Purification of Sequence‐Specific <scp>DNA</scp>‐Binding Proteins by Affinity Chromatography

Kerrigan, Leslie A.; Kadonaga, James T.

doi:10.1002/0471142727.mb1210s24

Cited by 7 publications

(7 citation statements)

References 26 publications

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“…Aliquots of 0.50 mg of GSTA1 ARE half-site (5Ј-GATCTAAT-GGTGACAAAGCAACTT-3Ј) and complementary sequence were annealed, phosphorylated with polynucleotide kinase, and filled-in with Klenow to provide a site-specific sequence suitable for affinity chromatography. DNA was purified and ligated as described by Kerrigan and Kadonaga (1993). Extracted DNA was coupled to a 1-ml N-hydroxysuccinamide-activated agarose Hi-trap column (Amersham Biosciences) for 30 min at 4°C in coupling buffer (0.2 M NaHCO 3 and 0.5 M NaCl, pH 8.3).…”

Section: Methodsmentioning

confidence: 99%

Identification of Albumin Precursor Protein, Phi AP3, and α-Smooth Muscle Actin as Novel Components of Redox Sensing Machinery in Vascular Smooth Muscle Cells

et al. 2002

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Aerobic organisms are continually subjected to environmental stressors that compromise redox homeostasis and induce cellular injury. In vascular smooth muscle cells (vSMCs), the activation/repression of redox-regulated genes after environmental stress often involves protein binding to cis-acting antioxidant response elements (AREs). The present study was conducted to identify proteins that participate in redox-regulated protein binding to human c-Ha-ras and mouse glutathione S-transferase A1 AREs in vSMCs after oxidant injury. Challenge of vSMCs with 0.3 or 3 M hydrogen peroxide, 3-methylcholanthrene, benzo[a]pyrene-7,8-diol, 3-hydroxy benzo[a]pyrene, and benzo[a]pyrene-3,6-quinone induced concentration-related increases in ARE protein binding. The profiles of ARE complex assembly were comparable, but exhibited chemical specificity. Pretreatment with 0.5 mM N-acetylcysteine inhibited activation of ARE protein binding in hydrogen peroxidetreated cells. Preparative electrophoretic mobility shift assays coupled to Western analysis identified NF-E2-related proteins 1 and 2 and JunD in complexes assembled on AREs. Polyethylenimine affinity and sequence-specific serial immobilized DNA affinity chromatography followed by N-terminal sequencing identified albumin precursor protein, phi AP3, and ␣-smooth muscle actin as members of the ARE signaling pathway. Sequence analysis of albumin protein revealed homology to the redox-regulated transcription factors Bach1 and 2, as well as cytoskeletal and molecular motor proteins. These results implicate albumin precursor protein, phi AP3, and ␣-smooth muscle actin as participants in redox sensing in vSMCs, and suggest that protein complex assembly involves interactions between leucine zipper and zinc finger transcription factors with cytoskeletal proteins.

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Section: Methodsmentioning

confidence: 99%

Identification of Albumin Precursor Protein, Phi AP3, and α-Smooth Muscle Actin as Novel Components of Redox Sensing Machinery in Vascular Smooth Muscle Cells

et al. 2002

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“…31 As a control, oligonucleotides containing the STAT3 binding site (5Ј-TCGACTCGTTCCCAGCAGCAC-3Ј) were coupled to the beads. A quantity of 100 L nuclear extracts was added to 100 L of 1x binding buffer (4% ficoll, 20 mM HEPES, pH 7.9, 50 mM KCl, 1 mM ethylenediaminetetraacetic acid [EDTA], 1 mM dithiothreitol [DTT], 0.25 mg/mL bovine serum albumin [BSA]) and precleared with 10 L of sepharose 4B beads at 4°C for 1 hour.…”

Section: Precipitation Of C-myb and Pax-5 By Dna-sepharosementioning

confidence: 99%

Cooperative binding of c-Myb and Pax-5 activates theRAG-2 promoter in immature B cells

Kishi

Jin

Wei

et al. 2002

Blood

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The recombination activating gene-1 (RAG-1) and RAG-2 are expressed specifically in immature lymphoid cells undergoing the recombination of antigen receptor genes. The regulation of murine RAG-2 promoter was studied and it was revealed that the ؊41/؊17 RAG-2 promoter region, which is conserved between humans and mice, was indispensable for the RAG-2 promoter activity in B IntroductionImmunoglobulin (Ig) and T-cell receptor (TCR) variable genes consist of germline variable (V), diversity (D), and joining (J) segments and are assembled during lymphocyte development by V(D)J recombination. The recombination activating gene-1 (RAG-1) and RAG-2 encode the essential and lymphocyte-specific components of V(D)J recombination machinery. Their products are sufficient for the recognition and initial cleavage of DNA containing recombination signal sequences that flank each coding segment. 1,2 During lymphocyte development, expression of RAG-1 and RAG-2 is tightly regulated. RAG genes are expressed in immature B-or T-lineage cells undergoing Ig or TCR gene rearrangements. [3][4][5][6] The failure of functional expression of RAG causes a defect in the formation of the functional antigen receptor of lymphocytes, and hence causes the block of lymphocyte development in mice and humans. [7][8][9][10] The transcription of RAG is regulated at different levels. At the chromatin level, Fuller and Storb 11 and Kitagawa et al 12 have demonstrated that alteration of chromatin structure detected by DNase I hypersensitivity was noted in the promoter region of mouse and human RAG-1 only in RAG-expressing lymphocytes, indicating that chromatin remodeling is one of the mechanisms for regulating RAG expression. At the cis-element level, Yu et al have demonstrated that about 10 kb 5Ј upstream region of RAG-2 is necessary for the expression of RAG in B-lineage cells and in CD4 Ϫ CD8 Ϫ thymocytes, and that further upstream region is required for the expression of RAG in CD4 ϩ CD8 ϩ thymocytes. 13 Monroe et al have demonstrated that about 10 kb 5Ј upstream region of RAG-2 is enough for rescuing B-and T-cell development in RAG2 Ϫ/Ϫ mice by using RAG-2 Ϫ/Ϫ blastocyst complementation. 14 These results suggest that expression of RAG is regulated by the cis-element, such as enhancer. At the promoter level, it was reported that the human RAG-1 promoter region does not confer the lymphocyte-specific expression of RAG-1. 12,[15][16][17] Regarding the promoter of RAG-2, about 300 bp 5Ј upstream region from the major transcription initiation site of mouse RAG-2 is conserved between mice and humans, [17][18][19] indicating that this region is important for the promoter activity of RAG-2. It was also demonstrated that the human RAG-2 promoter is activated not only in lymphoid cells but also in nonlymphoid cells. 17 Materials and methods Cells and cell cultureThe 18.8.1 pre-B cell line 18 and the BAL17 B cell line, 18 both of which express endogenous murine RAG-1 and RAG-2, were cultured in RPMI 1640 supplemented with 10% fetal calf serum, 100 U/mL penicill...

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“…Transcription factors that bind to specific promoters only account for Ͻ0.01% of the total cellular protein (27). Thus, the low abundance of transcription factors necessitates purification from nuclear extracts prepared from a large number of cultured cells to achieve the 10,000-to 100,000-fold enrichment, which is required to obtain sufficient amounts of protein for further chemical and functional analyses (28).…”

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confidence: 99%

Complementary Quantitative Proteomics Reveals that Transcription Factor AP-4 Mediates E-box-dependent Complex Formation for Transcriptional Repression of HDM2>

Chiu

Chen

et al. 2009

Molecular & Cellular Proteomics

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Transcription factor activating enhancer-binding protein 4 (AP-4) is a basic helix-loop-helix protein that binds to E-box elements. AP-4 has received increasing attention for its regulatory role in cell growth and development, including transcriptional repression of the human homolog of murine double minute 2 (HDM2), an important oncoprotein controlling cell growth and survival, by an unknown mechanism. Here we demonstrate that AP-4 binds to an E-box located in the HDM2-P2 promoter and represses HDM2 transcription in a p53-independent manner. Incremental truncations of AP-4 revealed that the C-terminal Gln/Pro-rich domain was essential for transcriptional repression of HDM2. To further delineate the molecular mechanism(s) of AP-4 transcriptional control and its potential implications, we used DNA-affinity purification followed by complementary quantitative proteomics, cICAT and iTRAQ labeling methods, to identify a previously unknown E-box-bound AP-4 protein complex containing 75 putative components. The two labeling methods complementarily quantified differentially AP-4-enriched proteins, including the most significant recruitment of DNA damage response proteins, followed by transcription factors, transcriptional repressors/corepressors, and histone-modifying proteins. Specific interaction of AP-4 with CCCTC binding factor, stimulatory protein 1, and histone deacetylase 1 (an AP-4 corepressor) was validated using AP-4 truncation mutants. Importantly, inclusion of trichostatin A did not alleviate AP-4-mediated repression of HDM2 transcription, suggesting a previously unidentified histone deacetylase-independent repression mechanism. In contrast, the complementary quantitative proteomics study suggested that transcription repression occurs via coordination of AP-4 with other transcription factors, histone methyltransferases, and/or a nucleosome remodeling SWI.SNF complex. In addition to previously known functions of AP-4, our data suggest that AP-4 participates in a transcriptional-regulating complex at the HDM2-P2 promoter in response to DNA damage.

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Purification of Sequence‐Specific DNA‐Binding Proteins by Affinity Chromatography

Cited by 7 publications

References 26 publications

Identification of Albumin Precursor Protein, Phi AP3, and α-Smooth Muscle Actin as Novel Components of Redox Sensing Machinery in Vascular Smooth Muscle Cells

Identification of Albumin Precursor Protein, Phi AP3, and α-Smooth Muscle Actin as Novel Components of Redox Sensing Machinery in Vascular Smooth Muscle Cells

Cooperative binding of c-Myb and Pax-5 activates theRAG-2 promoter in immature B cells

Complementary Quantitative Proteomics Reveals that Transcription Factor AP-4 Mediates E-box-dependent Complex Formation for Transcriptional Repression of HDM2>

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