E-box- and MEF-2-independent muscle-specific expression, positive autoregulation, and cross-activation of the chicken MyoD (CMD1) promoter reveal an indirect regulatory pathway

1994 ◽  
Vol 14 (8) ◽  
pp. 5474-5486
Author(s):  
C A Dechesne ◽  
Q Wei ◽  
J Eldridge ◽  
L Gannoun-Zaki ◽  
P Millasseau ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Primary Cultures ◽  
Regulatory Elements ◽  
The Other ◽  
Indirect Pathway ◽  
Specific Expression ◽  
Fibroblast Cultures ◽  
E Box ◽  
Myogenic Factors

Members of the MyoD family of gene-regulatory proteins (MyoD, myogenin, myf5, and MRF4) have all been shown not only to regulate the transcription of numerous muscle-specific genes but also to positively autoregulate and cross activate each other's transcription. In the case of muscle-specific genes, this transcriptional regulation can often be correlated with the presence of a DNA consensus in the regulatory region CANNTG, known as an E box. Little is known about the regulatory interactions of the myogenic factors themselves; however, these interactions are thought to be important for the activation and maintenance of the muscle phenotype. We have identified the minimal region in the chicken MyoD (CMD1) promoter necessary for muscle-specific transcription in primary cultures of embryonic chicken skeletal muscle. The CMD1 promoter is silent in primary chick fibroblast cultures and in muscle cell cultures treated with the thymidine analog bromodeoxyuridine. However, CMD1 and chicken myogenin, as well as, to a lesser degree, chicken Myf5 and MRF4, expressed in trans can activate transcription from the minimal CMD1 promoter in these primary fibroblast cultures. Here we show that the CMD1 promoter contains numerous E-box binding sites for CMD1 and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific expression, autoregulation, or cross activation depends upon the presence of of these E-box or MEF-2 binding sites in the CMD1 promoter. These results demonstrate that the autoregulation and cross activation of the chicken MyoD promoter through the putative direct binding of the myogenic basic helix-loop-helix regulatory factors is mediated through an indirect pathway that involves unidentified regulatory elements and/or ancillary factors.

scholarly journals E-box- and MEF-2-independent muscle-specific expression, positive autoregulation, and cross-activation of the chicken MyoD (CMD1) promoter reveal an indirect regulatory pathway.

1994 ◽  
Vol 14 (8) ◽  
pp. 5474-5486 ◽  
Author(s):  
C A Dechesne ◽  
Q Wei ◽  
J Eldridge ◽  
L Gannoun-Zaki ◽  
P Millasseau ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Primary Cultures ◽  
Regulatory Elements ◽  
The Other ◽  
Indirect Pathway ◽  
Specific Expression ◽  
Fibroblast Cultures ◽  
E Box ◽  
Myogenic Factors

Members of the MyoD family of gene-regulatory proteins (MyoD, myogenin, myf5, and MRF4) have all been shown not only to regulate the transcription of numerous muscle-specific genes but also to positively autoregulate and cross activate each other's transcription. In the case of muscle-specific genes, this transcriptional regulation can often be correlated with the presence of a DNA consensus in the regulatory region CANNTG, known as an E box. Little is known about the regulatory interactions of the myogenic factors themselves; however, these interactions are thought to be important for the activation and maintenance of the muscle phenotype. We have identified the minimal region in the chicken MyoD (CMD1) promoter necessary for muscle-specific transcription in primary cultures of embryonic chicken skeletal muscle. The CMD1 promoter is silent in primary chick fibroblast cultures and in muscle cell cultures treated with the thymidine analog bromodeoxyuridine. However, CMD1 and chicken myogenin, as well as, to a lesser degree, chicken Myf5 and MRF4, expressed in trans can activate transcription from the minimal CMD1 promoter in these primary fibroblast cultures. Here we show that the CMD1 promoter contains numerous E-box binding sites for CMD1 and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific expression, autoregulation, or cross activation depends upon the presence of of these E-box or MEF-2 binding sites in the CMD1 promoter. These results demonstrate that the autoregulation and cross activation of the chicken MyoD promoter through the putative direct binding of the myogenic basic helix-loop-helix regulatory factors is mediated through an indirect pathway that involves unidentified regulatory elements and/or ancillary factors.

scholarly journals Novel Interactions of ESCRT-III with LIP5 and VPS4 and their Implications for ESCRT-III Disassembly

2008 ◽  
Vol 19 (6) ◽  
pp. 2661-2672 ◽  
Author(s):  
Soomin Shim ◽  
Samuel A. Merrill ◽  
Phyllis I. Hanson
Keyword(s):  
Atpase Activity ◽  
Binding Site ◽  
Binding Sites ◽  
Essential Role ◽  
Multivesicular Body ◽  
The Other ◽  
Aaa Atpase ◽  
Direct Binding ◽  
Novel Interactions

The AAA+ ATPase VPS4 plays an essential role in multivesicular body biogenesis and is thought to act by disassembling ESCRT-III complexes. VPS4 oligomerization and ATPase activity are promoted by binding to LIP5. LIP5 also binds to the ESCRT-III like protein CHMP5/hVps60, but how this affects its function remains unclear. Here we confirm that LIP5 binds tightly to CHMP5, but also find that it binds well to additional ESCRT-III proteins including CHMP1B, CHMP2A/hVps2–1, and CHMP3/hVps24 but not CHMP4A/hSnf7–1 or CHMP6/hVps20. LIP5 binds to a different region within CHMP5 than within the other ESCRT-III proteins. In CHMP1B and CHMP2A, its binding site encompasses sequences at the proteins' extreme C-termini that overlap with “MIT interacting motifs” (MIMs) known to bind to VPS4. We find unexpected evidence of a second conserved binding site for VPS4 in CHMP2A and CHMP1B, suggesting that LIP5 and VPS4 may bind simultaneously to these proteins despite the overlap in their primary binding sites. Finally, LIP5 binds preferentially to soluble CHMP5 but instead to polymerized CHMP2A, suggesting that the newly defined interactions between LIP5 and ESCRT-III proteins may be regulated by ESCRT-III conformation. These studies point to a role for direct binding between LIP5 and ESCRT-III proteins that is likely to complement LIP5's previously described ability to regulate VPS4 activity.

scholarly journals A combination of MEF3 and NFI proteins activates transcription in a subset of fast-twitch muscles.

1997 ◽  
Vol 17 (2) ◽  
pp. 656-666 ◽  
Author(s):  
F Spitz ◽  
M Salminen ◽  
J Demignon ◽  
A Kahn ◽  
D Daegelen ◽  
...  
Keyword(s):  
Regulatory Elements ◽  
Specific Expression ◽  
Transgenic Lines ◽  
Slow Twitch ◽  
Myogenic Factors ◽  
Fast Twitch ◽  
Restricted Pattern ◽  
Aldolase A ◽  
Drive Reporter Gene Expression

The human aldolase A pM promoter is active in fast-twitch muscles. To understand the role of the different transcription factors which bind to this promoter and determine which ones are responsible for its restricted pattern of expression, we analyzed several transgenic lines harboring different combinations of pM regulatory elements. We show that muscle-specific expression can be achieved without any binding sites for the myogenic factors MyoD and MEF2 and that a 64-bp fragment comprising a MEF3 motif and an NFI binding site is sufficient to drive reporter gene expression in some but, interestingly, not all fast-twitch muscles. A result related to this pattern of expression is that some isoforms of NFI proteins accumulate differentially in fast- and slow-twitch muscles and in distinct fast-twitch muscles. We propose that these isoforms of NFI proteins might provide a molecular basis for skeletal muscle diversity.

scholarly journals Differential functional significance of AP-1 binding sites in the promoter of the gene encoding mouse tissue inhibitor of metalloproteinases-3

1997 ◽  
Vol 324 (2) ◽  
pp. 547-553 ◽  
Author(s):  
Hyungtae KIM ◽  
William D. PENNIE ◽  
Yi SUN ◽  
Nancy H. COLBURN
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Functional Significance ◽  
Regulatory Elements ◽  
Binding Activity ◽  
Model Systems ◽  
Mouse Tissue ◽  
Significant Activity ◽  
Tissue Inhibitor Of Metalloproteinases ◽  
Tissue Inhibitor

Tissue inhibitor of metalloproteinases-3 (TIMP-3) is an extracellular-matrix-associated protein that suppresses tumorigenicity or invasion in several model systems. We have identified, by in vitro footprinting, six AP-1 (activator protein-1) or AP-1-like binding sites in the mouse TIMP-3 promoter that bind purified c-Jun homodimers. Electrophoretic mobility shift assays revealed that the non-consensus fifth AP-1 binding site (AP-720; nt -720 to -714) had the strongest binding activity for recombinant c-Jun protein, and that the fourth binding site (AP-763; nt -763 to -754) and AP-720 showed strong binding activity for cellular nuclear proteins. Antibody supershift and blocking experiments suggest that AP-720, but not AP-763, binds authentic AP-1 components. Transient transfection reporter assays of deletion constructs showed that the region spanning AP-720 has the highest transcriptional activity, and that sequences 5′ to this region (nt -2846 to -747) may contain negative regulatory elements. The deletion construct containing about 500 nt 5′ to the transcriptional start, but no AP-1 sites, showed lower but significant activity, suggesting both AP-1-dependent and -independent regulation of the mouse TIMP-3 promoter. Mutational inactivation of AP-720 abolished the activity increment that distinguished the reporter construct containing both AP-720 and sixth AP-1 binding site (AP-617; nt -617 to -611) from that containing only AP-617. In summary, we report here that both AP-1 and non-AP-1 elements contribute to activity, with the non-consensus AP-1 site at -720 showing the greatest functional significance among the AP-1 sites.

scholarly journals A novel cis-acting element required for lipopolysaccharide-induced transcription of the murine interleukin-1 beta gene.

1995 ◽  
Vol 15 (1) ◽  
pp. 112-119 ◽  
Author(s):  
S A Godambe ◽  
D D Chaplin ◽  
T Takova ◽  
L M Read ◽  
C J Bellone
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Competitive Inhibition ◽  
Interleukin 1 ◽  
Sodium Dodecyl ◽  
Regulatory Elements ◽  
Nuclear Factor ◽  
Interleukin 1 Beta ◽  
Factor Binding ◽  
Beta Gene

Regulatory elements important for transcription of the murine interleukin-1 beta (IL-1 beta) gene lie within a DNase I-hypersensitive region located > 2,000 bp upstream from the transcription start site. We have identified within this region a novel positive regulatory element that is required for activation of an IL-1 beta promoter-chloramphenicol acetyltransferase (CAT) fusion gene in the murine macrophage line RAW264.7. Electrophoretic mobility shift analysis of the 3' portion (-2315 to -2106) of the hypersensitive region revealed at least two nuclear factor binding sites, one of which is located between positions -2285 and -2256. Competitive inhibition studies localized the binding site to a 15-bp sequence between -2285 and -2271. Nuclear factor binding was lost by mutation of the 6-bp sequence from -2280 to -2275. The specific retarded complex formed with RAW264.7 nuclear extract was not detected under similar conditions with nuclear extracts from RLM-11, a murine T-cell line which does not express IL-1 beta RNA. Mutation of the 6-bp sequence (-2280 to -2275) in the chimeric IL-1 beta promoter -4093 +I CAT plasmid virtually eliminated the activation of this reporter gene by lipopolysaccharide (LPS) in transfected RAW264.7 cells. Multimerization of the 15-bp sequence containing the core wild-type 6-bp sequence 5' of minimal homologous or heterologous promoters in CAT reporter plasmids resulted in significant enhancement of CAT expression compared with parallel constructs containing the mutant 6-bp core sequence. This element was LPS independent and position and orientation dependent. The multimerized 15-bp sequence did not enhance expression in RLM-11 cells. Methylation interference revealed contact residues from -2281 to -2271, CCAAAAAGGAA. Because a search of the NIH TFD data bank with the 11-bp binding site sequence found no homology to known nuclear factor binding sites, we have designated this sequence the IL1 beta -upstream nuclear factor 1 (IL1 beta -UNF1) target. UV cross-linking and sodium dodecyl sulfate-polyacrylamide electrophoresis identified an IL1 beta -UNF1-specific binding factor approximately 85 to 90 kDa in size.

Molecular Modelling of the Interaction between DCMU and the QB-Binding Site of Photosystem II

1993 ◽  
Vol 48 (3-4) ◽  
pp. 191-198 ◽  
Author(s):  
Simon P. Mackay ◽  
Patrick J. O ’Malley
Keyword(s):  
Hydrogen Bond ◽  
Photosystem Ii ◽  
Binding Site ◽  
Binding Sites ◽  
The Other ◽  
Reaction Centre ◽  
Protein Model ◽  
Intermolecular Energy ◽  
Herbicide Binding ◽  
Binding Orientations

Abstract The prefered binding orientations for the herbicide DCMU within the QB-binding site of the D 1 protein model from a photosystem II reaction centre have been determined. Calculation of the intermolecular energy between the herbicide and the binding site has been instrumental in obtaining optimum positions reinforced by experimental results from mutation studies and herbicide binding to analogous bacterial reaction centres. We have shown that two binding sites are possible, one involving a hydrogen bond to and the other to the Ser 264 residue. In both cases, which are more important for the stabilization of the interactions.

THE PHOSPHOLIPID-BINDING SITE OF FACTOR VIII IS LOCATED ON THE 80 kD LIGHT CHAIN

1987 ◽  
Author(s):  
G Kemball-Cook ◽  
S J A Edwards ◽  
K Sewerin ◽  
L-O Andersson ◽  
T W Barrowcliffe
Keyword(s):  
Binding Site ◽  
Light Chain ◽  
Binding Sites ◽  
Immunoaffinity Chromatography ◽  
The Other ◽  
Immunological Methods ◽  
Electrophoretic Separation ◽  
Antigenic Sites ◽  
Reducing Conditions ◽  
Sds Page

The binding of Factoi. VIII (F.VIII) peptides to phospholipid (PL) vesicles has been studied by two different methods involving the use of fractionated anti-F.VIII:C I-Fab123’pre viously reported, i-Fab123’ was fractionated by immunoadsorptionwith F.VIII-PL complexes into two pools:one binding only to PL-binding sites on F.VIIIsAg (PL-site antibody), the other directed against other antigenic sites (non-PL-site antibody).The first technique used was a modification of the method of Weinstein et al. (Proc.Natl.Acad.Sci.USA, 78, 5137-5141, 1981), and involved incubation of the two anti-F.VIII pool swith F.VIII-containing samples, followed by electrophoretic separation of the complexes on the basis of size in non-denaturing SDS gels: this technique allows qualitative analysis of antibody reactive peptides in highly impure samples. Non-PL-site pool reacted with a range of peptides with MrMapparent Mr 90 kD up to 280 kD, a similar pattern to that of ’heavy chain’(HC) peptides of F.VIII seen on SDS-PAGE under reducing conditions; the PL-site antibody, however, reacted only with peptides at apparent Mrs of 80 kD and sometimes150 kD, but not with bands of higher Mr a pattern more consistent with binding to light chain (LC) peptides. Thesame patterns with the two labels were seen in both plasma and F.VIII concentrateThe second approach employed the two labels described above in direct immunoradiometric assays (IFMA’s) on purified human F.VIII peptides prepared by immunoaffinity chromatography and ion exchange on Mono Q gel. Both PL-site and non-PL-site labels measured similar amounts of F.VIII m a sample containing both HC and LC peptides; however, on assaying a sample containing purified HC peptides alone, PL-site antibody measured only 2% of F.VIII:Ag found by non-PL-site label, indicating that PL-binding sites present in samples containing both HC and LC are absent in HC alone.Results from both these immunological methods indicate that the 80 kD LC peptide of F.VIII carries the PL-binding site.

Functional Differences in GATA-1 Affinity for Double Versus Single GATA-1 Binding Sites Dictate Synergistic Versus Antagonistic Interactions of PU.1 and GATA-1 in Myeloid Gene Transcription.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1608-1608
Author(s):  
Jian Du ◽  
Dharmesh Vyas ◽  
Qing Xi ◽  
Steven J. Ackerman
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Gene Transcription ◽  
Binding Sites ◽  
Zinc Fingers ◽  
Specific Expression ◽  
High Affinity ◽  
Dual Versus ◽  
P2 Promoter ◽  
Dual Site

Abstract Instructive roles for both GATA-1 and PU.1 have been demonstrated in hematopoiesis, and recent studies have identified both antagonistic and synergistic interactions between them in myeloid gene transcription and lineage development. In prior studies, we reported that PU.1 synergizes with rather than antagonizes GATA-1 for transactivation of a hallmark eosinophil gene, the major basic protein P2 promoter (MBP-P2), which possesses a novel dual (double) GATA-binding site, similar to the palindromic double site in the murine GATA-1 control locus that may specify eosinophil lineage-specific expression of GATA-1 and eosinophil development. To address the transcriptional mechanism for PU.1-GATA-1 synergy through the MBP-P2 dual GATA site, we investigated GATA-1 and PU.1 physical and functonal interactions via their binding sites in the MBP-P2 promoter. DNA binding affinities of GATA-1 and its C- versus N-terminal zinc fingers were assessed for single versus double GATA sites in the presence or absence of PU.1. Our results show that the dual GATA site strongly binds full length GATA-1 with higher affinity than either of the single sites, using both zinc fingers, but that mutant GATA-1 proteins with C-finger or N-finger deletions retain their ability to bind, albeit at lower affinity, to the dual site. DNA binding activities of the two zinc fingers with the dual GATA site were confirmed using peptides containing only the C-finger or N-finger region. Of note, formation of GATA-1 complexes with the dual GATA site was not inhibited by the addition of PU.1, whereas formation of binding complexes for mutants of GATA-1 containing only the C- or N-finger region could be completely inhibited in a dose-response fashion by PU.1. These unique features of PU.1/GATA-1 interactions on a dual versus single GATA-1 site were confirmed using peptides containing only the C- or N-finger regions of GATA-1. Our findings indicate that both zinc fingers of GATA-1 are involved in formation of the high-affinity GATA-1 complex with the dual site. Importantly, we show that the higher affinity dual GATA-1 site complex is not affected by the addition of PU.1, whereas formation of the binding complex with a single GATA-1 site is eliminated by PU.1, emphasizing the different mechanisms of GATA-1/PU.1 interactions on dual versus single GATA binding sites. Functional analyses by transactivation confirmed that synergistic activation of the MBP-P2 promoter by GATA-1 and PU.1 is mediated by their protein-protein interactions through this unique high affinity dual GATA-1 binding site. We suggest two possible mechanisms for PU.1/GATA-1 synergy on dual GATA sites: (1) PU.1 may change GATA-1 conformation and its high affinity for the dual site, enhancing its availability for interaction with the basal transcriptional machinery. Alternatively, (2) PU.1 could impede interactions of GATA-1 with a co-repressor, e.g. FOG-1, which we and others have shown represses GATA-1 function in the eosinophil lineage.

scholarly journals Functional Cooperation between Multiple Regulatory Elements in the Untranslated Exon 1 Stimulates the Basal Transcription of the Human GnRH-II Gene

2003 ◽  
Vol 17 (7) ◽  
pp. 1175-1191 ◽  
Author(s):  
Chi Keung Cheng ◽  
Ruby L. C. Hoo ◽  
Billy K. C. Chow ◽  
Peter C. K. Leung
Keyword(s):  
Gene Transcription ◽  
Binding Sites ◽  
Molecular Mechanisms ◽  
Mutational Analysis ◽  
Regulatory Elements ◽  
Start Codon ◽  
Basal Transcription ◽  
Untranslated Exon ◽  
E Box ◽  
Exon 1

Abstract The wide distribution of GnRH-II and conservation of its structure over all vertebrate classes suggest that the neuropeptide possesses vital biological functions. Although recent studies have shown that the expression of the human GnRH-II gene is regulated by cAMP and estrogen, the molecular mechanisms governing its basal transcription remain poorly understood. Using the neuronal TE-671 and placental JEG-3 cells, we showed that the minimal human GnRH-II promoter was located between nucleotide −1124 and −750 (relative to the translation start codon) and that the untranslated exon 1 was important to produce full promoter activity. Two putative E-box binding sites and one Ets-like element were identified within the first exon, and mutational analysis demonstrated that these cis-acting elements functioned cooperatively to stimulate the human GnRH-II gene transcription. EMSAs, UV cross-linking, and Southwestern blot analyses indicated that the basic helix-loop-helix transcription factor AP-4 bound specifically to the two E-box binding sites, whereas an unidentified protein bound to the Ets-like element. The functional importance of AP-4 in controlling human GnRH-II gene transcription was demonstrated by overexpression of sense and antisense full-length AP-4 cDNAs. Taken together, our present data demonstrate a novel mechanism in stimulating basal human GnRH-II gene transcription mediated by cooperative actions of multiple regulatory elements within the untranslated first exon of the gene.

scholarly journals Structure-aided prediction of mammalian transcription factor complexes in conserved non-coding elements

2013 ◽  
Vol 368 (1632) ◽  
pp. 20130029 ◽  
Author(s):  
Harendra Guturu ◽  
Andrew C. Doxey ◽  
Aaron M. Wenger ◽  
Gill Bejerano
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Protein Interactions ◽  
Binding Sites ◽  
Three Dimensional ◽  
Structural Data ◽  
Regulatory Elements ◽  
Computational Method ◽  
Web Resource ◽  
Three Dimensional Models

Mapping the DNA-binding preferences of transcription factor (TF) complexes is critical for deciphering the functions of cis -regulatory elements. Here, we developed a computational method that compares co-occurring motif spacings in conserved versus unconserved regions of the human genome to detect evolutionarily constrained binding sites of rigid TF complexes. Structural data were used to estimate TF complex physical plausibility, explore overlapping motif arrangements seldom tackled by non-structure-aware methods, and generate and analyse three-dimensional models of the predicted complexes bound to DNA. Using this approach, we predicted 422 physically realistic TF complex motifs at 18% false discovery rate, the majority of which (326, 77%) contain some sequence overlap between binding sites. The set of mostly novel complexes is enriched in known composite motifs, predictive of binding site configurations in TF–TF–DNA crystal structures, and supported by ChIP-seq datasets. Structural modelling revealed three cooperativity mechanisms: direct protein–protein interactions, potentially indirect interactions and ‘through-DNA’ interactions. Indeed, 38% of the predicted complexes were found to contain four or more bases in which TF pairs appear to synergize through overlapping binding to the same DNA base pairs in opposite grooves or strands. Our TF complex and associated binding site predictions are available as a web resource at http://bejerano.stanford.edu/complex .

Sign in / Sign up

Export Citation Format

Share Document