Specific DNA-binding Proteins Research Articles

Abstract 1840: MYBBP1A AMPylated by SELO to influence its function

Abstract Introduction: MYBBP1A, named Myb-binding protein 1A, is a protein that mainly locates in nucleolar and shuttles between the nucleus and cytoplasm, nuclear import may be mediated by KPNA2, however export depend partially on XPO1/CRM1. It activates or represses transcription via interactions with sequence specific DNA-binding proteins. Repression may be mediated at least in part by histone deacetylase activity (HDAC activity). Acts as a corepressor and in concert with CRY1, represses the transcription of the core circadian clock component PER2. Preferentially binds to di-methylated histone H3 'Lys-9' (H3K9me2) on the PER2 promoter. MYBBP1A overexpressed in tumor than normal and have a significantly negative correlation with patient overall survival. We aim to explore the role of MYBBP1A in PDAC progression and underlying mechanism. In our research, we find and prove the novel AMPylating enzyme SELO-AMPylate MYBBP1A inhibits PDAC progression and modified site in S1353 and further influence downstream relative RNA, such as 18s rRNA, 28S rRNA. Methods and Materials: 1. The function of MYBBP1A in tumorigenesis at the organismal and cellular levels. MYBBP1A is widely expressed in different tissues. Firstly, we explored the correlation between MYBBP1A expression level and PDAC through TCGA database and Kaplan-Meier plotter. We then established MYBBP1A gene knockout mice mating with PDX/P53K172H/KRASG12C to induced a primary PDAC model to analyze the progression of pancreatic cancer. The effect of MYBBP1A on tumor cell growth and proliferation was explored through cell proliferation analysis and a nude mouse tumor xenograft model. 2. The MYBBP1A AMPylated by SELO influence MYBBP1A function in cell proliferation was investigated at the molecular and cellular levels. AMPylation-specific antibodies were used to confirmed MYBBP1A AMPylated by SELO in both cell lysates and purified proteins through experiments such as CO-IP and WB. Stably expressed MYBBP1A WT/S1353A were constructed to verify the function in cell proliferation or migration by SELO. The signaling molecules were further explored through CO-IP and WB to identify the changed molecular by SELO-AMPylate-MYBBP1A, revealing the entire signaling pathways of AMPylation-mediated regulation of PDAC proliferation. Conclusion: We have demonstrated the function of MYBBP1A in the formation of pancreatic cancer, and further revealed SELO-AMPylate-MYBBP1A importance in the process of reversing MYBBP1A’s role, which has played a certain significance for the treatment of pancreatic cancer and the exploration of related therapeutic targets. Citation Format: Wu Li. MYBBP1A AMPylated by SELO to influence its function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1840.

DTL is a Novel Downstream Gene of E2F1 that Promotes the Progression of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC), one of the world's most prevalent malignancies, accounts for 90% of primary liver cancer cases. Recent studies have shown an increased expression of denticles E3 ubiquitin protein ligase homolog (DTL) in several different tumor types, but its function and regulatory mechanisms remain unclear. This study aimed to investigate the expressions of the Cullin4 (CUL4) complex in HCC and elucidate the roles of DTL in HCC cells. The relative expression of the CUL4 complex and its clinical significance were analyzed with The Cancer Genome Atlas (TCGA) data, and the level of DTL was confirmed by immunohistochemistry. The functions of DTL1 and upstream E2F1 were evaluated by a Western blot, MTT, transwell, and xenograft in HCC cell lines. The elevated mRNA expression of the CUL4 complex, including CUL4B, DDB1 (Damage Specific DNA Binding Protein 1), and DTL, was associated with the overall survival of HCC patients. We also found that the DTL protein was elevated in HCC tissues, and patients with highly expressed DTL and nucleus-located DTL had a poorer survival time. DTL knockdown significantly inhibited cancer proliferation, migration, and invasion. Further experiments showed that E2F1 was an upstream regulatory molecule of DTL, which was bound to the promoter of DTL, promoting the expression of DTL. The study results demonstrate that E2F1-DTL signaling promotes the growth, migration, and invasion of HCC cells, which provides new insights and a potential biological target for future HCC therapies.

DDB2-associated incidence of squamous cell carcinoma in Haflingers: risk minimization by genotyping

SCC (squamous cell carinomas) are among the most common eye neoplasms in horses. In recent studies Haflinger horses with a homozygous genotype for a missense variant in the DDB2 gene (damage specific DNA binding protein 2) had a significant increased risk of developing ocular SCC. The aims of this study were to determine the frequency of the SCC-associated risk allele in the DDB2 gene in Swiss and Austrian Haflinger populations and to validate the previously described phenotypic correlation. For this purpose, Haflingers presented at various horse clinics in Switzerland (n = 21, including 11 SCC cases), privately kept Haflingers (n = 52, including 1 SCC case), and Haflingers from a stud farm in the Austrian Tyrol (n = 53) were recruited. The individual DDB2 genotype of the animals was determined using a polymerase chain ceaction (PCR) test using hair follicle or whole blood samples. Of the 12 horses suffering from SCC, nine had ocular SCC and three had non-ocular SCC. Six of the nine Haflingers with ocular SCC and one of the three Haflingers with non-ocular SCC were homozygous for the DDB2 variant. Of the 113 clinically normal animals, 7/113 were homozygous (6 %) and 42/113 were heterozygous (37 %), which corresponds to an allele frequency of 24,8 % in the control cohort. The risk of ocular SCC occurring in Haflingers is significantly increased with the homozygous DDB2 genotype. However, not all animals with SCC carry this gene variant and not all DDB2 homozygous animals develop SCC, which can be explained by the multifactorial genesis of the disease. Due to the high frequency of the undesirable allele, we recommend taking the individual DDB2 genotype of breeding animals into account in order to avoid homozygous offspring with a greatly increased SCC risk by excluding high-risk matings.

Mutual promotion of co-condensation of KRAS G-quadruplex and a well-folded protein HMGB1.

Liquid-liquid phase separation (LLPS) of G-quadruplex (GQ) is involved in many crucial cellular processes, while the quadruplex-folding and their functions are typically modulated by specific DNA-binding proteins. However, the regulatory mechanism of binding proteins, particularly the well-folded proteins, on the LLPS of GQs is largely unknown. Here, we investigated the effect of HMGB1 on the condensation of a G-quadruplex of KRAS promoter (GQKRAS). The results show that these two rigid macro-biomolecules undergo co-condensation through a mutual promotion manner, while neither of them can form LLPS alone. Fluidity measurements confirm that the liquid-like droplets are highly dynamic. HMGB1 facilitates and stabilizes the quadruplex folding of GQKRAS, and this process enhances their co-condensation. The KRAS promoter DNA retains quadruplex folding in the droplets; interference with the GQ-folding disrupts the co-condensation of GQKRAS/HMGB1. Mechanistic studies reveal that electrostatic interaction is a key driving force of the interaction and co-condensation of GQKRAS/HMGB1; meanwhile, the recognition of two macro-biomolecules plays a crucial role in this process. This result indicates that the phase separation of GQs can be modulated by DNA binding proteins, and this process could also be an efficient way to recruit specific DNA binding proteins.

REPIN1 regulates iron metabolism and osteoblast apoptosis in osteoporosis

Osteoporosis is not well treated due to the difficulty of finding commonalities between the various types of it. Iron homeostasis is a vital component in supporting biochemical functions, and iron overload is recognized as a common risk factor for osteoporosis. In this research, we found that there is indeed evidence of iron accumulation in the bone tissue of patients with osteoporosis and REPIN1, as an origin specific DNA binding protein, may play a key role in this process. We revealed that sh-Repin1 therapy can rescue bone loss in an iron-overload-induced osteoporosis mouse model. Knockdown of Repin1 can inhibit apoptosis and enhance the resistance of osteoblasts to iron overload toxicity. REPIN1 promoted apoptosis by regulating iron metabolism in osteoblasts. Mechanistically, knockdown of Repin1 decreased the expression of Lcn2, which ameliorated the toxic effects of intracellular iron overload. The anti-iron effect of lentivirus sh-Repin1 was partially reversed or replicated by changing LCN2 expression level via si-RNA or plasmid, which indirectly verified the key regulatory role of LCN2 as a downstream target. Furthermore, the levels of BCL2 and BAX, which play a key role in the mitochondrial apoptosis pathway, were affected. In summary, based on the results of clinical specimens, animal models and in vitro experiments, for the first time, we proved the key role of REPIN1 in iron metabolism-related osteoporosis.

Expression of DDB1 is associated with subtypes of epithelial ovarian cancer and predicts clinical outcomes

BackgroundOvarian cancer is the most lethal gynaecological malignancy. Damage specific DNA-binding protein 1 (DDB1) functions in nucleotide-excision repair and has been reported to be involved in cancer development. In this study, we aimed to determine the expression levels of DDB1 and their association with the clinical outcomes of patients with ovarian cancer. MethodsTissue arrays were performed on 54 epithelial ovarian cancer (EOC) samples. Immunohistochemistry was performed to determine DDB1 expression. DDB1 expression levels among different EOC subtypes were analysed via one-way analysis of variance using SPSS Statistics 19.0. Correlation between DDB1 expression and chemotherapy course/progression-free survival (PFS) of patients was determined via Kaplan–Meier survival analysis using GraphPad Prism 5. Moreover, knockdown of DDB1 in ovarian cancer cells ES2 and OVCAR3 was used to preliminarily validate the role of DDB1. ResultsDDB1 was detected in the cytoplasm, especially in the nucleus, of all subtypes of EOC. However, DDB1 expression levels were significantly different between clear cell carcinoma and low-grade serous carcinoma (P = 0.022) and clear cell carcinoma and endometrioid cancer (P = 0.016). In addition, DDB1 expression was not significantly correlated with chemotherapy course (P = 0.433) or PFS (P = 0.566). High expression levels of DDB1 were correlated with significantly worse overall survival (P = 0.017) in patients with EOC. In addition, DDB1 knockdown in ovarian cancer cells decreased their proliferation in vitro. ConclusionOur results revealed that DDB1 expression is heterogeneous in ovarian cancer, suggesting its use as a potential biomarker for poor survival in ovarian cancer.

UV irradiation remodels the specificity landscape of transcription factors

Somatic mutations are highly enriched at transcription factor (TF) binding sites, with the strongest trend being observed for ultraviolet light (UV)-induced mutations in melanomas. One of the main mechanisms proposed for this hypermutation pattern is the inefficient repair of UV lesions within TF-binding sites, caused by competition between TFs bound to these lesions and the DNA repair proteins that must recognize the lesions to initiate repair. However, TF binding to UV-irradiated DNA is poorly characterized, and it is unclear whether TFs maintain specificity for their DNA sites after UV exposure. We developed UV-Bind, a high-throughput approach to investigate the impact of UV irradiation on protein-DNA binding specificity. Weapplied UV-Bind to ten TFs from eight structural families, and found that UV lesions significantly altered the DNA-binding preferences of all the TFs tested. The main effect was a decrease in binding specificity, but the precise effects and their magnitude differ across factors. Importantly, we found that despite the overall reduction in DNA-binding specificity in the presence of UV lesions, TFs can still compete with repair proteins for lesion recognition, in a manner consistent with their specificity for UV-irradiated DNA. In addition, for a subset of TFs, we identified a surprising but reproducible effect at certain nonconsensus DNA sequences, where UV irradiation leads to a high increase in the level of TF binding. These changes in DNA-binding specificity after UV irradiation, at both consensus and nonconsensus sites, have important implications for the regulatory and mutagenic roles of TFs in the cell.

Sensing of protein and DNA complexes using solid-state nanopores.

The field of nanopores has garnered increased interest for nucleic acid and peptide sensing and sequencing to develop targeted therapies for various diseases, including cancer, due to versatile pore sizes, measurement conditions, signal-to-noise ratio, and integration into electronics. We utilize a SiNx solid-state nanopore platform for studying the interaction and specificity of DNA-binding proteins with DNA. These DNA-binding proteins include various transcription factors, nuclear receptors, polymerases, and nucleases, which are responsible for several essential cellular processes and intra- and intercellular signaling cascades. Some of these DNA-binding proteins are known to be diagnostic and therapeutic targets for various diseases, specifically breast, ovarian, and prostate cancers. We seek to use these DNA-binding proteins as a clinically relevant model to detect their binding sites on the DNA and identify the protein mutations that would have a detrimental effect on its functionality, as they are highly dependent on accurate post-translational modifications. Using our nanopores, we successfully observe the binding of DNA-binding proteins onto the conventional binding sites on the target DNA and correlate these with the gold-standard Electrophoretic Mobility Shift Assay (EMSA) carried out parallelly. These proteins are observed as a pronounced local spike over and above the DNA strand translocating through the nanopore, signifying their binding to the DNA. The protein binding is stable and unaffected at a relatively high salt concentration (1 M LiCl, pH 8). Furthermore, the isoelectronic point of the protein is higher than this pH, imparting a positive charge on the protein and slowing down the translocation of such nuclear receptor-DNA complex, leading to a better signal resolution. We believe studying DNA binding proteins, including nuclear receptors, using nanopores could improve understanding of these proteins on a single molecule level, furthering the development of novel targeted treatment and diagnostic strategies.

AIDmut-Seq: a Three-Step Method for Detecting Protein-DNA Binding Specificity.

Transcriptional factors (TFs) and their regulons make up the gene regulatory networks. Here, we developed a method based on TF-directed activation-induced cytidine deaminase (AID) mutagenesis in combination with genome sequencing, called AIDmut-Seq, to detect TF targets on the genome. AIDmut-Seq involves only three simple steps, including the expression of the AID-TF fusion protein, whole-genome sequencing, and single nucleotide polymorphism (SNP) profiling, making it easy for junior and interdisciplinary researchers to use. Using AIDmut-Seq for the major quorum sensing regulator LasR in Pseudomonas aeruginosa, we confirmed that a few TF-guided C-T (or G-A) conversions occurred near their binding boxes on the genome, and a number of previously characterized and uncharacterized LasR-binding sites were detected. Further verification of AIDmut-Seq using various transcriptional regulators demonstrated its high efficiency for most transcriptional activators (FleQ, ErdR, GacA, ExsA). We confirmed the binding of LasR, FleQ, and ErdR to 100%, 50%, and 86% of their newly identified promoters by using in vitro protein-DNA binding assay. And real-time RT-PCR data validated the intracellular activity of these TFs to regulate the transcription of those newly found target promoters. However, AIDmut-Seq exhibited low efficiency for some small transcriptional repressors such as RsaL and AmrZ, with possible reasons involving fusion-induced TF dysfunction as well as low transcription rates of target promoters. Although there are false-positive and false-negative results in the AIDmut-Seq data, preliminary results have demonstrated the value of AIDmut-Seq to act as a complementary tool for existing methods. IMPORTANCE Protein-DNA interactions (PDI) play a central role in gene regulatory networks (GRNs). However, current techniques for studying genome-wide PDI usually involve complex experimental procedures, which prevent their broad use by scientific researchers. In this study, we provide a in vivo method called AIDmut-Seq. AIDmut-Seq involves only three simple steps that are easy to operate for researchers with basic skills in molecular biology. The efficiency of AIDmut-Seq was tested and confirmed using multiple transcription factors in Pseudomonas aeruginosa. Although there are still some defects regarding false-positive and false-negative results, AIDmut-Seq will be a good choice in the early stage of PDI study.

DDB2 represses epithelial-to-mesenchymal transition and sensitizes pancreatic ductal adenocarcinoma cells to chemotherapy.

Damage specific DNA binding protein 2 (DDB2) is an UV-indiced DNA damage recognition factor and regulator of cancer development and progression. DDB2 has dual roles in several cancers, either as an oncogene or as a tumor suppressor gene, depending on cancer localization. Here, we investigated the unresolved role of DDB2 in pancreatic ductal adenocarcinoma (PDAC). The expression level of DDB2 in pancreatic cancer tissues and its correlation with patient survival were evaluated using publicly available data. Two PDAC cell models with CRISPR-modified DDB2 expression were developed: DDB2 was repressed in DDB2-high T3M4 cells (T3M4 DDB2-low) while DDB2 was overexpressed in DDB2-low Capan-2 cells (Capan-2 DDB2-high). Immunofluorescence and qPCR assays were used to investigate epithelial-to-mesenchymal transition (EMT) in these models. Migration and invasion properties of the cells were also determined using wound healing and transwell assays. Sensitivity to 5-fluorouracil (5-FU), oxaliplatin, irinotecan and gemcitabine were finally investigated by crystal violet assays. DDB2 expression level was reduced in PDAC tissues compared to normal ones and DDB2-low levels were correlated to shorter disease-free survival in PDAC patients. DDB2 overexpression increased expression of E-cadherin epithelial marker, and decreased levels of N-cadherin mesenchymal marker. Conversely, we observed opposite effects in DDB2 repression and enhanced transcription of SNAIL, ZEB1, and TWIST EMT transcription factors (EMT-TFs). Study of migration and invasion revealed that these properties were negatively correlated with DDB2 expression in both cell models. DDB2 overexpression sensitized cells to 5-fluorouracil, oxaliplatin and gemcitabine. Our study highlights the potential tumor suppressive effects of DDB2 on PDAC progression. DDB2 could thus represent a promising therapeutic target or biomarker for defining prognosis and predicting chemotherapy response in patients with PDAC.

The miRNA125a-5p and miRNA125b-1-5p cluster induces cell invasion by down-regulating DDB2-reduced epithelial-to-mesenchymal transition (EMT) in colorectal cancer

MicroRNA (miRNA) is a kind of non-coding RNA that regulates gene expression and is involved in tumor development. MiRNA-125 is reportedly aberrantly expressed in colorectal cancer tissue; however, its potential function and underlying mechanism remain unclear. The present study aimed to investigate the expression level and potential role of the miRNA-125 family in the invasion and migration of colorectal cancer. To further understand the role of the miRNA-125 family in metastatic colorectal cancer, we overexpressed miRNA-125 in the SW480 cell line by transfection with the miRNA-125 family mimics or a sponge. Methyl thiazolyl tetrazolium (MTT) assay was performed to identify the effect of the miRNA-125 family on cell proliferation, and a Transwell filter assay was used to detect the role of the miRNA-125 family in migration and invasion. A luciferase assay was carried out to confirm the binding site of miRNA-125 and the target gene, damage specific DNA binding protein 2 (DDB2). Western blot was applied to detect the expression levels of DDB2 and the markers of epithelial-to-mesenchymal transition (EMT) in colorectal cancer cells. The real-time polymerase chain reaction (PCR) results showed that miR-125a-5p and miR-125b-1-5p were up-regulated in metastatic colorectal cancer tissues. The Transwell filter assay results appeared that miR-125a-5p and miR-125b-1-5p could promote the invasion and migration of colorectal cancer cells. The luciferase assay data confirmed the binding site of miR-125a-5p and miR-125b-1-5p on the 3' untranslated region (3'UTR) of DDB2 messenger RNA (mRNA). The real-time PCR and Western blot results indicated that miR-125a-5p and miR-125b-1-5p could regulate the expression levels of DDB2 and EMT markers, and lower DDB2 expression was observed in metastatic tissues. Our findings illustrated that miRNA125a-5p and miRNA125b-1-5p could reduce the expression of DDB2 by binding to the 3'UTR region, and then regulate the expression levels of EMT markers, leading to the enhanced invasion and metastasis of colorectal cancer cells. Thus, miRNA125a-5p and miRNA125b-1-5p might be novel markers of colorectal cancer migration and potential therapeutic targets to treat metastatic colorectal cancer patients.

66P DDB2 influences epithelial-to-mesenchymal transition and sensitivity to chemotherapy in pancreatic ductal adenocarcinoma cells

Damage specific DNA binding protein 2 (DDB2) is originally implied in the recognition of ultraviolet-induced DNA damage and the initiation of nucleotide excision repair (NER) process. This protein also demonstrated dual roles in several cancers, acting either as an oncogene or a tumor suppressor gene depending on cancer localization. In this study, we investigated the unresolved role of DDB2 in pancreatic ductal adenocarcinoma (PDAC).

Effects of Global and Specific DNA-Binding Proteins on Transcriptional Regulation of the E. coli bgl Operon.

Using reporter gene (lacZ) transcriptional fusions, we examined the transcriptional dependencies of the bgl promoter (Pbgl) and the entire operon regulatory region (Pbgl-bglG) on eight transcription factors as well as the inducer, salicin, and an IS5 insertion upstream of Pbgl. Crp-cAMP is the primary activator of both Pbgl and the bgl operon, while H-NS is a strong dominant operon repressor but only a weak repressor of Pbgl. H-NS may exert its repressive effect by looping the DNA at two binding sites. StpA is a relatively weak repressor in the absence of H-NS, while Fis also has a weak repressive effect. Salicin has no effect on Pbgl activity but causes a 30-fold induction of bgl operon expression. Induction depends on the activity of the BglF transporter/kinase. IS5 insertion has only a moderate effect on Pbgl but causes a much greater activation of the bgl operon expression by preventing the full repressive effects of H-NS and StpA. While several other transcription factors (BglJ, RcsB, and LeuO) have been reported to influence bgl operon transcription when overexpressed, they had little or no effect when present at wild type levels. These results indicate the important transcriptional regulatory mechanisms operative on the bgl operon in E. coli.

Theoretical insights into water network of B-DNA duplex with Watson-Crick and Hoogsteen base pairing geometries

Solvation is essential for the proper structure and functioning of biomolecules. It has a major role in establishing the B-form as the dominant structure of DNA and strongly affects its biological functions. In our genome, two modes of base pairing (Watson-Crick (WC) and Hoogsteen (HG)) exist in equilibrium and are related to the functional properties of the DNA including ligand recognition and gene regulation. Here, we use molecular dynamics simulation along with topological analysis of the H-bond network to investigate the hydration patterns of DNA structure with WC and HG base pairings. We showed that DNA most significantly disturbs the H-bonding properties of the first hydration shell and slightly the second shell compared to bulk water and that the water network is considerably different in the minor and major grooves. We found that HG base pairing leads to increased solvation of the major groove and a less ordered water structure in the minor groove accompanied with increasing hydrophobicity of the minor groove which could also contribute to the site recognition of specific DNA binding proteins. Our newly developed method to selectively study the solvation of the minor and major grooves opens the door to study in finer details the binding between various form of DNA and different ligands such drugs, RNA and proteins.

Abstract 2640: Targeted CUL4A inhibition synergizes with cisplatin to yield long-term survival in models of head and neck squamous cell carcinoma through a DDB2-mediated mechanism

Abstract Patients with late-stage and human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) continue to have a very poor prognosis. The development of more effective novel therapies that improve overall survival and overcome drug resistance is an urgent priority. We, and others, have reported that HNSCC tumors significantly overexpress NEDD8 and exhibit high sensitivity to the first-in-class NEDD8 activating enzyme (NAE) inhibitor pevonedistat. Here, we report that disruption of NEDD8-mediated protein turnover with pevonedistat dramatically augments cisplatin-induced DNA damage and apoptosis in HNSCC models. Further analysis revealed that the specific pevonedistat target, CUL4A, played an essential role in driving the synergy of the pevonedistat and cisplatin combination. Targeted inhibition of CUL4A resulted in significant downregulation in Damage Specific DNA binding protein 2 (DDB2), a DNA-damage recognition protein that promotes nucleotide excision repair and resistance to cisplatin. Silencing of CUL4A enhanced cisplatin-induced DNA damage and apoptosis in a manner similar to that of pevonedistat demonstrating that targeted inhibition of CUL4A may be a novel approach to augment cisplatin therapy. Administration of pevonedistat to mice bearing HNSCC tumors significantly decreased DDB2 expression in tumor cells, increased DNA damage and potently enhanced the activity of cisplatin to yield tumor regression and long-term survival of all animals. Our findings provide strong rationale for clinical investigation of CUL4A inhibition with pevonedistat as a novel strategy to augment the efficacy of cisplatin therapy for patients with HNSCC and identify loss of DDB2 as a key pharmacodynamic mediator controlling sensitivity to this regimen. Citation Format: Trace M. Jones, Claudia M. Espitia, Aikseng Ooi, Julie E. Bauman, Jennifer S. Carew, Steffan T. Nawrocki. Targeted CUL4A inhibition synergizes with cisplatin to yield long-term survival in models of head and neck squamous cell carcinoma through a DDB2-mediated mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2640.

The soybean PLATZ transcription factor GmPLATZ17 suppresses drought tolerance by interfering with stress-associated gene regulation of GmDREB5

Plant AT-rich sequence and zinc binding (PLATZ) transcription factors are a class of plant specific zinc-dependent DNA-binding proteins that function in abiotic stress response and plant development. In this study, 31 GmPLATZ genes were identified in soybean. GmPLATZ17 was down-regulated by drought and exogenous abscisic acid. Transgenic Arabidopsis and soybean hairy roots overexpressing GmPLATZ17 showed drought sensitivity and inhibition of stress-associated gene transcription. In contrast, suppressed expression of GmPLATZ17 led to increased drought tolerance in transgenic soybean hairy roots. The GmPLATZ17 protein was verified to interact physically with the GmDREB5 transcription factor, and overexpression of GmDREB5 increased drought tolerance in soybean hairy roots. Interaction of GmPLATZ17 with GmDREB5 was shown to interfere with the DRE-binding activity of GmDREB5, suppressing downstream stress-associated gene expression. These results show that GmPLATZ17 inhibits drought tolerance by interacting with GmDREB5. This study sheds light on PLATZ transcription factors and the function of GmPLATZ17 in regulating drought sensitivity.

Targeted CUL4A inhibition synergizes with cisplatin to yield long-term survival in models of head and neck squamous cell carcinoma through a DDB2-mediated mechanism

Patients with late-stage and human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) continue to have a very poor prognosis. The development of more effective novel therapies that improve overall survival and overcome drug resistance is an urgent priority. Here we report that HNSCC tumors significantly overexpress NEDD8 and exhibit high sensitivity to the first-in-class NEDD8-activating enzyme (NAE) inhibitor pevonedistat. Additional studies established that disruption of NEDD8-mediated protein turnover with pevonedistat dramatically augmented cisplatin-induced DNA damage and apoptosis in HNSCC models. Further analysis revealed that the specific pevonedistat target CUL4A played an essential role in driving the synergy of the pevonedistat and cisplatin combination. Targeted inhibition of CUL4A resulted in significant downregulation in Damage Specific DNA binding protein 2 (DDB2), a DNA-damage recognition protein that promotes nucleotide excision repair and resistance to cisplatin. Silencing of CUL4A or DDB2 enhanced cisplatin-induced DNA damage and apoptosis in a manner similar to that of pevonedistat demonstrating that targeted inhibition of CUL4A may be a novel approach to augment cisplatin therapy. Administration of pevonedistat to mice bearing HNSCC tumors significantly decreased DDB2 expression in tumor cells, increased DNA damage and potently enhanced the activity of cisplatin to yield tumor regression and long-term survival of all animals. Our findings provide strong rationale for clinical investigation of CUL4A inhibition with pevonedistat as a novel strategy to augment the efficacy of cisplatin therapy for patients with HNSCC and identify loss of DDB2 as a key pharmacodynamic mediator controlling sensitivity to this regimen.

Proteins That Read DNA Methylation.

Covalent modification of DNA via deposition of a methyl group at the 5' position on cytosine residues alters the chemical groups available for interaction in the major groove of DNA. This modification, thereby, alters the affinity and specificity of DNA-binding proteins; some of them favor interaction with methylated DNA, and others disfavor it. Molecular recognition of cytosine methylation by proteins often initiates sequential regulatory events that impact gene expression and chromatin structure. The known methyl-DNA-binding proteins have unique domains responsible for DNA methylation recognition: (1) the methyl-CpG-binding domain (MBD), (2) the SET- and RING finger-associated domain (SRA), and (3) some of TF families, such as the C2H2 zinc finger domain, basic helix-loop-helix (bHLH), basic leucine-zipper (bZIP), and homeodomain proteins. Structural analyses have revealed that each domain has a characteristic methylated DNA-binding pattern, and the difference in the recognition mechanisms renders the DNA methylation mark able to transmit complicated biological information. Recent genetic and genomic studies have revealed novel functions of methyl-DNA-binding proteins. These emerging data have also provided glimpses into how methyl-DNA-binding proteins possess unique features and, presumably, functions. In this chapter, we summarize structural and biochemical analyses elucidating the mechanisms for recognition of DNA methylation and correlate this information with emerging genomic and functional data.

An Inducible System for Silencing Establishment Reveals a Stepwise Mechanism in Which Anchoring at the Nuclear Periphery Precedes Heterochromatin Formation.

In eukaryotic cells, silent chromatin is mainly found at the nuclear periphery forming subnuclear compartments that favor silencing establishment. Here, we set up an inducible system to monitor silencing establishment at an ectopic locus in relation with its subnuclear localization in budding yeast. We previously showed that introducing LacI bound lacO arrays in proximity to gene flanked by HML silencers favors the recruitment of the yeast silencing complex SIR at this locus, leading to its silencing and anchoring at the nuclear periphery. Using an inducible version of this system, we show that silencing establishment is a stepwise process occurring over several cell cycles, with the progressive recruitment of the SIR complex. In contrast, we observed a rapid, SIR-independent perinuclear anchoring, induced by the high amount of LacI binding at the lacO array leading to nucleosome eviction at this array and to the phosphorylation of H2A in the neighboring nucleosomes by Mec1 kinase. While the initial phosphorylation of H2A (H2A-P) and perinuclear anchoring are independent of the SIR complex, its latter recruitment stabilizes H2A-P and reinforces the perinuclear anchoring. Finally, we showed that Sir3 spreading stabilizes nucleosomes and limits the access of specific DNA-binding protein to DNA.

RANK- NFATc1 signaling forms positive feedback loop on rank gene expression via functional NFATc1 responsive element in rank gene promoter

Receptor Activator of NF-κB (RANK) expressed on osteoclasts and their precursors is a receptor for RANK ligand (RANKL). Signals transduced by RANKL-RANK interaction induce genes essential for the differentiation and function of osteoclasts, partly through the direct binding of NFATc1, to target gene promoters. We have previously cloned a 6-kb fragment containing the 5′-flanking region of the mouse RANK gene and have demonstrated the presence of binding elements of hematological transcription factors, such as MITF, PU.1 and AP-1. Here, we demonstrated the presence of the functional NFATc1 responsive element on the RANK gene promoter. Transfection of an NFATc1-expression vector increased RANK mRNA that was subsequently nullified by NFATc1 knockdown. With the use of electrophoretic mobility shift assay (EMSA), an oligonucleotide (−388/-353) showed specific protein-DNA binding that was blockshifted with an anti-NFATc1 antibody and washed out with excess amounts of the cold consensus sequence. Co-transfection studies with the use of an NFATc1-expression vector and RANK promoter-reporter constructs showed that NFATc1 increased promoter activity 2-fold in RAW264.7 cells that was again nullified as disclosed by mutagenesis studies. Taken together, these results indicate that RANK transcription is positively regulated by the RANKL signal through the direct binding of NFATc1 to its specific binding site of the RANK gene promoter, and suggest the presence of a crucial positive feedback mechanism of gene expression that promotes accelerated terminal differentiation of RANK-positive committed precursors to mature osteoclasts.