LEF1 isoforms regulate cellular senescence and aging.

The study of aging and its mechanisms, such as cellular senescence, has provided valuable insights into age-related pathologies, thus contributing to their prevention and treatment. The current abundance of high-throughput data combined with the surge of robust analysis algorithms has facilitated novel ways of identifying underlying pathways that may drive these pathologies. For the purpose of identifying key regulators of lung aging, we performed comparative analyses of transcriptional profiles of aged versus young human subjects and mice, focusing on the common age-related changes in the transcriptional regulation in lung macrophages, T cells, and B immune cells. Importantly, we validated our findings in cell culture assays and human lung samples. Our analysis identified lymphoid enhancer binding factor 1 (LEF1) as an important age-associated regulator of gene expression in all three cell types across different tissues and species. Follow-up experiments showed that the differential expression of long and short LEF1 isoforms is a key regulatory mechanism of cellular senescence. Further examination of lung tissue from patients with idiopathic pulmonary fibrosis, an age-related disease with strong ties to cellular senescence, revealed a stark dysregulation of LEF1. Collectively, our results suggest that LEF1 is a key factor of aging, and its differential regulation is associated with human and murine cellular senescence.

LEF1 isoforms regulate cellular senescence and aging.

Background: The study of aging and its mechanisms, such as cellular senescence, has provided valuable insights into age-related pathologies, thus contributing to their prevention and treatment. The current abundance of high throughput data combined with the surge of robust analysis algorithms has facilitated novel ways of identifying underlying pathways that may drive these pathologies. Methods: With the focus on identifying key regulators of lung aging, we performed comparative analyses of transcriptional profiles of aged versus young human subjects and mice, focusing on the common age-related changes in the transcriptional regulation in lung macrophages, T cells, and B immune cells. Importantly, we validated our findings in cell culture assays and human lung samples. Results: We identified Lymphoid Enhancer Binding Factor 1 (LEF1) as an important age-associated regulator of gene expression in all three cell types across different tissues and species. Follow-up experiments showed that the differential expression of long and short LEF1 isoforms is a key regulatory mechanism of cellular senescence. Further examination of lung tissue from patients with Idiopathic Pulmonary Fibrosis (IPF), an age-related disease with strong ties to cellular senescence, we demonstrated a stark dysregulation of LEF1. Conclusions: Collectively, our results suggest that the LEF1 is a key factor of aging, and its differential regulation is associated with human and murine cellular senescence.

Early events marking lung fibroblast transition to profibrotic state in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF) is an age-associated progressive lung disease with accumulation of scar tissue impairing gas exchange. Previous high-throughput studies elucidated the role of cellular heterogeneity and molecular pathways in advanced disease. However, critical pathogenic pathways occurring in the transition of fibroblasts from normal to profibrotic have been largely overlooked. METHODS: We used single cell transcriptomics (scRNA-seq) from lungs of healthy controls and IPF patients (lower and upper lobes). We identified fibroblast subclusters, genes and pathways associated with early disease. Immunofluorescence assays validated the role of MOXD1 early in fibrosis. RESULTS: We identified four distinct fibroblast subgroups, including one marking the normal-to-profibrotic state transition. Our results show for the first time that global downregulation of ribosomal proteins and significant upregulation of the majority of copper-binding proteins, including MOXD1, mark the IPF transition. We find no significant differences in gene expression in IPF upper and lower lobe samples, which were selected to have low and high degree of fibrosis, respectively. CONCLUSIONS: Early events during IPF onset in fibroblasts include dysregulation of ribosomal and copper-binding proteins. Fibroblasts in early stage IPF may have already acquired a profibrotic phenotype while hallmarks of advanced disease, including fibroblast foci and honeycomb formation, are still not evident. The new transitional fibroblasts we discover could prove very important for studying the role of fibroblast plasticity in disease progression and help develop early diagnosis tools and therapeutic interventions targeting earlier disease states.

Heme Induces IL-6 and Cardiac Hypertrophy Genes Transcripts in Sickle Cell Mice.

Emerging data indicate that free heme promotes inflammation in many different disease settings, including in sickle cell disease (SCD). Although free heme, proinflammatory cytokines, and cardiac hypertrophy are co-existing features of SCD, no mechanistic links between these features have been demonstrated. We now report significantly higher levels of IL-6 mRNA and protein in hearts of the Townes sickle cell disease (SS) mice (2.9-fold, p ≤ 0.05) than control mice expressing normal human hemoglobin (AA). We find that experimental administration of heme 50 µmoles/kg body weight induces IL-6 expression directly in vivo and induces gene expression markers of cardiac hypertrophy in SS mice. We administered heme intravenously and found that within three hours plasma IL-6 protein significantly increased in SS mice compared to AA mice (3248 ± 275 vs. 2384 ± 255 pg/ml, p ≤ 0.05). In the heart, heme induced a 15-fold increase in IL-6 transcript in SS mice heart compared to controls. Heme simultaneously induced other markers of cardiac stress and hypertrophy, including atrial natriuretic factor (Nppa; 14-fold, p ≤ 0.05) and beta myosin heavy chain (Myh7; 8-fold, p ≤ 0.05) in SS mice. Our experiments in Nrf2-deficient mice indicate that the cardiac IL-6 response to heme does not require Nrf2, the usual mediator of transcriptional response to heme for heme detoxification by heme oxygenase-1. These data are the first to show heme-induced IL-6 expression in vivo, suggesting that hemolysis may play a role in the elevated IL-6 and cardiac hypertrophy seen in patients and mice with SCD. Our results align with published evidence from rodents and humans without SCD that suggest a causal relationship between IL-6 and cardiac hypertrophy.

Nrf2 deficiency in mice attenuates erythropoietic stress-related macrophage hypercellularity.

Erythropoiesis in the bone marrow and spleen depends on intricate interactions between the resident macrophages and erythroblasts. Our study focuses on identifying the role of nuclear factor erythroid 2-related factor 2 (Nrf2) during recovery from stress erythropoiesis. To that end, we induced stress erythropoiesis in Nrf2+/+ and Nrf2-null mice and evaluated macrophage subsets known to support erythropoiesis and erythroid cell populations. Our results confirm macrophage and erythroid hypercellularity after acute blood loss. Importantly, Nrf2 depletion results in a marked numerical reduction of F4/80+/CD169+/CD11b+ macrophages, which is more prominent under the induction of stress erythropoiesis. The observed macrophage deficiency is concomitant to a significantly impaired erythroid response to acute stress erythropoiesis in both murine bone marrow and murine spleen. Additionally, peripheral blood reticulocyte count as a response to acute blood loss is delayed in Nrf2-deficient mice compared with age-matched controls (11.0 ± 0.6% vs. 14.8 ± 0.6%, p ≤ 0.001). Interestingly, we observe macrophage hypercellularity in conjunction with erythroid hyperplasia in the bone marrow during stress erythropoiesis in Nrf2+/+ controls, with both impaired in Nrf2-/- mice. We further confirm the finding of macrophage hypercellularity in another model of erythroid hyperplasia, the transgenic sickle cell mouse, characterized by hemolytic anemia and chronic stress erythropoiesis. Our results revealed the role of Nrf2 in stress erythropoiesis in the bone marrow and that macrophage hypercellularity occurs concurrently with erythroid expansion during stress erythropoiesis. Macrophage hypercellularity is a previously underappreciated feature of stress erythropoiesis in sickle cell disease and recovery from blood loss.

The Worst Things in Life are Free: The Role of Free Heme in Sickle Cell Disease.

Hemolysis is a pathological feature of several diseases of diverse etiology such as hereditary anemias, malaria, and sepsis. A major complication of hemolysis involves the release of large quantities of hemoglobin into the blood circulation and the subsequent generation of harmful metabolites like labile heme. Protective mechanisms like haptoglobin-hemoglobin and hemopexin-heme binding, and heme oxygenase-1 enzymatic degradation of heme limit the toxicity of the hemolysis-related molecules. The capacity of these protective systems is exceeded in hemolytic diseases, resulting in high residual levels of hemolysis products in the circulation, which pose a great oxidative and proinflammatory risk. Sickle cell disease (SCD) features a prominent hemolytic anemia which impacts the phenotypic variability and disease severity. Not only is circulating heme a potent oxidative molecule, but it can act as an erythrocytic danger-associated molecular pattern (eDAMP) molecule which contributes to a proinflammatory state, promoting sickle complications such as vaso-occlusion and acute lung injury. Exposure to extracellular heme in SCD can also augment the expression of placental growth factor (PlGF) and interleukin-6 (IL-6), with important consequences to enthothelin-1 (ET-1) secretion and pulmonary hypertension, and potentially the development of renal and cardiac dysfunction. This review focuses on heme-induced mechanisms that are implicated in disease pathways, mainly in SCD. A special emphasis is given to heme-induced PlGF and IL-6 related mechanisms and their role in SCD disease progression.

Cardiac expression of HMOX1 and PGF in sickle cell mice and haem-treated wild type mice dominates organ expression profiles via Nrf2 (Nfe2l2).

Haemolysis is a major feature of sickle cell disease (SCD) that contributes to organ damage. It is well established that haem, a product of haemolysis, induces expression of the enzyme that degrades it, haem oxygenase-1 (HMOX1). We have also shown that haem induces expression of placental growth factor (PGF), but the organ specificity of these responses has not been well-defined. As expected, we found high level expression of Hmox1 and Pgf transcripts in the reticuloendothelial system organs of transgenic sickle cell mice, but surprisingly strong expression in the heart (P < 0·0001). This pattern was largely replicated in wild type mice by intravenous injection of exogenous haem. In the heart, haem induced unexpectedly strong mRNA responses for Hmox1 (18-fold), Pgf (4-fold), and the haem transporter Slc48a1 (also termed Hrg1; 2·4-fold). This was comparable to the liver, the principal known haem-detoxifying organ. The NFE2L2 (also termed NRF2) transcription factor mediated much of the haem induction of Hmox1 and Hrg1 in all organs, but less so for Pgf. Our results indicate that the heart expresses haem response pathway genes at surprisingly high basal levels and shares with the liver a similar transcriptional response to circulating haem. The role of the heart in haem response should be investigated further.

Free heme regulates placenta growth factor through NRF2-antioxidant response signaling.

Free heme activates erythroblasts to express and secrete Placenta Growth Factor (PlGF), an angiogenic peptide of the VEGF family. High circulating levels of PlGF have been associated in experimental animals and in patients with sickle cell disease with echocardiographic markers of pulmonary hypertension, a life-limiting complication associated with more intense hemolysis. We now show that the mechanism of heme regulation of PlGF requires the contribution of the key antioxidant response regulator NRF2. Mimicking the effect of heme, the NRF2 agonist sulforaphane stimulates the PlGF transcript level nearly 30-fold in cultured human erythroblastoid cells. Heme and sulforaphane also induce transcripts for NRF2 itself, its partners MAFF and MAFG, and its competitor BACH1. Furthermore, heme induction of the PlGF transcript is significantly diminished by the NRF2 inhibitor brusatol and by siRNA knockdown of the NRF2 and/or MAFG transcription factors. Chromatin immunoprecipitation experiments show that heme induces NRF2 to bind directly to the PlGF promoter region. In complementary in vivo experiments, mice injected with heme show a significant increase in their plasma PlGF protein as early as 3 h after treatment. Our results reveal an important mechanism of PlGF regulation, adding to the growing literature that supports the pivotal importance of the NRF2 axis in the pathobiology of sickle cell disease.

PARP1 rs1805407 Increases Sensitivity to PARP1 Inhibitors in Cancer Cells Suggesting an Improved Therapeutic Strategy.

Personalized cancer therapy relies on identifying patient subsets that benefit from a therapeutic intervention and suggest alternative regimens for those who don't. A new data integrative approach, based on graphical models, was applied on our multi-modal -omics, and clinical data cohort of metastatic melanoma patients. We found that response to chemotherapy is directly linked to ten gene expression, four methylation variables and PARP1 SNP rs1805407. PARP1 is a DNA repair gene critical for chemotherapy response and for which FDA-approved inhibitors are clinically available (olaparib). We demonstrated that two PARP inhibitors (ABT-888 and olaparib) make SNP carrier cancer cells of various histologic subtypes more sensitive to alkylating agents, but they have no effect in wild-type cells. Furthermore, PARP1 inhibitors act synergistically with chemotherapy in SNP carrier cells (especially in ovarian cancer for which olaparib is FDA-approved), but they are additive at best in wild-type cancer cells. Taken together, our results suggest that the combination of chemotherapy and PARP1 inhibition may benefit the carriers of rs1805407 in the future and may be used in personalized therapy strategies to select patients that are more likely to respond to PARP inhibitors.

Modified mesenchymal stem cells using miRNA transduction alter lung injury in a bleomycin model.

Although different preclinical models have demonstrated a favorable role for bone marrow-derived mesenchymal stem cells (B-MSC) in preventing fibrosis, this protective effect is not observed with late administration of these cells, when fibrotic changes are consolidated. We sought to investigate whether the late administration of B-MSCs overexpressing microRNAs (miRNAs) let-7d (antifibrotic) or miR-154 (profibrotic) could alter lung fibrosis in a murine bleomycin model. Using lentiviral vectors, we transduced miRNAs (let-7d or miR-154) or a control sequence into human B-MSCs. Overexpression of let-7d or miR-154 was associated with changes in the mesenchymal properties of B-MSCs and in their cytokine expression. Modified B-MSCs were intravenously administered to mice at day 7 after bleomycin instillation, and the mice were euthanized at day 14 Bleomycin-injured animals that were treated with let-7d cells were found to recover quicker from the initial weight loss compared with the other treatment groups. Interestingly, animals treated with miR-154 cells had the lowest survival rate. Although a slight reduction in collagen mRNA levels was observed in lung tissue from let-7d mice, no significant differences were observed in Ashcroft score and OH-proline. However, the distinctive expression in cytokines and CD45-positive cells in the lung suggests that the differential effects observed in both miRNA mice groups were related to an effect on the immunomodulation function. Our results establish the use of miRNA-modified mesenchymal stem cells as a potential future research in lung fibrosis.

Aging and Lung Disease. Clinical Impact and Cellular and Molecular Pathways.

With the expected rapid growth of the aging population worldwide, there is a clear need to understand the complex process of aging to develop interventions that might extend the health span in this group of patients. Aging is associated with increased susceptibility to a variety of chronic diseases, and lung pathologies are no exception. The prevalence of lung diseases such as idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease has been found to increase considerably with age. In October 2014, the Division of Pulmonary, Allergy, and Critical Care of the University of Pittsburgh cohosted the Pittsburgh-Munich Lung Conference focused in aging and lung disease with the Comprehensive Pneumology Center, Institute of Lung Biology and Disease, Ludwig-Maximilians University and Helmholtz Zentrum Munich Germany. The purpose of the conference was to disseminate novel concepts in aging mechanisms that have an impact in lung physiology and pathogenesis of pulmonary diseases that commonly occur in older populations. The conference included 28 presentations on diverse topics, which are summarized in this report. The participants identified priorities for future basic and translational investigations that will assist in the identification of molecular insights involved in the pathogenesis of age-related pulmonary diseases and the design of therapeutic interventions for these lung conditions.

Aging mesenchymal stem cells fail to protect because of impaired migration and antiinflammatory response.

RATIONALE: Aging is characterized by functional impairment and reduced capacity to respond appropriately to environmental stimuli and injury. With age, there is an increase in the incidence and severity of chronic and acute lung diseases. However, the relationship between age and the lung's reduced ability to repair is far from established and necessitates further research in the field. OBJECTIVES: Little is currently known about age-related phenomena in mesenchymal stem cells (MSCs). On account of their ability to protect the endothelium and the alveolar epithelium through multiple paracrine mechanisms, we looked for adverse effects that aging might cause in MSC biology. Such age-related changes might partly account for the increased susceptibility of the aging lung to injury. MEASUREMENTS AND MAIN RESULTS: We demonstrated that old mice have more inflammation in response to acute lung injury. To investigate the causes, we compared the global gene expression of aged and young bone marrow-derived MSCs (B-MSCs). Our results revealed that the expression levels of inflammatory response genes depended on the age of the B-MSCs. We demonstrated that the age-dependent decrease in expression of several cytokine and chemokine receptors is important for the migration and activation of B-MSCs. Finally, we showed by adoptive transfer of aged B-MSCs to young endotoxemic mice that aged cells lacked the antiinflammatory protective effect of their young counterparts. CONCLUSIONS: Taken together, the decreased expression of cytokine and chemokine receptors in aged B-MSCs compromises their protective role by perturbing the potential of B-MSCs to become activated and mobilize to the site of injury.

Expression of regulatory platelet microRNAs in patients with sickle cell disease.

BACKGROUND: Increased platelet activation in sickle cell disease (SCD) contributes to a state of hypercoagulability and confers a risk of thromboembolic complications. The role for post-transcriptional regulation of the platelet transcriptome by microRNAs (miRNAs) in SCD has not been previously explored. This is the first study to determine whether platelets from SCD exhibit an altered miRNA expression profile. METHODS AND FINDINGS: We analyzed the expression of miRNAs isolated from platelets from a primary cohort (SCD = 19, controls = 10) and a validation cohort (SCD = 7, controls = 7) by hybridizing to the Agilent miRNA microarrays. A dramatic difference in miRNA expression profiles between patients and controls was noted in both cohorts separately. A total of 40 differentially expressed platelet miRNAs were identified as common in both cohorts (p-value 0.05, fold change>2) with 24 miRNAs downregulated. Interestingly, 14 of the 24 downregulated miRNAs were members of three families - miR-329, miR-376 and miR-154 - which localized to the epigenetically regulated, maternally imprinted chromosome 14q32 region. We validated the downregulated miRNAs, miR-376a and miR-409-3p, and an upregulated miR-1225-3p using qRT-PCR. Over-expression of the miR-1225-3p in the Meg01 cells was followed by mRNA expression profiling to identify mRNA targets. This resulted in significant transcriptional repression of 1605 transcripts. A combinatorial approach using Meg01 mRNA expression profiles following miR-1225-3p overexpression, a computational prediction analysis of miRNA target sequences and a previously published set of differentially expressed platelet transcripts from SCD patients, identified three novel platelet mRNA targets: PBXIP1, PLAGL2 and PHF20L1. CONCLUSIONS: We have identified significant differences in functionally active platelet miRNAs in patients with SCD as compared to controls. These data provide an important inventory of differentially expressed miRNAs in SCD patients and an experimental framework for future studies of miRNAs as regulators of biological pathways in platelets.

Influence of age on wound healing and fibrosis.

The incidence and severity of fibrotic lung diseases increase with age, but very little is known about how age-related changes affect the mechanisms that underlie disease emergence and progression. Normal ageing includes accumulation of DNA mutations, oxidative and cell stresses, mitochondria dysfunction, increased susceptibility to apoptosis, telomere length dysfunction and differential gene expression as a consequence of epigenetic changes and miR regulation. These inevitable ageing-related phenomena may cause dysfunction and impaired repair capacity of lung epithelial cells, fibroblasts and MSCs. As a consequence, the composition of the extracellular matrix changes and the dynamic interaction between cells and their environment is damaged, resulting ultimately in predisposition for several diseases. This review summarizes what is known about age-related molecular changes that are implicated in the pathobiology of lung fibrosis in lung tissue.

Global methylation patterns in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF) is characterized by profound changes in the lung phenotype including excessive extracellular matrix deposition, myofibroblast foci, alveolar epithelial cell hyperplasia and extensive remodeling. The role of epigenetic changes in determining the lung phenotype in IPF is unknown. In this study we determine whether IPF lungs exhibit an altered global methylation profile. METHODOLOGY/PRINCIPAL FINDINGS: Immunoprecipitated methylated DNA from 12 IPF lungs, 10 lung adenocarcinomas and 10 normal histology lungs was hybridized to Agilent human CpG Islands Microarrays and data analysis was performed using BRB-Array Tools and DAVID Bioinformatics Resources software packages. Array results were validated using the EpiTYPER MassARRAY platform for 3 CpG islands. 625 CpG islands were differentially methylated between IPF and control lungs with an estimated False Discovery Rate less than 5%. The genes associated with the differentially methylated CpG islands are involved in regulation of apoptosis, morphogenesis and cellular biosynthetic processes. The expression of three genes (STK17B, STK3 and HIST1H2AH) with hypomethylated promoters was increased in IPF lungs. Comparison of IPF methylation patterns to lung cancer or control samples, revealed that IPF lungs display an intermediate methylation profile, partly similar to lung cancer and partly similar to control with 402 differentially methylated CpG islands overlapping between IPF and cancer. Despite their similarity to cancer, IPF lungs did not exhibit hypomethylation of long interspersed nuclear element 1 (LINE-1) retrotransposon while lung cancer samples did, suggesting that the global hypomethylation observed in cancer was not typical of IPF. CONCLUSIONS/SIGNIFICANCE: Our results provide evidence that epigenetic changes in IPF are widespread and potentially important. The partial similarity to cancer may signify similar pathogenetic mechanisms while the differences constitute IPF or cancer specific changes. Elucidating the role of these specific changes will potentially allow better understanding of the pathogenesis of IPF.

Monoubiquitinated histone H2A destabilizes photolesion-containing nucleosomes with concomitant release of UV-damaged DNA-binding protein E3 ligase.

How the nucleotide excision repair (NER) machinery gains access to damaged chromatinized DNA templates and how the chromatin structure is modified to promote efficient repair of the non-transcribed genome remain poorly understood. The UV-damaged DNA-binding protein complex (UV-DDB, consisting of DDB1 and DDB2, the latter of which is mutated in xeroderma pigmentosum group E patients, is a substrate-recruiting module of the cullin 4B-based E3 ligase complex, DDB1-CUL4B(DDB2). We previously reported that the deficiency of UV-DDB E3 ligases in ubiquitinating histone H2A at UV-damaged DNA sites in the xeroderma pigmentosum group E cells contributes to the faulty NER in these skin cancer-prone patients. Here, we reveal the mechanism by which monoubiquitination of specific H2A lysine residues alters nucleosomal dynamics and subsequently initiates NER. We show that DDB1-CUL4B(DDB2) E3 ligase specifically binds to mononucleosomes assembled with human recombinant histone octamers and nucleosome-positioning DNA containing cyclobutane pyrimidine dimers or 6-4 photoproducts photolesions. We demonstrate functionally that ubiquitination of H2A Lys-119/Lys-120 is necessary for destabilization of nucleosomes and concomitant release of DDB1-CUL4B(DDB2) from photolesion-containing DNA. Nucleosomes in which these lysines are replaced with arginines are resistant to such structural changes, and arginine mutants prevent the eviction of H2A and dissociation of polyubiquitinated DDB2 from UV-damaged nucleosomes. The partial eviction of H3 from the nucleosomes is dependent on ubiquitinated H2A Lys-119/Lys-120. Our results provide mechanistic insight into how post-translational modification of H2A at the site of a photolesion initiates the repair process and directly affects the stability of the human genome.

Mesenchymal stem cells enhance survival and bacterial clearance in murine Escherichia coli pneumonia.

RATIONALE: Bacterial pneumonia is the most common infectious cause of death worldwide and treatment is increasingly hampered by antibiotic resistance. Mesenchymal stem cells (MSCs) have been demonstrated to provide protection against acute inflammatory lung injury; however, their potential therapeutic role in the setting of bacterial pneumonia has not been well studied. OBJECTIVE: This study focused on testing the therapeutic and mechanistic effects of MSCs in a mouse model of Gram-negative pneumonia. METHODS AND RESULTS: Syngeneic MSCs from wild-type mice were isolated and administered via the intratracheal route to mice 4 h after the mice were infected with Escherichia coli. 3T3 fibroblasts and phosphate-buffered saline (PBS) were used as controls for all in vivo experiments. Survival, lung injury, bacterial counts and indices of inflammation were measured in each treatment group. Treatment with wild-type MSCs improved 48 h survival (MSC, 55%; 3T3, 8%; PBS, 0%; p<0.05 for MSC vs 3T3 and PBS groups) and lung injury compared with control mice. In addition, wild-type MSCs enhanced bacterial clearance from the alveolar space as early as 4 h after administration, an effect that was not observed with the other treatment groups. The antibacterial effect with MSCs was due, in part, to their upregulation of the antibacterial protein lipocalin 2. CONCLUSIONS: Treatment with MSCs enhanced survival and bacterial clearance in a mouse model of Gram-negative pneumonia. The bacterial clearance effect was due, in part, to the upregulation of lipocalin 2 production by MSCs.

The cullin 4B-based UV-damaged DNA-binding protein ligase binds to UV-damaged chromatin and ubiquitinates histone H2A.

By removing UV-induced lesions from DNA, the nucleotide excision repair (NER) pathway preserves the integrity of the genome. The UV-damaged DNA-binding (UV-DDB) protein complex is involved in the recognition of chromatin-embedded UV-damaged DNA, which is the least understood step of NER. UV-DDB consists of DDB1 and DDB2, and it is a component of the cullin 4A (CUL4A)-based ubiquitin ligase, DDB1-CUL4A(DDB2). We previously showed that DDB1-CUL4A(DDB2) ubiquitinates histone H2A at the sites of UV lesions in a DDB2-dependent manner. Mutations in DDB2 cause a cancer prone syndrome, xeroderma pigmentosum group E (XP-E). CUL4A and its paralog, cullin 4B (CUL4B), copurify with the UV-DDB complex, but it is unclear whether CUL4B has a role in NER as a separate E3 ubiquitin ligase. Here, we present evidence that CUL4A and CUL4B form two individual E3 ligases, DDB1-CUL4A(DDB2) and DDB1-CUL4B(DDB2). To investigate CUL4B's possible role in NER, we examined its subcellular localization in unirradiated and irradiated cells. CUL4B colocalizes with DDB2 at UV-damaged DNA sites. Furthermore, CUL4B binds to UV-damaged chromatin as a part of the DDB1-CUL4B(DDB2) E3 ligase in the presence of functional DDB2. In contrast to CUL4A, CUL4B is localized in the nucleus and facilitates the transfer of DDB1 into the nucleus independently of DDB2. Importantly, DDB1-CUL4B(DDB2) is more efficient than DDB1-CUL4A(DDB2) in monoubiquitinating histone H2A in vitro. Overall, this study suggests that DDB1-CUL4B(DDB2) E3 ligase may have a distinctive function in modifying the chromatin structure at the site of UV lesions to promote efficient NER.

The DNA transposon Minos as a tool for transgenesis and functional genomic analysis in vertebrates and invertebrates.

Transposons are powerful tools for conducting genetic manipulation and functional studies in organisms that are of scientific, economic, or medical interest. Minos, a member of the Tc1/mariner family of DNA transposons, exhibits a low insertional bias and transposes with high frequency in vertebrates and invertebrates. Its use as a tool for transgenesis and genome analysis of rather different animal species is described.

The DDB1-CUL4ADDB2 ubiquitin ligase is deficient in xeroderma pigmentosum group E and targets histone H2A at UV-damaged DNA sites.

Xeroderma pigmentosum (XP) is a heritable human disorder characterized by defects in nucleotide excision repair (NER) and the development of skin cancer. Cells from XP group E (XP-E) patients have a defect in the UV-damaged DNA-binding protein complex (UV-DDB), involved in the damage recognition step of NER. UV-DDB comprises two subunits, products of the DDB1 and DDB2 genes, respectively. Mutations in the DDB2 gene account for the underlying defect in XP-E. The UV-DDB complex is a component of the newly identified cullin 4A-based ubiquitin E3 ligase, DDB1-CUL4A(DDB2). The E3 ubiquitin ligases recognize specific substrates and mediate their ubiquitination to regulate protein activity or target proteins for degradation by the proteasomal pathway. In this study, we have addressed the role of the UV-DDB-based E3 in NER and sought a physiological substrate. We demonstrate that monoubiquitinated histone H2A in native chromatin coimmunoprecipitates with the endogenous DDB1-CUL4A(DDB2) complex in response to UV irradiation. Further, mutations in DDB2 alter the formation and binding activity of the DDB1-CUL4A(DDB2) ligase, accompanied by impaired monoubiquitination of H2A after UV treatment of XP-E cells, compared with repair-proficient cells. This finding indicates that DDB2, as the substrate receptor of the DDB1-CUL4A-based ligase, specifically targets histone H2A for monoubiquitination in a photolesion-binding-dependent manner. Given that the loss of monoubiquitinated histone H2A at the sites of UV-damaged DNA is associated with decreased global genome repair in XP-E cells, this study suggests that histone modification, mediated by the XPE factor, facilitates the initiation of NER.