The ability of regulatory factors to access their nucleosomal targets is modulated by nuclear proteins such as histone H1 and HMGN (previously named HMG‐14/‐17 family) that bind to nucleosomes and ...either stabilize or destabilize the higher‐order chromatin structure. We tested whether HMGN proteins affect the interaction of histone H1 with chromatin. Using microinjection into living cells expressing H1–GFP and photobleaching techniques, we found that wild‐type HMGN, but not HMGN point mutants that do not bind to nucleosomes, inhibits the binding of H1 to nucleosomes. HMGN proteins compete with H1 for nucleosome sites but do not displace statically bound H1 from chromatin. Our results provide evidence for in vivo competition among chromosomal proteins for binding sites on chromatin and suggest that the local structure of the chromatin fiber is modulated by a dynamic interplay between nucleosomal binding proteins.
High mobility group nucleosome-binding protein 5 (HMGN5) is a chromatin architectural protein that binds specifically to nucleosomes and reduces the compaction of the chromatin fiber. The protein is ...present in most vertebrate tissues however the physiological function of this protein is unknown. To examine the function of HMGN5 in vivo, mice lacking the nucleosome-binding domain of HMGN5 were generated and characterized. Serological analysis revealed that compared to wild-type littermates (Hmgn5(+/Y)), mice with a targeted mutation in the HMGN5 gene (Hmgn5(tm1/Y)), had elevated serum albumin, non-HDL cholesterol, triglycerides, and alanine transaminase, suggesting mild hepatic abnormalities. Metabolomics analysis of liver extracts and urine revealed clear differences in metabolites between Hmgn5(tm1/Y) and their Hmgn5(+/Y) littermates. Hmgn5(tm1/Y) mice had a significant increase in hepatic glutathione levels and decreased urinary concentrations of betaine, phenylacetylglycine, and creatine, all of which are metabolically related to the glutathione precursor glycine. Microarray and qPCR analysis revealed that expression of two genes affecting glutathione metabolism, glutathione peroxidase 6 (Gpx6) and hexokinase 1 (Hk1), was significantly decreased in Hmgn5(tm1/Y) mouse liver tissue. Analysis of chromatin structure by DNase I digestion revealed alterations in the chromatin structure of these genes in the livers of Hmgn5(tm1/Y) mice. Thus, functional loss of HMGN5 leads to changes in transcription of Gpx6 and Hk1 that alter glutathione metabolism.
We have demonstrated that levels of specific modification in histone H3 are modulated by members of the nucleosome-binding high mobility group N (HMGN) protein family in a variant-specific manner. ...HMGN1 (but not HMGN2) inhibits the phosphorylation of both H3S10 and H3S28, whereas HMGN2 enhances H3K14 acetylation more robustly than HMGN1. Two HMGN domains are necessary for modulating chromatin modifications, a non-modification-specific domain necessary for chromatin binding and a modification-specific domain localized in the C terminus of the HMGNs. Thus, chromatin-binding structural proteins such as HMGNs affect the levels of specific chromatin modifications and therefore may play a role in epigenetic regulation.
Directed cell migration is a property central to multiple basic biological processes. Here, we show that directed cell migration is associated with global changes in the chromatin fiber. Polarized ...posttranslational changes in histone H1 along with a transient decrease in H1 mobility were detected in cells facing the scratch in a wound healing assay. In parallel to the changes in H1, the levels of the heterochromatin marker histone H3 lysine 9 tri-methylation were elevated. Interestingly, reduction of the chromatin-binding affinity of H1 altered the cell migration rates. Moreover, migration-associated changes in histone H1 were observed during nuclear motility in the simple multicellular organism Neurospora crassa. Our studies suggest that dynamic reorganization of the chromatin fiber is an early event in the cellular response to migration cues.
HMGNs are nucleosome-binding proteins that alter the pattern of histone modifications and modulate the binding of linker histones to chromatin. The HMGN3 family member exists as two splice forms, ...HMGN3a which is full-length and HMGN3b which lacks the C-terminal RD (regulatory domain). In the present study, we have used the Glyt1 (glycine transporter 1) gene as a model system to investigate where HMGN proteins are bound across the locus in vivo, and to study how the two HMGN3 splice variants affect histone modifications and gene expression. We demonstrate that HMGN1, HMGN2, HMGN3a and HMGN3b are bound across the Glyt1 gene locus and surrounding regions, and are not enriched more highly at the promoter or putative enhancer. We conclude that the peaks of H3K4me3 (trimethylated Lys(4) of histone H3) and H3K9ac (acetylated Lys(9) of histone H3) at the active Glyt1a promoter do not play a major role in recruiting HMGN proteins. HMGN3a/b binding leads to increased H3K14 (Lys(14) of histone H3) acetylation and stimulates Glyt1a expression, but does not alter the levels of H3K4me3 or H3K9ac enrichment. Acetylation assays show that HMGN3a stimulates the ability of PCAF p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor to acetylate nucleosomal H3 in vitro, whereas HMGN3b does not. We propose a model where HMGN3a/b-stimulated H3K14 acetylation across the bodies of large genes such as Glyt1 can lead to more efficient transcription elongation and increased mRNA production.
The high mobility group N (HMGN) proteins are a family of nuclear proteins that binds to nucleosomes, changes the architecture of chromatin, and enhances transcription and replication from chromatin ...templates. The intracellular organization of the HMGN (previously known as HMG-14/17) proteins is dynamic and is related to both cell-cycle and transcriptional events. These proteins roam the nucleus, perhaps as part of multiprotein complexes, and their target interactions are modulated by posttranslational modifications. Functional studies on HMGN proteins provide insights into the molecular mechanisms by which structural proteins affect DNA-dependent activities in the context of chromatin.
We report that loss of HMGN1, a nucleosome-binding protein that alters the compaction of the chromatin fiber, increases the cellular sensitivity to ionizing radiation and the tumor burden of mice. ...The mortality and tumor burden of ionizing radiation-treated Hmgn1-/- mice is higher than that of their Hmgn1+/+ littermates. Hmgn1-/- fibroblasts have an altered G2-M checkpoint activation and are hypersensitive to ionizing radiation. The ionizing radiation hypersensitivity and the aberrant G2-M checkpoint activation of Hmgn1-/- fibroblasts can be reverted by transfections with plasmids expressing wild-type HMGN1, but not with plasmids expressing mutant HMGN proteins that do not bind to chromatin. Transformed Hmgn1-/- fibroblasts grow in soft agar and produce tumors in nude mice with a significantly higher efficiency than Hmgn1+/+ fibroblasts, suggesting that loss of HMGN1 protein disrupts cellular events controlling proliferation and growth. Hmgn1-/- mice have a higher incidence of multiple malignant tumors and metastases than their Hmgn1+/+ littermates. We suggest that HMGN1 optimizes the cellular response to ionizing radiation and to other tumorigenic events; therefore, loss of this protein increases the tumor burden in mice.
Chromatin architecture proteins HMGNs (High Mobility Group Nucleosome-binding) affect local and global chromatin structure and colocalize with cell-specific regulatory sites and regulate cell ...identity. The role of HMGN proteins in adipocyte function and energy metabolism is yet to be investigated. Here we report that HMGN1 and HMGN2 double knock-out (DKO) mice on chow diet exhibited decreased weight gain (10%) and total activity with increased food intake and energy expenditure compared to control mice. DKO mice on a high-fat diet also displayed increased food intake and decreased weight gain. We have observed that DKO mice possess less fat mass, which is correlated with decreased WAT size, but not BAT. Histological analysis of WAT from mice revealed smaller adipocytes and increased numbers of cells in DKO, while immunofluorescence analysis indicated more UCP1 in DKO, suggestive of increased WAT browning in DKO mice. Investigations on white preadipocytes or MEF cells differentiation by adipogenic cocktail revealed faster differentiation and elevated UCP1 in DKO cells. Time course RNA seq analysis on in-vitro differentiated adipocytes also confirmed enhanced browning in DKO cells. We further investigated the distribution of active histone marks such as H3K9 acetylation and H3K27 acetylation in adipocytes differentiated from preadipocytes. Our time-course ChIP seq analyses indicated that HMGN’s absence altered both H3K9 and H3K27 acetylation patterns in DKO cells compared to WT. Interestingly, correlation analysis of ChIP seq and RNA seq suggests that DKO preadipocytes and adipocytes lost active chromatin marks at certain white adipocyte-specific enhancers.
In summary, loss of HMGN attributes thermogenic character to WAT in DKO mice, thereby contributing to increased energy expenditure, which might protect mice from metabolic disorders—increased white adipocyte browning in DKO is partly by reducing the active histone marks at white specific enhancers, thereby altering white adipocyte identity.
Disclosure
R. Nanduri: None. T. Furusawa: None. A. Lobanov: None. O. Gavrilova: None. M. Bustin: None.
Funding
National Cancer Institute
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.
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