RESUMO
High-throughput (HT) in vitro methods for measuring protein-DNA binding have become invaluable for characterizing transcription factor (TF) complexes and modeling gene regulation. However, current methods do not utilize endogenous proteins and, therefore, do not quantify the impact of cell-specific post-translational modifications (PTMs) and cooperative cofactors. We introduce the HT nextPBM (nuclear extract protein-binding microarray) approach to study DNA binding of native cellular TFs that accounts for PTMs and cell-specific cofactors. We integrate immune-depletion and phosphatase treatment steps into our nextPBM pipeline to characterize the impact of cofactors and phosphorylation on TF binding. We analyze binding of PU.1/SPI1 and IRF8 from human monocytes, delineate DNA-sequence determinants for their cooperativity, and show how PU.1 affinity correlates with enhancer status and the presence of cooperative and collaborative cofactors. We describe how nextPBMs, and our accompanying computational framework, can be used to discover cell-specific cofactors, screen for synthetic cooperative DNA elements, and characterize TF cooperativity.
Assuntos
Núcleo Celular/química , Redes Reguladoras de Genes , Análise Serial de Proteínas/métodos , Fatores de Transcrição/análise , Fatores de Transcrição/metabolismo , Extratos Celulares/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Especificidade de Órgãos/genética , Ligação Proteica , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Células THP-1RESUMO
Non-coding DNA variants (NCVs) impact gene expression by altering binding sites for regulatory complexes. New high-throughput methods are needed to characterize the impact of NCVs on regulatory complexes. We developed CASCADE (Customizable Approach to Survey Complex Assembly at DNA Elements), an array-based high-throughput method to profile cofactor (COF) recruitment. CASCADE identifies DNA-bound transcription factor-cofactor (TF-COF) complexes in nuclear extracts and quantifies the impact of NCVs on their binding. We demonstrate CASCADE sensitivity in characterizing condition-specific recruitment of COFs p300 and RBBP5 (MLL subunit) to the CXCL10 promoter in lipopolysaccharide (LPS)-stimulated human macrophages and quantify the impact of all possible NCVs. To demonstrate applicability to NCV screens, we profile TF-COF binding to ~1,700 single-nucleotide polymorphism quantitative trait loci (SNP-QTLs) in human macrophages and identify perturbed ETS domain-containing complexes. CASCADE will facilitate high-throughput testing of molecular mechanisms of NCVs for diverse biological applications.
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The effectiveness of cell-based therapy to treat muscle disease has been hampered by difficulties in isolating, maintaining and propagating the stem cells that are needed for treatment. Here we report the isolation of muscle-derived stem cells from both young and old mice and their propagation over extended periods of time in culture as "free-floating" myospheres. Analysis of these sphere-forming cells showed that they express stem cell antigen-1 (Sca-1), beta1 integrin (CD29), Thy-1 (CD90), and CD34, but did not express CD45, CD31, or myogenic markers (Pax7, Myf5, and MyoD). We found that cells derived from myospheres and then grown adherently (MDACs) behaved similar to primary myoblasts, in that these cells expressed myogenic markers and were able to easily form multinucleated myotubes. Unlike the parental myospheres but analogous to primary myoblasts, MDACs expressed Pax7, Myf5, and MyoD, indicating that the parent myosphere cells were a more primitive type of cell. In support of this we demonstrated that myospheres were also able to differentiate into adipogenic and osteogenic cells in culture, as well as being able to contribute to injured muscle in vivo. In summary, we report that primitive adult muscle stem cells can be easily isolated and sustained in culture as myospheres.
Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Mioblastos/citologia , Animais , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Mioblastos/metabolismoRESUMO
The type II nuclear receptors (NRs) function as heterodimeric transcription factors with the retinoid X receptor (RXR) to regulate diverse biological processes in response to endogenous ligands and therapeutic drugs. DNA-binding specificity has been proposed as a primary mechanism for NR gene regulatory specificity. Here we use protein-binding microarrays (PBMs) to comprehensively analyze the DNA binding of 12 NR:RXRα dimers. We find more promiscuous NR-DNA binding than has been reported, challenging the view that NR binding specificity is defined by half-site spacing. We show that NRs bind DNA using two distinct modes, explaining widespread NR binding to half-sites in vivo. Finally, we show that the current models of NR specificity better reflect binding-site activity rather than binding-site affinity. Our rich dataset and revised NR binding models provide a framework for understanding NR regulatory specificity and will facilitate more accurate analyses of genomic datasets.
Assuntos
DNA/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores X de Retinoides/metabolismo , Proteínas de Ligação a DNA , Humanos , Receptores X do Fígado , Mutagênese Sítio-Dirigida , Receptores Ativados por Proliferador de Peroxissomo , Receptor de Pregnano X , Receptores de Calcitriol , Receptores do Ácido Retinoico , Receptores dos Hormônios TireóideosRESUMO
Transcription factor NF-κB plays a central role in immunity from fruit flies to humans, and NF-κB activity is altered in many human diseases. To investigate a role for NF-κB in immunity and disease on a broader evolutionary scale we have characterized NF-κB in a sea anemone (Exaiptasia pallida; called Aiptasia herein) model for cnidarian symbiosis and dysbiosis (i.e., "bleaching"). We show that the DNA-binding site specificity of Aiptasia NF-κB is similar to NF-κB proteins from a broad expanse of organisms. Analyses of NF-κB and IκB kinase proteins from Aiptasia suggest that non-canonical NF-κB processing is an evolutionarily ancient pathway, which can be reconstituted in human cells. In Aiptasia, NF-κB protein levels, DNA-binding activity, and tissue expression increase when loss of the algal symbiont Symbiodinium is induced by heat or chemical treatment. Kinetic analysis of NF-κB levels following loss of symbiosis show that NF-κB levels increase only after Symbiodinium is cleared. Moreover, introduction of Symbiodinium into naïve Aiptasia larvae results in a decrease in NF-κB expression. Our results suggest that Symbiodinium suppresses NF-κB in order to enable establishment of symbiosis in Aiptasia. These results are the first to demonstrate a link between changes in the conserved immune regulatory protein NF-κB and cnidarian symbiotic status.
Assuntos
NF-kappa B/metabolismo , Anêmonas-do-Mar/metabolismo , Animais , DNA/metabolismo , Humanos , Simbiose/fisiologiaRESUMO
Protein-DNA binding is central to specificity in gene regulation, and methods for characterizing transcription factor (TF)-DNA binding remain crucial to studies of regulatory specificity. High-throughput (HT) technologies have revolutionized our ability to characterize protein-DNA binding by significantly increasing the number of binding measurements that can be performed. Protein-binding microarrays (PBMs) are a robust and powerful HT platform for studying DNA-binding specificity of TFs. Analysis of PBM-determined DNA-binding profiles has provided new insight into the scope and mechanisms of TF binding diversity. In this review, we focus specifically on the PBM technique and discuss its application to the study of TF specificity, in particular, the binding diversity of TF homologs and multi-protein complexes.
Assuntos
Complexos Multiproteicos/metabolismo , Análise Serial de Proteínas/métodos , Fatores de Transcrição/metabolismo , Animais , DNA/metabolismo , Humanos , Ligação Proteica , Isoformas de Proteínas/metabolismoRESUMO
NF-κB transcription factors control a wide array of important cellular and organismal processes in eukaryotes. All NF-κB transcription factors bind to DNA target sites as dimers. In vertebrates, there are five NF-κB subunits, p50, p52, RelA (p65), c-Rel, and RelB, that can form almost all combinations of homodimers and heterodimers, which recognize distinct, but overlapping, target sequences. In this chapter, we describe the use of protein-binding microarrays (PBMs), a high-throughput method to measure the binding of proteins to different DNA sequences. PBM datasets allow for sensitive comparisons of NF-κB dimer DNA-binding differences and can aid in the computational and experimental prediction of NF-κB target genes.
Assuntos
Sítios de Ligação , DNA/genética , DNA/metabolismo , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Ligação ProteicaRESUMO
A myosphere cell is a unique type of muscle stem cell that is able to maintain its pre-myogenic state in culture over time. These cells are propagated in culture as free-floating, non-adherent spheres. We believe that the 3-dimensional adhesive cell-cell interactions involved in maintaining the sphere-like myosphere structures are also involved in maintaining their longevity in culture. We found that Sca-1, which is highly expressed by myosphere cells, plays a role in the growth and the formation of the myospheres. In comparing adhesion molecules expressed by 3-dimensionally grown myosphere cells to those expressed by 2-dimensionally grown primary myoblasts, we found that there was a distinct difference in the expression of ß3 integrin. Upon further investigation we discovered that there is an adhesive interaction between Sca-1(+) cells and αVß3 integrin. Here we show that Sca-1(+) cells (myosphere cells and NIH3T3 cells) adhere to αVß3 integrin and that Sca-1(-) cells (primary myoblasts) do not adhere. The interaction between Sca-1 and αVß3 integrin was confirmed using antibody blocking, shRNA knockdown of Sca-1 in Sca-1(+) cells, and by expressing Sca-1 cDNA in Sca-1(-) cells, which demonstrated that the level of adhesion of these cells to αVß3 integrin was dependent on the presence of Sca-1. Additionally, we found that the co-expression of Sca-1 and ß3 resulted in significantly greater adhesion of Sca-1(+) cells to αVß3 integrin. In conclusion, our data indicate that Sca-1 is involved in maintaining the 3-dimensional myosphere cell-cell contacts and that Sca-1 is involved in the binding of cells to αVß3 integrin.
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Ligament and tendon repair is an important topic in orthopedic tissue engineering; however, the cell source for tissue regeneration has been a controversial issue. Until now, scientists have been split between the use of primary ligament fibroblasts or marrow-derived mesenchymal stem cells (MSCs). The objective of this study was to show that a co-culture of anterior cruciate ligament (ACL) cells and MSCs has a beneficial effect on ligament regeneration that is not observed when utilizing either cell source independently. Autologous ACL cells (ACLcs) and MSCs were isolated from Yorkshire pigs, expanded in vitro, and cultured in multiwell plates in varying %ACLcs/%MSCs ratios (100/0, 75/25, 50/50, 25/75, and 0/100) for 2 and 4 weeks. Quantitative mRNA expression analysis and immunofluorescent staining for ligament markers Collagen type I (Collagen-I), Collagen type III (Collagen-III), and Tenascin-C were performed. We show that Collagen-I and Tenascin-C expression is significantly enhanced over time in 50/50 co-cultures of ACLcs and MSCs (p≤0.03), but not in other groups. In addition, Collagen-III expression was significantly greater in MSC-only cultures (p≤0.03), but the Collagen-I-to-Collagen-III ratio in 50% co-culture was closest to native ligament levels. Finally, Tenascin-C expression at 4 weeks was significantly higher (p≤0.02) in ACLcs and 50% co-culture groups compared to all others. Immunofluorescent staining results support our mRNA expression data. Overall, 50/50 co-cultures had the highest Collagen-I and Tenascin-C expression, and the highest Collagen-I-to-Collagen-III ratio. Thus, we conclude that using a 50% co-culture of ACLcs and MSCs, instead of either cell population alone, may better maintain or even enhance ligament marker expression and improve healing.