Expression patterns and function of chromatin protein HMGB2 during mesenchymal stem cell differentiation

The superficial zone (SZ) of articular cartilage is critical in maintaining tissue function and homeostasis and represents the site of the earliest changes in osteoarthritis (OA). The expression of chromatin protein HMGB2 is restricted to the SZ, which contains cells expressing mesenchymal stem cell (MSC) markers. Age-related loss of HMGB2 and gene deletion are associated with reduced SZ cellularity and early onset OA. This study addressed HMGB2 expression patterns in MSC and its role during differentiation. HMGB2 was detected at higher levels in human MSC as compared with human articular chondrocytes, and its expression declined during chondrogenic differentiation of MSC. Lentiviral HMGB2 transduction of MSC suppressed chondrogenesis as reflected by an inhibition of Col2a1 and Col10a1 expression. Conversely, in bone marrow MSC from Hmgb2(-/-) mice, Col10a1 was more strongly expressed than in wild-type MSC. This is consistent with in vivo results from mouse growth plates showing that Hmgb2 is expressed in proliferating and prehypertrophic zones but not in hypertrophic cartilage where Col10a1 is strongly expressed. Osteogenesis was also accelerated in Hmgb2(-/-) MSC. The expression of Runx2, which plays a major role in late stage chondrocyte differentiation, was enhanced in Hmgb2(-/-) MSC, and HMGB2 negatively regulated the stimulatory effect of Wnt/β-catenin signaling on the Runx2 proximal promoter. These results demonstrate that HMGB2 expression is inversely correlated with the differentiation status of MSC and that HMGB2 suppresses chondrogenic differentiation. The age-related loss of HMGB2 in articular cartilage may represent a mechanism responsible for the decline in adult cartilage stem cell populations.     

HMGB1, an architectural chromatin protein and extracellular signalling factor,has a spatially and temporally restricted expression pattern in mouse brain

HMGB1 is an abundant chromatin component, so far considered ubiquitous. HMGB1 also has an extracellular signalling role: when passively released by necrotic cells, it triggers inflammation; moreover, it can be actively secreted by myeloid cells, neurons and neuronal cancer cells. We show here that HMGB1 protein is undetectable in most cells in adult mouse brain, and is present in a subset of brain cells during development, with a very complex temporal, spatial and subcellular expression pattern. HMGB1 is expressed in the cortical plate of E14.5 embryos, predominantly in the nucleus, although roughly 1% of cells show a cytoplasmic localization as well. In E16 embryos, HMGB1 is nuclearly expressed in scattered cells apparently moving from the ventricular zone to the cortical plate. HMGB1 expression is strongly down-regulated at later developmental stages; in adult mice significant expression is maintained only in areas of continuing neurogenesis. Finally, HMGB1 subcellular localization changes during retinoic acid induced differentiation of P19 neuroblastoma cells.     

Role of the Acidic Tail of High Mobility Group Protein B1 (HMGB1) in Protein Stability and DNA Bending

High mobility group box (HMGB) proteins are abundant nonhistone proteins found in all eukaryotic nuclei and are capable of binding/bending DNA. The human HMGB1 is composed of two binding motifs, known as Boxes A and B, are L-shaped alpha-helix structures, followed by a random-coil acidic tail that consists of 30 Asp and Glu residues. This work aimed at evaluating the role of the acidic tail of human HMGB1 in protein stability and DNA interactions. For this purpose, we cloned, expressed and purified HMGB1 and its tailless form, HMGB1ΔC, in E. coli strain. Tryptophan fluorescence spectroscopy and circular dichroism (CD) experiments clearly showed an increase in protein stability promoted by the acidic tail under different conditions, such as the presence of the chemical denaturant guanidine hydrochloride (Gdn.HCl), high temperature and low pH. Folding intermediates found at low pH for both proteins were denatured only in the presence of chemical denaturant, thus showing a relatively high stability. The acidic tail did not alter the DNA-binding properties of the protein, although it enhanced the DNA bending capability from 76° (HMGB1ΔC) to 91° (HMGB1), as measured using the fluorescence resonance energy transfer technique. A model of DNA bending in vivo was proposed, which might help to explain the interaction of HMGB1 with DNA and other proteins, i.e., histones, and the role of that protein in chromatin remodeling.     

Stage-specific secretion of HMGB1 in cartilage regulates endochondral ossification

High mobility group box 1 protein (HMGB1) is a chromatin protein that has a dual function as a nuclear factor and as an extracellular factor. Extracellular HMGB1 released by damaged cells acts as a chemoattractant, as well as a proinflammatory cytokine, suggesting that HMGB1 is tightly connected to the process of tissue organization. However, the role of HMGB1 in bone and cartilage that undergo remodeling during embryogenesis, tissue repair, and disease is largely unknown. We show here that the stage-specific secretion of HMGB1 in cartilage regulates endochondral ossification. We analyzed the skeletal development of Hmgb1(-/-) mice during embryogenesis and found that endochondral ossification is significantly impaired due to the delay of cartilage invasion by osteoclasts, osteoblasts, and blood vessels. Immunohistochemical analysis revealed that HMGB1 protein accumulated in the cytosol of hypertrophic chondrocytes at growth plates, and its extracellular release from the chondrocytes was verified by organ culture. Furthermore, we demonstrated that the chondrocyte-secreted HMGB1 functions as a chemoattractant for osteoclasts and osteoblasts, as well as for endothelial cells, further supporting the conclusion that Hmgb1(-/-) mice are defective in cell invasion. Collectively, these findings suggest that HMGB1 released from differentiating chondrocytes acts, at least in part, as a regulator of endochondral ossification during osteogenesis.     

Interplay between human high mobility group protein 1 and replication protein A on psoralen-cross-linked DNA

Human high mobility group box (HMGB) 1 and -2 proteins are highly conserved and abundant chromosomal proteins that regulate chromatin structure and DNA metabolism. HMGB proteins bind preferentially to DNA that is bent or underwound and to DNA damaged by agents such as cisplatin, UVC radiation, and benzo[a]pyrenediol epoxide (BPDE). Binding of HMGB1 to DNA adducts is thought to inhibit nucleotide excision repair (NER), leading to cell death, but the biological roles of these proteins remain obscure. We have used psoralen-modified triplex-forming oligonucleotides (TFOs) to direct a psoralen-DNA interstrand cross-link (ICL) to a specific site to determine the effect of HMGB proteins on recognition of these lesions. Our results reveal that human HMGB1 (but not HMGB2) binds with high affinity and specificity to psoralen ICLs, and interacts with the essential NER protein, replication protein A (RPA), at these lesions. RPA, shown previously to bind tightly to these lesions, also binds in the presence of HMGB1, without displacing HMGB1. A discrete ternary complex is formed, containing HMGB1, RPA, and psoralen-damaged DNA. Thus, HMGB1 has the ability to recognize ICLs, can cooperate with RPA in doing so, and likely modulates their repair by the NER machinery. The abundance of HMGB1 suggests that it may play an important role in determining the sensitivity of cells to DNA damage under physiological, experimental, and therapeutic conditions.     

Comment on: HMGB1-dependent and -independent autophagy

HMGB1 (high mobility group box 1), a ubiquitously expressed DNA-binding nucleoprotein, has not only been attributed with important functions in the regulation of gene expression but is thought to function as an important damage-associated molecular pattern in the extracellular space. Recently, conditional Hmgb1 deletion strategies have been employed to overcome the perinatal mortality of global Hmgb1 deletion and to understand HMGB1 functions under disease conditions. From these studies, it has become evident that HMGB1 is not required for normal organ function. However, the different conditional ablation strategies have yielded contradictory results in some disease models. With nearly complete recombination in all transgenic mouse models, the main reason for opposite results is likely to lie within different targeting strategies. In summary, different targeting strategies need to be taken into account when interpreting HMGB1 functions, and further efforts need to be undertaken to compare these models side by side.We appreciate the thoughtful analysis on HMGB1-dependent and -independent autophagy by Sun and Tang.¹ However, we disagree with several statements in this review. Sun and Tang write “Mice with hepatocyte-specific deletion of Hmgb1 from Robert Schwabe''s lab are not complete conditional knockout mice; the protein level of HMGB1 in the liver is decreased by about 70%,” as well as “a major difference between Robert Schwabe''s engineered HMGB1 mice and other groups is the tissue-level expression of HMGB1 after knockout.”¹We would like to point out that livers are not solely composed of hepatocytes and that albumin-Cre mediated deletion of target genes in the liver cannot result in complete loss of hepatic mRNA or protein of target genes due to the presence of unrecombined nonparenchymal cells, unless the target gene is exclusively expressed in hepatocytes and/or cholangiocytes. The reduction of hepatic HMGB1 in our studies—reaching 90% and 72% at the mRNA and protein level, respectively—is precisely at the expected level for this conditional strategy, and similar to other studies that employed albumin-Cre for hepatocyte-specific knockout of other target genes.^2-5 Hepatocytes account only for approximately 52% of cells in the liver, with other cell types including Kupffer cells (∼18% of liver cells), hepatic stellate cells (˜8% of liver cells), endothelial cells (∼22% cells of liver cells) and cholangiocytes (<1 % of liver cells) contributing to the remainder.⁶ Accordingly, albumin-Cre-mediated reduction of mRNA and protein levels of target genes (i.e., Hmgb1 and HMGB1 in our study) in the liver cannot exceed the amount of mRNA and protein expressed by hepatocytes and cholangiocytes (which is typically about 70–90%,^2-5 due to higher mRNA and protein levels in hepatocytes than in other hepatic cell types). The high efficacy of our conditional approach is best demonstrated by almost complete loss of HMGB1 expression in the hepatocellular compartment of albumin-Cre mice—as evidenced by loss of HMGB1 expression in all HNF4α-positive cells and in isolated primary hepatocytes—whereas HMGB1 expression is retained in nonparenchymal cells, as demonstrated by costaining for Kupffer cell marker F4/80, endothelial cell marker endomucin, and hepatic stellate cell marker desmin.^7,8 The nearly perfect recombination rate in our mice was further confirmed by experiments that employed Mx1Cre for Hmgb1 deletion, which resulted in almost complete loss of hepatic Hmgb1 mRNA and HMGB1 protein.^7,8 Moreover, our transgenic mice show early postnatal mortality when bred with a germline Cre deleter,⁷ thus reproducing the phenotype of the global HMGB1 knockout.⁹In summary, our transgenic mouse model results in nearly perfect recombination efficiency with virtually complete loss of Hmgb1 mRNA and HMGB1 protein in all targeted cell types, and constitutes a valid tool for the assessment of HMGB1 functions in vivo. Findings from this model need to be taken into account for proper interpretation of the role of HMGB1 in the normal and diseased liver, and cannot be interpreted as a result of incomplete deletion efficiency. Hence, differences in targeting strategies (exons 2–4 by our approach, exons 2–3 in mice from Tang and colleagues) are likely to explain opposite findings, e.g. improvement of ischemia-reperfusion injury in our hands, but aggravation of liver damage in the study by Huang et al.^8,10 Further analysis needs to be performed to determine whether ablation of exons 2–3 versus exons 2–4 leads to complete loss of HMGB1 function.     

Cooperative recruitment of HMGB1 during V(D)J recombination through interactions with RAG1 and DNA

During V(D)J recombination, recombination activating gene (RAG)1 and RAG2 bind and cleave recombination signal sequences (RSSs), aided by the ubiquitous DNA-binding/-bending proteins high-mobility group box protein (HMGB)1 or HMGB2. HMGB1/2 play a critical, although poorly understood, role in vitro in the assembly of functional RAG–RSS complexes, into which HMGB1/2 stably incorporate. The mechanism of HMGB1/2 recruitment is unknown, although an interaction with RAG1 has been suggested. Here, we report data demonstrating only a weak HMGB1–RAG1 interaction in the absence of DNA in several assays, including fluorescence anisotropy experiments using a novel Alexa488-labeled HMGB1 protein. Addition of DNA to RAG1 and HMGB1 in fluorescence anisotropy experiments, however, results in a substantial increase in complex formation, indicating a synergistic binding effect. Pulldown experiments confirmed these results, as HMGB1 was recruited to a RAG1–DNA complex in a RAG1 concentration-dependent manner and, interestingly, without strict RSS sequence specificity. Our finding that HMGB1 binds more tightly to a RAG1–DNA complex over RAG1 or DNA alone provides an explanation for the stable integration of this typically transient architectural protein in the V(D)J recombinase complex throughout recombination. These findings also have implications for the order of events during RAG–DNA complex assembly and for the stabilization of sequence-specific and non-specific RAG1–DNA interactions.     

Interaction of wheat high-mobility-group proteins with four-way-junction DNA and characterization of the structure and expression of HMGA gene

Plant high-mobility-group (HMG) chromosomal proteins are the most abundant and ubiquitous nonhistone proteins found in the nuclei of higher eukaryotes. There are only two families of HMG proteins, namely, HMGA and HMGB in plants. The cDNA encoding wheat HMGa protein was isolated and characterized. Wheat HMGA cDNA encodes a protein of 189 amino acid residues. At its N terminus, there is a histone H1-like structure, which is a common feature of plant HMGA proteins, followed by four AT-hook motifs. Polymerase chain reaction results show that the gene contains a single intron of 134 bp. All four AT-hook motifs are encoded by the second exon. Northern blot results show that the expression of HMGA gene is much higher in organs undergoing active cell proliferation. Gel retardation analysis show that wheat HMGa, b, c and histone H1 bind to four-way-junction DNA with high binding affinity, but affinity is dramatically reduced with increasing Mg(2+) and Na(+) ion concentration. Competition binding studies show that proteins share overlapping binding sites on four-way-junction DNA. HMGd does not bind to four-way-junction DNA.     

Post-synthetic acetylation of HMGB1 protein modulates its interactions with supercoiled DNA

High mobility group box (HMGB) proteins 1 and 2 are abundant non-histone nuclear proteins that regulate chromatin structure because of their structure-specific binding to DNA. Here, we have investigated how the post-synthetic acetylation of HMGB1 affects its interaction with negatively supercoiled DNA by employing monoacetylated at Lys2 protein, isolated from butyrate-treated cells. Our data reveal that this modification enhances three reaction parameters: binding affinity, supercoiling activity and capacity to protect the supercoiled DNA from relaxation by topoisomerase I. We show that monoacetylation at Lys2 mimics the effect of acidic tail removal but to a lesser extent thus demonstrating that in vivo acetylated HMGB1 is capable of modulating its interaction with negatively supercoiled DNA.