ABSTRACT
Gene duplication results in two identical paralogs that diverge through mutation, leading to loss or gain of interactions with other biomolecules. Here, we comprehensively characterize such network rewiring for C. elegans transcription factors (TFs) within and across four newly delineated molecular networks. Remarkably, we find that even highly similar TFs often have different interaction degrees and partners. In addition, we find that most TF families have a member that is highly connected in multiple networks. Further, different TF families have opposing correlations between network connectivity and phylogenetic age, suggesting that they are subject to different evolutionary pressures. Finally, TFs that have similar partners in one network generally do not in another, indicating a lack of pressure to retain cross-network similarity. Our multiparameter analyses provide unique insights into the evolutionary dynamics that shaped TF networks.
Subject(s)
Caenorhabditis elegans Proteins/physiology , Caenorhabditis elegans/genetics , Gene Expression Regulation , Gene Regulatory Networks , Transcription Factors/physiology , Animals , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Evolution, Molecular , Phylogeny , Promoter Regions, Genetic , Transcription Factors/metabolismABSTRACT
How tumour suppressor p53 bifurcates cell cycle arrest and apoptosis and executes these distinct pathways is not clearly understood. We show that BAX and PUMA promoters harbour an identical MAR element and are transcriptional targets of SMAR1. On mild DNA damage, SMAR1 selectively represses BAX and PUMA through binding to the MAR independently of inducing p53 deacetylation through HDAC1. This generates an anti-apoptotic response leading to cell cycle arrest. Importantly, knockdown of SMAR1 induces apoptosis, which is abrogated in the absence of p53. Conversely, apoptotic DNA damage results in increased size and number of promyelocytic leukaemia (PML) nuclear bodies with consequent sequestration of SMAR1. This facilitates p53 acetylation and restricts SMAR1 binding to BAX and PUMA MAR leading to apoptosis. Thus, our study establishes MAR as a damage responsive cis element and SMAR1-PML crosstalk as a switch that modulates the decision between cell cycle arrest and apoptosis in response to DNA damage.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Apoptosis/physiology , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Matrix Attachment Regions , Nuclear Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , bcl-2-Associated X Protein/metabolism , Acetylation , Animals , Apoptosis/genetics , Apoptosis Regulatory Proteins/genetics , Base Sequence , Cell Cycle , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Line , DNA/genetics , DNA/metabolism , DNA Damage , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , Histone Deacetylase 1/metabolism , Humans , Mice , Models, Biological , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Promoter Regions, Genetic , Promyelocytic Leukemia Protein , Proto-Oncogene Proteins/genetics , RNA, Small Interfering/genetics , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic , Tumor Suppressor Proteins/antagonists & inhibitors , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , bcl-2-Associated X Protein/geneticsABSTRACT
A major challenge in systems biology is to understand the gene regulatory networks that drive development, physiology and pathology. Interactions between transcription factors and regulatory genomic regions provide the first level of gene control. Gateway-compatible yeast one-hybrid (Y1H) assays present a convenient method to identify and characterize the repertoire of transcription factors that can bind a DNA sequence of interest. To delineate genome-scale regulatory networks, however, large sets of DNA fragments need to be processed at high throughput and high coverage. Here we present enhanced Y1H (eY1H) assays that use a robotic mating platform with a set of improved Y1H reagents and automated readout quantification. We demonstrate that eY1H assays provide excellent coverage and identify interacting transcription factors for multiple DNA fragments in a short time. eY1H assays will be an important tool for mapping gene regulatory networks in Caenorhabditis elegans and other model organisms as well as in humans.
Subject(s)
Gene Regulatory Networks , High-Throughput Screening Assays , Two-Hybrid System Techniques , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , DNA/genetics , Gene Expression Regulation , Humans , Reproducibility of Results , Systems Biology , Transcription Factors/metabolismABSTRACT
BACKGROUND: GAD65 (Glutamic acid decarboxylase 65 KDa isoform) is one of the most important auto-antigens involved in Type 1 diabetes induction. Although it serves as one of the first injury markers of ß-islets, the mechanisms governing GAD65 expression remain poorly understood. Since the regulation of GAD65 is crucial for the proper functioning of insulin secreting cells, we investigated the stress induced regulation of GAD65 transcription. RESULTS: The present study shows that SMAR1 regulates GAD65 expression at the transcription level. Using a novel protein-DNA pull-down assay, we show that SMAR1 binding is very specific to GAD65 promoter but not to the other isoform, GAD67. We show that Streptozotocin (STZ) mediated DNA damage leads to upregulation of SMAR1 and p53 expression, resulting in elevated levels of GAD65, in both cell lines as well as mouse ß-islets. SMAR1 and p53 act synergistically to up-regulate GAD65 expression upon STZ treatment. CONCLUSION: We propose a novel mechanism of GAD65 regulation by synergistic activities of SMAR1 and p53.
Subject(s)
Cell Cycle Proteins/metabolism , DNA Damage , DNA-Binding Proteins/metabolism , DNA/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Glutamate Decarboxylase/metabolism , Nuclear Proteins/metabolism , Streptozocin/pharmacology , Tumor Suppressor Protein p53/metabolism , Animals , Base Sequence , Cell Line , Chromatin Immunoprecipitation , DNA Primers/genetics , Electrophoretic Mobility Shift Assay , Flow Cytometry , Gene Expression Regulation, Enzymologic/genetics , Glutamate Decarboxylase/genetics , Immunoblotting , Luciferases , Mice , Molecular Sequence Data , Promoter Regions, Genetic/genetics , Protein Isoforms/metabolism , Sequence AlignmentABSTRACT
An amendment to this paper has been published and can be accessed via the original article.
ABSTRACT
Tetrapeptides derived from glycine and beta-alanine were hooked at the C-3beta position of the modified cholic acid to realize novel linear tetrapeptide-linked cholic acid derivatives. All the synthesized compounds were tested against a wide variety of microorganisms (gram-negative bacteria, gram-positive bacteria and fungi) and their cytotoxicity was evaluated against human embryonic kidney (HEK293) and human mammary adenocarcinoma (MCF-7) cell lines. While relatively inactive by themselves, these compounds interact synergistically with antibiotics such as fluconazole and erythromycin to inhibit growth of fungi and bacteria, respectively, at 1-24 microg/mL. The synergistic effect shown by our novel compounds is due to their inherent amphiphilicity. The fractional inhibitory concentrations reported are comparable to those reported for Polymyxin B derivatives.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Cholic Acid/chemistry , Peptides/chemistry , Anti-Bacterial Agents/pharmacology , Cell Line , Cell Line, Tumor , Chemistry, Pharmaceutical/methods , Drug Design , Erythromycin/pharmacology , Fluconazole/pharmacology , Glycine/chemistry , Humans , Models, Chemical , Molecular Conformation , Polymyxin B/analogs & derivatives , Polymyxin B/pharmacology , beta-Alanine/chemistryABSTRACT
We report herein the synthesis and biological evaluation of bile acid dimers linked through 1,2,3-triazole and bis-beta-lactam. The dimers were synthesized using 1,3-dipolar cycloaddition reaction of diazido bis-beta-lactams , and terminal alkynes derived from cholic acid/deoxycholic acid in the presence of Cu(i) catalyst (click chemistry). These novel molecules were evaluated in vitro for their antifungal and antibacterial activity. Most of the compounds exhibited significant antifungal as well as antibacterial activity against all the tested fungal and bacterial strains. Moreover, their in vitro cytotoxicities towards HEK-293 and MCF-7 cells were also established.