Metabolism-focused CRISPR screen unveils mitochondrial pyruvate carrier 1 as a critical driver for PARP inhibitor resistance in lung cancer.

Homologous recombination (HR) and poly ADP-ribosylation are partially redundant pathways for the repair of DNA damage in normal and cancer cells. In cell lines that are deficient in HR, inhibition of poly (ADP-ribose) polymerase (poly (ADP-ribose) polymerase [PARP]1/2) is a proven target with several PARP inhibitors (PARPis) currently in clinical use. Resistance to PARPi often develops, usually involving genetic alterations in DNA repair signaling cascades, but also metabolic rewiring particularly in HR-proficient cells. We surmised that alterations in metabolic pathways by cancer drugs such as Olaparib might be involved in the development of resistance to drug therapy. To test this hypothesis, we conducted a metabolism-focused clustered regularly interspaced short palindromic repeats knockout screen to identify genes that undergo alterations during the treatment of tumor cells with PARPis. Of about 3000 genes in the screen, our data revealed that mitochondrial pyruvate carrier 1 (MPC1) is an essential factor in desensitizing nonsmall cell lung cancer (NSCLC) lung cancer lines to PARP inhibition. In contrast to NSCLC lung cancer cells, triple-negative breast cancer cells do not exhibit such desensitization following MPC1 loss and reprogram the tricarboxylic acid cycle and oxidative phosphorylation pathways to overcome PARPi treatment. Our findings unveil a previously unknown synergistic response between MPC1 loss and PARP inhibition in lung cancer cells.

Epigenetic regulation of white adipose tissue plasticity and energy metabolism by nucleosome binding HMGN proteins.

White adipose tissue browning is a key metabolic process controlled by epigenetic factors that facilitate changes in gene expression leading to altered cell identity. We find that male mice lacking the nucleosome binding proteins HMGN1 and HMGN2 (DKO mice), show decreased body weight and inguinal WAT mass, but elevated food intake, WAT browning and energy expenditure. DKO white preadipocytes show reduced chromatin accessibility and lower FRA2 and JUN binding at Pparγ and Pparα promoters. White preadipocytes and mouse embryonic fibroblasts from DKO mice show enhanced rate of differentiation into brown-like adipocytes. Differentiating DKO adipocytes show reduced H3K27ac levels at white adipocyte-specific enhancers but elevated H3K27ac levels at brown adipocyte-specific enhancers, suggesting a faster rate of change in cell identity, from white to brown-like adipocytes. Thus, HMGN proteins function as epigenetic factors that stabilize white adipocyte cell identity, thereby modulating the rate of white adipose tissue browning and affecting energy metabolism in mice.

Multiple epigenetic factors co-localize with HMGN proteins in A-compartment chromatin.

BACKGROUND: Nucleosomal binding proteins, HMGN, is a family of chromatin architectural proteins that are expressed in all vertebrate nuclei. Although previous studies have discovered that HMGN proteins have important roles in gene regulation and chromatin accessibility, whether and how HMGN proteins affect higher order chromatin status remains unknown. RESULTS: We examined the roles that HMGN1 and HMGN2 proteins play in higher order chromatin structures in three different cell types. We interrogated data generated in situ, using several techniques, including Hi-C, Promoter Capture Hi-C, ChIP-seq, and ChIP-MS. Our results show that HMGN proteins occupy the A compartment in the 3D nucleus space. In particular, HMGN proteins occupy genomic regions involved in cell-type-specific long-range promoter-enhancer interactions. Interestingly, depletion of HMGN proteins in the three different cell types does not cause structural changes in higher order chromatin, i.e., in topologically associated domains (TADs) and in A/B compartment scores. Using ChIP-seq combined with mass spectrometry, we discovered protein partners that are directly associated with or neighbors of HMGNs on nucleosomes. CONCLUSIONS: We determined how HMGN chromatin architectural proteins are positioned within a 3D nucleus space, including the identification of their binding partners in mononucleosomes. Our research indicates that HMGN proteins localize to active chromatin compartments but do not have major effects on 3D higher order chromatin structure and that their binding to chromatin is not dependent on specific protein partners.

H3K27ac nucleosomes facilitate HMGN localization at regulatory sites to modulate chromatin binding of transcription factors.

Nucleosomes containing acetylated H3K27 are a major epigenetic mark of active chromatin and identify cell-type specific chromatin regulatory regions which serve as binding sites for transcription factors. Here we show that the ubiquitous nucleosome binding proteins HMGN1 and HMGN2 bind preferentially to H3K27ac nucleosomes at cell-type specific chromatin regulatory regions. HMGNs bind directly to the acetylated nucleosome; the H3K27ac residue and linker DNA facilitate the preferential binding of HMGNs to the modified nucleosomes. Loss of HMGNs increases the levels of H3K27me3 and the histone H1 occupancy at enhancers and promoters and alters the interaction of transcription factors with chromatin. These experiments indicate that the H3K27ac epigenetic mark enhances the interaction of architectural protein with chromatin regulatory sites and identify determinants that facilitate the localization of HMGN proteins at regulatory sites to modulate cell-type specific gene expression.

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.

Biological Functions of HMGN Chromosomal Proteins.

Chromatin plays a key role in regulating gene expression programs necessary for the orderly progress of development and for preventing changes in cell identity that can lead to disease. The high mobility group N (HMGN) is a family of nucleosome binding proteins that preferentially binds to chromatin regulatory sites including enhancers and promoters. HMGN proteins are ubiquitously expressed in all vertebrate cells potentially affecting chromatin function and epigenetic regulation in multiple cell types. Here, we review studies aimed at elucidating the biological function of HMGN proteins, focusing on their possible role in vertebrate development and the etiology of disease. The data indicate that changes in HMGN levels lead to cell type-specific phenotypes, suggesting that HMGN optimize epigenetic processes necessary for maintaining cell identity and for proper execution of specific cellular functions. This manuscript contains tables that can be used as a comprehensive resource for all the English written manuscripts describing research aimed at elucidating the biological function of the HMGN protein family.

Epigenetic regulation of REX1 expression and chromatin binding specificity by HMGNs.

HMGN proteins localize to chromatin regulatory sites and modulate the cell-type specific transcription profile; however, the molecular mechanism whereby these ubiquitous nucleosome binding proteins affect gene expression is not fully understood. Here, we show that HMGNs regulate the expression of Rex1, one of the most highly transcribed genes in mouse embryonic stem cells (ESCs), by recruiting the transcription factors NANOG, OCT4 and SOX2 to an ESC-specific super enhancer located in the 5' region of Rex1. HMGNs facilitate the establishment of an epigenetic landscape characteristic of active chromatin and enhancer promoter interactions, as seen by chromatin conformation capture. Loss of HMGNs alters the local epigenetic profile, increases histone H1 occupancy, decreases transcription factors binding and reduces enhancer promoter interactions, thereby downregulating, but not abolishing Rex1 expression. ChIP-seq analyses show high colocalization of HMGNs and of REX1, a zinc finger protein, at promoters and enhancers. Loss of HMGNs preferentially reduces the specific binding of REX1 to these chromatin regulatory sites. Thus, HMGNs affects both the expression and the chromatin binding specificity of REX1. We suggest that HMGNs affect cell-type specific gene expression by modulating the binding specificity of transcription factors to chromatin.

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.

Elevated HMGN4 expression potentiates thyroid tumorigenesis.

Thyroid cancer originates from genetic and epigenetic changes that alter gene expression and cellular signaling pathways. Here, we report that altered expression of the nucleosome-binding protein HMGN4 potentiates thyroid tumorigenesis. Bioinformatics analyses reveal increased HMGN4 expression in thyroid cancer. We find that upregulation of HMGN4 expression in mouse and human cells, and in the thyroid of transgenic mice, alters the cellular transcription profile, downregulates the expression of the tumor suppressors Atm, Atrx and Brca2, and elevates the levels of the DNA damage marker γH2AX. Mouse and human cells overexpressing HMGN4 show increased tumorigenicity as measured by colony formation, by tumor generation in nude mice, and by the formation of preneoplastic lesions in the thyroid of transgenic mice. Our study identifies a novel epigenetic factor that potentiates thyroid oncogenesis and raises the possibility that HMGN4 may serve as an additional diagnostic marker, or therapeutic target in certain thyroid cancers.

HMGN proteins modulate chromatin regulatory sites and gene expression during activation of naïve B cells.

The activation of naïve B lymphocyte involves rapid and major changes in chromatin organization and gene expression; however, the complete repertoire of nuclear factors affecting these genomic changes is not known. We report that HMGN proteins, which bind to nucleosomes and affect chromatin structure and function, co-localize with, and maintain the intensity of DNase I hypersensitive sites genome wide, in resting but not in activated B cells. Transcription analyses of resting and activated B cells from wild-type and Hmgn(-/-) mice, show that loss of HMGNs dampens the magnitude of the transcriptional response and alters the pattern of gene expression during the course of B-cell activation; defense response genes are most affected at the onset of activation. Our study provides insights into the biological function of the ubiquitous HMGN chromatin binding proteins and into epigenetic processes that affect the fidelity of the transcriptional response during the activation of B cell lymphocytes.

Functional compensation among HMGN variants modulates the DNase I hypersensitive sites at enhancers.

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.

Chromatin decompaction by the nucleosomal binding protein HMGN5 impairs nuclear sturdiness.

In most metazoan nuclei, heterochromatin is located at the nuclear periphery in contact with the nuclear lamina, which provides mechanical stability to the nucleus. We show that in cultured cells, chromatin decompaction by the nucleosome binding protein HMGN5 decreases the sturdiness, elasticity and rigidity of the nucleus. Mice overexpressing HMGN5, either globally or only in the heart, are normal at birth but develop hypertrophic heart with large cardiomyoctyes, deformed nuclei and disrupted lamina and die of cardiac malfunction. Chromatin decompaction is seen in cardiomyocytes of newborn mice but misshaped nuclei with disrupted lamina are seen only in adult cardiomyocytes, suggesting that loss of heterochromatin diminishes the ability of the nucleus to withstand the mechanical forces of the contracting heart. Thus, heterochromatin enhances the ability of the nuclear lamina to maintain the sturdiness and shape of the eukaryotic nucleus; a structural role for chromatin that is distinct from its genetic functions.

The roles of C-terminal residues on the thermal stability and local heme environment of cytochrome c' from the thermophilic purple sulfur bacterium Thermochromatium tepidum.

A soluble cytochrome (Cyt) c' from thermophilic purple sulfur photosynthetic bacterium Thermochromatium (Tch.) tepidum exhibits marked thermal tolerance compared with that from the closely related mesophilic counterpart Allochromatium vinosum. Here, we focused on the difference in the C-terminal region of the two Cyts c' and examined the effects of D131 and R129 mutations on the thermal stability and local heme environment of Cyt c' by differential scanning calorimetry (DSC) and resonance Raman (RR) spectroscopy. In the oxidized forms, D131K and D131G mutants exhibited denaturing temperatures significantly lower than that of the recombinant control Cyt c'. In contrast, R129K and R129A mutants denatured at nearly identical temperatures with the control Cyt c', indicating that the C-terminal D131 is an important residue maintaining the enhanced thermal stability of Tch. tepidum Cyt c'. The control Cyt c' and all of the mutants increased their thermal stability upon the reduction. Interestingly, D131K exhibited narrow DSC curves and unusual thermodynamic parameters in both redox states. The RR spectra of the control Cyt c' exhibited characteristic bands at 1,635 and 1,625 cm(-1), ascribed to intermediate spin (IS) and high spin (HS) states, respectively. The IS/HS distribution was differently affected by the D131 and R129 mutations and pH changes. Furthermore, R129 mutants suggested the lowering of their redox potentials. These results strongly indicate that the D131 and R129 residues play significant roles in maintaining the thermal stability and modulating the local heme environment of Tch. tepidum Cyt c'.

Metabolomics reveals a role for the chromatin-binding protein HMGN5 in glutathione metabolism.

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.

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

UNLABELLED: 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. IMPLICATIONS: Loss of HMGN1 leads to accelerated progression of DEN-induced hepatocarcinogenesis in mice.

High mobility group N proteins modulate the fidelity of the cellular transcriptional profile in a tissue- and variant-specific manner.

The nuclei of most vertebrate cells contain members of the high mobility group N (HMGN) protein family, which bind specifically to nucleosome core particles and affect chromatin structure and function, including transcription. Here, we study the biological role of this protein family by systematic analysis of phenotypes and tissue transcription profiles in mice lacking functional HMGN variants. Phenotypic analysis of Hmgn1(tm1/tm1), Hmgn3(tm1/tm1), and Hmgn5(tm1/tm1) mice and their wild type littermates with a battery of standardized tests uncovered variant-specific abnormalities. Gene expression analysis of four different tissues in each of the Hmgn(tm1/tm1) lines reveals very little overlap between genes affected by specific variants in different tissues. Pathway analysis reveals that loss of an HMGN variant subtly affects expression of numerous genes in specific biological processes. We conclude that within the biological framework of an entire organism, HMGNs modulate the fidelity of the cellular transcriptional profile in a tissue- and HMGN variant-specific manner.

High-mobility group nucleosome-binding protein 1 acts as an alarmin and is critical for lipopolysaccharide-induced immune responses.

Alarmins are endogenous mediators capable of promoting the recruitment and activation of antigen-presenting cells (APCs), including dendritic cells (DCs), that can potentially alert host defense against danger signals. However, the relevance of alarmins to the induction of adaptive immune responses remains to be demonstrated. In this study, we report the identification of HMGN1 (high-mobility group nucleosome-binding protein 1) as a novel alarmin and demonstrate that it contributes to the induction of antigen-specific immune responses. HMGN1 induced DC maturation via TLR4 (Toll-like receptor 4), recruitment of APCs at sites of injection, and activation of NF-κB and multiple mitogen-activated protein kinases in DCs. HMGN1 promoted antigen-specific immune response upon co-administration with antigens, and Hmgn1(-/-) mice developed greatly reduced antigen-specific antibody and T cell responses when immunized with antigens in the presence of lipopolysaccharide (LPS). The impaired ability of Hmgn1(-/-) mice to mount antigen-specific immune responses was accompanied by both deficient DC recruitment at sites of immunization and reduced production of inflammatory cytokines. Bone marrow chimera experiments revealed that HMGN1 derived from nonleukocytes was critical for the induction of antigen-specific antibody and T cell responses. Thus, extracellular HMGN1 acts as a novel alarmin critical for LPS-induced development of innate and adaptive immune responses.

Developmental function of HMGN proteins.

High mobility group N (HMGN) proteins are the only nuclear proteins known to specifically recognize the generic structure of the 147-bp nucleosome core particle. Both in vitro and in vivo experiments demonstrate that HMGN proteins are involved in epigenetic regulation by modulating chromatin structure and levels of posttranslational modifications of nucleosomal histones. Expression of HMGN proteins is developmentally regulated, and the loss or overexpression of these proteins can lead to developmental abnormalities. This review will focus on the role and on the possible molecular mechanism whereby HMGN proteins affect cellular differentiation and development.

The nucleosome binding protein HMGN3 is expressed in pancreatic alpha-cells and affects plasma glucagon levels in mice.

Glucose homeostasis requires the coordinated actions of various organs and is critically dependent on the proper functioning of the various cell types present in the pancreatic Langerhans islets. Here we report that chromatin architectural protein HMGN3 is highly expressed in all pancreatic endocrine islet cells, and that Hmgn3-/- mice which have a mild diabetic phenotype, have reduced glucagon levels in their blood. To elucidate the mechanism leading to altered glucagon secretion of Hmgn3-/- mice, we tested whether HMGN3 affect glucagon synthesis and secretion in alphaTC1-9 cells, a glucagon secreting cell line that is used to study pancreatic alpha-cell function. We find that in these cells deletion of either HMGN3 or other HMGN variants, does not significantly affect glucagon gene expression or glucagon secretion. Our studies demonstrate a link between HMGN3 and glucagon blood levels that is not directly dependent of the function of pancreatic alpha-cells.