Asymmetric Recognition of Nonconsensus AP-1 Sites by Fos-Jun and Jun-Jun Influences Transcriptional Cooperativity with NFAT1

ABSTRACT Many regulatory elements in eukaryotic promoters do not correspond to optimal recognition sequences for the transcription factors that regulate promoter function by binding to the elements. The sequence of the binding site may influence the structural and functional properties of regulatory protein complexes. Fos-Jun heterodimers were found to bind nonconsensus AP-1 sites in a preferred orientation. Oriented Fos-Jun heterodimer binding was attributed to nonidentical recognition of the two half-sites by Fos and Jun. Jun bound preferentially to the consensus half-site, whereas Fos was able to bind nonconsensus half-sites. The orientation of heterodimer binding affected the transcriptional cooperativity of Fos-Jun-NFAT1 complexes at composite regulatory elements in mammalian cells. Jun dimerization with Fos versus ATF2 caused it to bind opposite half-sites at nonconsensus AP-1 elements. Similarly, ATF2 bound to opposite half-sites in Fos-ATF2-NFAT1 and ATF2-Jun-NFAT1 complexes. The orientations of nonconsensus AP-1 sites within composite regulatory elements affected the cooperativity of Fos-Jun as well as Jun-Jun binding with NFAT1. Since Jun homodimers cannot bind to AP-1 sites in a preferred orientation, the effects of the orientations of nonconsensus AP-1 sites on the stabilities of Jun-Jun-NFAT1 complexes are likely to be due to asymmetric conformational changes in the two subunits of the homodimer. Nonconsensus AP-1 site orientation also affected the synergy of transcription activation between Jun homodimers and NFAT1 at composite regulatory elements. The asymmetric recognition of nonconsensus AP-1 sites can therefore influence the transcriptional activities of Fos and Jun both through effects on the orientation of heterodimer binding and through differential conformational changes in the two subunits of the dimer.

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Regulation of Clathrin-Mediated Endocytosis

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-062917-012644 ◽

2018 ◽

Vol 87 (1) ◽

pp. 871-896 ◽

Cited By ~ 108

Author(s):

Marcel Mettlen ◽

Ping-Hung Chen ◽

Saipraveen Srinivasan ◽

Gaudenz Danuser ◽

Sandra L. Schmid

Keyword(s):

Conformational Changes ◽

Posttranslational Modifications ◽

Mammalian Cells ◽

Environmental Changes ◽

Protein Complexes ◽

Adaptor Protein ◽

Endocytic Pathway ◽

Coat Proteins ◽

Membrane Composition ◽

Coated Pits

Clathrin-mediated endocytosis (CME) is the major endocytic pathway in mammalian cells. It is responsible for the uptake of transmembrane receptors and transporters, for remodeling plasma membrane composition in response to environmental changes, and for regulating cell surface signaling. CME occurs via the assembly and maturation of clathrin-coated pits that concentrate cargo as they invaginate and pinch off to form clathrin-coated vesicles. In addition to the major coat proteins, clathrin triskelia and adaptor protein complexes, CME requires a myriad of endocytic accessory proteins and phosphatidylinositol lipids. CME is regulated at multiple steps—initiation, cargo selection, maturation, and fission—and is monitored by an endocytic checkpoint that induces disassembly of defective pits. Regulation occurs via posttranslational modifications, allosteric conformational changes, and isoform and splice-variant differences among components of the CME machinery, including the GTPase dynamin. This review summarizes recent findings on the regulation of CME and the evolution of this complex process.

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Identification of regulatory regions and regulatory protein complexes of the Spirulina desD gene under temperature stress conditions: Role of thioredoxin as an inactivator of a transcriptional repressor GntR under low-temperature stress

Biochemistry and Cell Biology ◽

10.1139/o2012-017 ◽

2012 ◽

Vol 90 (5) ◽

pp. 621-635 ◽

Cited By ~ 2

Author(s):

Pavinee Kurdrid ◽

Phuttawadee Phuengcharoen ◽

Rayakorn Yutthanasirikul ◽

Sittiruk Roytrakul ◽

Atchara Paemanee ◽

...

Keyword(s):

Low Temperature ◽

Temperature Stress ◽

Protein Complex ◽

Protein Complexes ◽

Regulatory Protein ◽

Regulatory Elements ◽

Stress Conditions ◽

Upstream Region ◽

Regulatory Regions ◽

Temperature Responsive

In the present study, electrophoretic mobility shift assays were used to identify temperature responsive elements in the 5′ upstream region (5′ UTR) of the Spirulina desD gene. Overlapping, synthetic oligonucleotides of both sense and anti-sense strands that spanned the entire 5′ UTR of the gene were analyzed. The responsive DNA-binding protein complexes were identified using liquid chromatography–tandem mass spectrometry. The results indicated that the cold-responsive elements were located at –453 to –247, –197 to –151, –105 to –76, and –50 to –1, whereas the low-temperature specific regulatory regions were located at –372 to –352. Moreover, the heat-responsive elements were located at –347 to –243, –197 to –151, and –124 to –1, whereas the high-temperature specific elements were located between –130 to –101 and –30 to –1. In terms of regulatory protein complexes under the two stress conditions, Trx was only detected in the low-temperature responsive protein complex, and divalent cations were essential for the binding of the protein complex to the regulatory elements. Furthermore, Trx was shown to play a critical role as a reducing agent that inactivates the Spirulina desD repressor, GntR. Consequently, the desD gene expression is induced under the low-temperature condition.

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Genetic information from discordant sibling pairs points to ESRP2 as a candidate trans-acting regulator of the CF modifier gene SCNN1B

Scientific Reports ◽

10.1038/s41598-020-79804-y ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Tim Becker ◽

Andreas Pich ◽

Stephanie Tamm ◽

Silke Hedtfeld ◽

Mohammed Ibrahim ◽

...

Keyword(s):

Multiple Testing ◽

Genomic Sequence ◽

Protein Complexes ◽

Regulatory Protein ◽

Regulatory Elements ◽

Snp Markers ◽

Mass Spectrometry Data ◽

Mobility Shift ◽

Sibling Pairs ◽

Protein Mass

AbstractSCNN1B encodes the beta subunit of the epithelial sodium channel ENaC. Previously, we reported an association between SNP markers of SCNN1B gene and disease severity in cystic fibrosis-affected sibling pairs. We hypothesized that factors interacting with the SCNN1B genomic sequence are responsible for intrapair discordance. Concordant and discordant pairs differed at six SCNN1B markers (Praw = 0.0075, Pcorr = 0.0397 corrected for multiple testing). To identify the factors binding to these six SCNN1B SNPs, we performed an electrophoretic mobility shift assay and captured the DNA–protein complexes. Based on protein mass spectrometry data, the epithelial splicing regulatory protein ESRP2 was identified when using SCNN1B-derived probes and the ESRP2-SCNN1B interaction was independently confirmed by coimmunoprecipitation assays. We observed an alternative SCNN1B transcript and demonstrated in 16HBE14o− cells that levels of this transcript are decreased upon ESRP2 silencing by siRNA. Furthermore, we confirmed that mildly and severely affected siblings have different ESPR2 genetic backgrounds and that ESRP2 markers are linked to the response of CF patients’ nasal epithelium to amiloride, indicating ENaC involvement (Pbest = 0.0131, Pcorr = 0.068 for multiple testing). Our findings demonstrate that sibling pairs clinically discordant for CF can be used to identify meaningful DNA regulatory elements and interacting factors.

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CXCR7 heterodimerizes with CXCR4 and regulates CXCL12-mediated G protein signaling

Blood ◽

10.1182/blood-2008-12-196618 ◽

2009 ◽

Vol 113 (24) ◽

pp. 6085-6093 ◽

Cited By ~ 385

Author(s):

Angélique Levoye ◽

Karl Balabanian ◽

Françoise Baleux ◽

Françoise Bachelerie ◽

Bernard Lagane

Keyword(s):

Conformational Changes ◽

Mammalian Cells ◽

Protein Complexes ◽

G Protein Signaling ◽

Protein Signaling ◽

Protein Activation ◽

Conformational Rearrangements ◽

Stromal Cell Derived Factor ◽

Pathologic Processes ◽

Gαi Protein

AbstractThe stromal cell-derived factor-1/CXCL12 chemokine engages the CXCR4 and CXCR7 receptors and regulates homeostatic and pathologic processes, including organogenesis, leukocyte homeostasis, and tumorigenesis. Both receptors are widely expressed in mammalian cells, but how they cooperate to respond to CXCL12 is not well understood. Here, we show that CXCR7 per se does not trigger Gαi protein–dependent signaling, although energy transfer assays indicate that it constitutively interacts with Gαi proteins and undergoes CXCL12-mediated conformational changes. Moreover, when CXCR4 and CXCR7 are coexpressed, we show that receptor heterodimers form as efficiently as receptor homodimers, thus opening the possibility that CXCR4/CXCR7 heterodimer formation has consequences on CXCL12-mediated signals. Indeed, expression of CXCR7 induces conformational rearrangements within preassembled CXCR4/Gαi protein complexes and impairs CXCR4-promoted Gαi-protein activation and calcium responses. Varying CXCR7 expression levels and blocking CXCL12/CXCR7 interactions in primary T cells suggest that CXCR4/CXCR7 heterodimers form in primary lymphocytes and regulate CXCL12-promoted chemotaxis. Taken together, these results identify CXCR4/CXCR7 heterodimers as distinct functional units with novel properties, which can contribute to the functional plasticity of CXCL12.

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A Winged-Helix Family Member Is Involved in a Steroid Hormone-Triggered Regulatory Circuit**This work was supported by NIH Grants RO1-DK-40082 (to M.M.S.) and T32-DK-0703 (to D.M.D.).

Endocrinology ◽

10.1210/endo.139.12.6363 ◽

1998 ◽

Vol 139 (12) ◽

pp. 4967-4975 ◽

Cited By ~ 2

Author(s):

Diane M. Dean ◽

Ryan R. Berger ◽

Michel M. Sanders

Keyword(s):

Transcription Factors ◽

Steroid Hormone ◽

Regulatory Element ◽

Regulatory Protein ◽

Regulatory Elements ◽

Response To Treatment ◽

Winged Helix ◽

Sequence Recognition ◽

Steroid Dependent ◽

Essential Components

Abstract A common theme emerging in eukaryotic gene regulation is that maximal gene induction requires several transcription factors acting in concert to regulate the activation of critical genes. Increasingly, nuclear receptors play key roles in orchestrating this regulation, often by integrating additional signaling pathways, through complex regulatory elements known as hormone response units. The ovalbumin gene contains one such unit, known as the steroid-dependent regulatory element. The binding of the chicken ovalbumin induced regulatory protein-I (Chirp-I) to this element occurs only in response to treatment with estrogen and glucocorticoid. Evidence presented herein demonstrates that Chirp-I has many features in common with the winged-helix (W-H) family of transcription factors. The binding sites for Chirp-I and for the W-H proteins have similar sequence recognition requirements. Northern blots establish that members of the W-H family are expressed in oviduct. Most convincing, the Chirp-I complex interacts with two different antibodies specific to W-H family members. The culmination of this work supports the hypothesis that Chirp-I is a member of the W-H family, and it lends credence to the idea that W-H proteins are essential components of some steroid hormone regulatory circuits.

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Regulatory elements in the FBP1 promoter respond differently to glucose-dependent signals in Saccharomyces cerevisiae

Biochemical Journal ◽

10.1042/bj3590193 ◽

2001 ◽

Vol 359 (1) ◽

pp. 193-201 ◽

Cited By ~ 8

Author(s):

Oscar ZARAGOZA ◽

Olivier VINCENT ◽

Juana M. GANCEDO

Keyword(s):

Saccharomyces Cerevisiae ◽

Carbon Source ◽

Fusion Gene ◽

Protein Complexes ◽

Regulatory Protein ◽

Regulatory System ◽

Regulatory Elements ◽

Nuclear Extracts ◽

Fructose 1,6 Bisphosphatase

In Saccharomyces cerevisiae expression of the fructose-1,6-bisphosphatase-encoding gene, FBP1, is controlled by glucose through the upstream activating sequences UAS1 and UAS2 and the upstream repressing sequence URS1 in its promoter. We have studied the regulation of the proteins that could bind to these elements. We have investigated the role of the putative transcription factors Cat8 and Sip4 in the formation of specific DNA–protein complexes with UAS1 and UAS2, and in the expression of UAS1-lacZ and UAS2-lacZ. The expression of CAT8-lacZ and SIP4-lacZ has been also measured in mig1, tup1 or hxk2 mutants, partially refractory to catabolite repression. We conclude that there is no strict correlation between Cat8 and Sip4 expression or in vitro formation of DNA–protein complexes and expression of UAS1-lacZ and UAS2-lacZ. The URS1 element binds the regulatory protein Mig1, which blocks transcription by recruiting the proteins Cyc8 and Tup1. The pattern of complexes of URS1 with nuclear extracts was dependent on the carbon source and on Cyc8, but not on Tup1; it was also affected by the protein kinase Snf1 and by the exportin Msn5. The repression caused by URS1 in a fusion gene was dependent on Mig1, Cyc8 and Tup1, and on the carbon source in the medium; in a snf1 strain the repression observed was independent of the carbon source. Expression of Mig1 could occur in the absence of Snf1 and was moderately sensitive to glucose. We present data showing that different elements of the regulatory system controlling FBP1 responded differently to the concentration of glucose in the medium.

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Identification of candidate master transcription factors within enhancer-centric transcriptional regulatory networks

10.1101/345413 ◽

2018 ◽

Cited By ~ 1

Author(s):

Alexander J. Federation ◽

Donald R. Polaski ◽

Christopher J. Ott ◽

Angela Fan ◽

Charles Y. Lin ◽

...

Keyword(s):

Transcription Factors ◽

Mammalian Cells ◽

Regulatory Networks ◽

Transcriptional Regulatory Network ◽

Regulation Of Gene Expression ◽

Cell Types ◽

Regulatory Elements ◽

Lymphocytic Leukemia ◽

Comparable Efficacy ◽

Transcriptional Regulatory

AbstractRegulation of gene expression through binding of transcription factors (TFs) to cis-regulatory elements is highly complex in mammalian cells. Genome-wide measurement technologies provide new means to understand this regulation, and models of TF regulatory networks have been built with the goal of identifying critical factors. Here, we report a network model of transcriptional regulation between TFs constructed by integrating genomewide identification of active enhancers and regions of focal DNA accessibility. Network topology is confirmed by published TF ChIP-seq data. By considering multiple methods of TF prioritization following network construction, we identify master TFs in well-studied cell types, and these networks provide better prioritization than networks only considering promoter-proximal accessibility peaks. Comparisons between networks from similar cell types show stable connectivity of most TFs, while master regulator TFs show dramatic changes in connectivity and centrality. Applying this method to study chronic lymphocytic leukemia, we prioritized several network TFs amenable to pharmacological perturbation and show that compounds targeting these TFs show comparable efficacy in CLL cell lines to FDA-approved therapies. The construction of transcriptional regulatory network (TRN) models can predict the interactions between individual TFs and predict critical TFs for development or disease.

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