relied on T cell-mediated immunity because it was abolished by in vivo depletion of CD4(+) and CD8(+) T cells. Moreover, mice vaccinated with a DNA vector expressing an HMGN1-gp100 fusion protein manifested gp100-specific, Th1-polarized immune responses, acquiring resistance to challenge with mouse B16F1 melanoma. Overall, our findings show that HMGN1 contributes to antitumor immunity and it may offer an effective adjuvant to heighten responses to cancer vaccines.

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

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

The Nucleosome Binding Protein HMGN1 Interacts with PCNA and Facilitates Its Binding to Chromatin

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

Genomic Profiling of HMGN1 Reveals an Association with Chromatin at Regulatory Regions

The interaction of architectural proteins such as the linker histone H1 and high-mobility-group (HMG) proteins with nucleosomes leads to changes in chromatin structure and histone modifications and alters the cellular transcription profile. The interaction of HMG proteins with chromatin is dynamic. However, it is not clear whether the proteins are constantly and randomly redistributed among all the nucleosomes or whether they preferentially associate with, and turn over at, specific regions in chromatin. To address this question, we examined the genome-wide distribution of the nucleosome binding protein HMGN1 and compared it to that of regulatory chromatin marks. We find that HMGN1 is not randomly distributed throughout the genome. Instead, the protein preferentially localizes to DNase I hypersensitive (HS) sites, promoters, functional enhancers, and transcription factor binding sites. Our results suggest that HMGN1 is part of the cellular machinery that modulates transcriptional fidelity by generating, maintaining, or preferentially interacting with specific sites in chromatin.

Activation of ATM depends on chromatin interactions occurring before induction of DNA damage

Efficient and correct responses to double stranded breaks (DSB) in chromosomal DNA are critical for maintaining genomic stability and preventing chromosomal alterations leading to cancer1. The generation of DSB is associated with structural changes in chromatin and the activation of the protein kinase ataxia-telangiectasia mutated (ATM), a key regulator of the signaling network of the cellular response to DSB 2,3. The interrelationship between DSB-induced changes in chromatin architecture and the activation of ATM is unclear 3. Here we show that the nucleosome-binding protein HMGN1 modulates the interaction of ATM with chromatin both prior to and after DSB formation thereby optimizing its activation. Loss of HMGN1, or ablation of its ability to bind to chromatin, reduces the levels of IR-induced ATM autophosphorylation and the activation of several ATM targets. IR treatments lead to a global increase in the acetylation of Lys14 of histone H3 (H3K14) in an HMGN1 dependent manner and treatment of cells with a histone deacetylase inhibitor bypasses the HMGN1 requirement for efficient ATM activation. Thus, by regulating the levels of histone modifications, HMGN1 affects ATM activation. Our studies identify a new mediator of ATM activation and demonstrate a direct link between the steady-state intranuclear organization of ATM and the kinetics of its activation following DNA damage.

Chromosomal Protein HMGN1 Enhances the Heat Shock-induced Remodeling of Hsp70 Chromatin

The nucleosome-binding protein HMGN1 affects the structure and function of chromatin; however, its role in regulating specific gene expression in living cells is not fully understood. Here we use embryonic fibroblasts from Hmgn1(+/+) and Hmgn1(-/-) mice to examine the effect of HMGN1 on the heat shock-induced transcriptional activation of Hsp70, a well characterized gene known to undergo a rapid chromatin re-structuring during transcriptional activation. We find that loss of HMGN1 decreases the levels of Hsp70 transcripts at the early stages of heat shock. HMGN1 enhances the rate of heat shockinduced changes in the Hsp70 chromatin but does not affect the chromatin structure before induction, an indication that it does not predispose the gene to rapid activation. Heat shock elevates the levels of H3K14 acetylation in the Hsp70 chromatin of wild type cells more efficiently than in the chromatin of Hmgn1(-/-) cells, whereas treatment with histone deacetylase inhibitors abrogates the effects of HMGN1 on the heat shock response. We suggest that HMGN1 enhances the rate of heat shock-induced H3K14 acetylation in the Hsp70 promoter, thereby enhancing the rate of chromatin remodeling and the subsequent transcription during the early rounds of Hsp70 activation when the gene is still associated with histones in a nucleosomal conformation.