scholarly journals Transcriptional repression by Msx-1 does not require homeodomain DNA-binding sites.

1995 ◽  
Vol 15 (2) ◽  
pp. 861-871 ◽  
Author(s):  
K M Catron ◽  
H Zhang ◽  
S C Marshall ◽  
J A Inostroza ◽  
J M Wilson ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Binding Domain ◽  
Specific Cell ◽  
Homeodomain Protein ◽  
Dna Binding Domain ◽  
Dna Binding Sites

This study investigates the transcriptional properties of Msx-1, a murine homeodomain protein which has been proposed to play a key role in regulating the differentiation and/or proliferation state of specific cell populations during embryogenesis. We show, using basal and activated transcription templates, that Msx-1 is a potent repressor of transcription and can function through both TATA-containing and TATA-less promoters. Moreover, repression in vivo and in vitro occurs in the absence of DNA-binding sites for the Msx-1 homeodomain. Utilizing a series of truncated Msx-1 polypeptides, we show that multiple regions of Msx-1 contribute to repression, and these are rich in alanine, glycine, and proline residues. When fused to a heterologous DNA-binding domain, both N- and C-terminal regions of Msx-1 retain repressor function, which is dependent upon the presence of the heterologous DNA-binding site. Moreover, a polypeptide consisting of the full-length Msx-1 fused to a heterologous DNA-binding domain is a more potent repressor than either the N- or C-terminal regions alone, and this fusion retains the ability to repress transcription in the absence of the heterologous DNA site. We further show that Msx-1 represses transcription in vitro in a purified reconstituted assay system and interacts with protein complexes composed of TBP and TFIIA (DA) and TBP, TFIIA, and TFIIB (DAB) in gel retardation assays, suggesting that the mechanism of repression is mediated through interaction(s) with a component(s) of the core transcription complex. We speculate that the repressor function of Msx-1 is critical for its proposed role in embryogenesis as a regulator of cellular differentiation.

scholarly journals Functionally distinct roles for T and Tbx6 during mouse development

Biology Open ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. bio054692
Author(s):  
Amy K. Wehn ◽  
Deborah R. Farkas ◽  
Carly E. Sedlock ◽  
Dibya Subedi ◽  
Deborah L. Chapman
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Transcriptional Activity ◽  
Es Cells ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Mouse Development ◽  
T Box

ABSTRACTThe mouse T-box transcription factors T and Tbx6 are co-expressed in the primitive streak and have unique domains of expression; T is expressed in the notochord, while Tbx6 is expressed in the presomitic mesoderm. T-box factors are related through a shared DNA binding domain, the T-domain, and can therefore bind to similar DNA sequences at least in vitro. We investigated the functional similarities and differences of T and Tbx6 DNA binding and transcriptional activity in vitro and their interaction genetically in vivo. We show that at one target, Dll1, the T-domains of T and Tbx6 have different affinities for the binding sites present in the mesoderm enhancer. We further show using in vitro assays that T and Tbx6 differentially affect transcription with Tbx6 activating expression tenfold higher than T, that T and Tbx6 can compete at target gene enhancers, and that this competition requires a functional DNA binding domain. Next, we addressed whether T and Tbx6 can compete in vivo. First, we generated embryos that express Tbx6 at greater than wild-type levels embryos and show that these embryos have short tails, resembling the T heterozygous phenotype. Next, using the dominant-negative TWis allele, we show that Tbx6+/− TWis/+ embryos share similarities with embryos homozygous for the Tbx6 hypomorphic allele rib-vertebrae, specifically fusions of several ribs and malformation of some vertebrae. Finally, we tested whether Tbx6 can functionally replace T using a knockin approach, which resulted in severe T null-like phenotypes in chimeric embryos generated with ES cells heterozygous for a Tbx6 knockin at the T locus. Altogether, our results of differences in affinity for DNA binding sites and transcriptional activity for T and Tbx6 provide a potential mechanism for the failure of Tbx6 to functionally replace T and possible competition phenotypes in vivo.

Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain

1994 ◽  
Vol 14 (9) ◽  
pp. 6056-6067
Author(s):  
M Tanaka ◽  
W Herr
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Pou Domain

The POU domain activator Oct-2 contains an N-terminal glutamine-rich transcriptional activation domain. An 18-amino-acid segment (Q18III) from this region reconstituted a fully functional activation domain when tandemly reiterated and fused to either the Oct-2 or GAL4 DNA-binding domain. A minimal transcriptional activation domain likely requires three tandem Q18III segments, because one or two tandem Q18III segments displayed little activity, whereas three to five tandem segments were active and displayed increasing activity with increasing copy number. As with natural Oct-2 activation domains, in our assay a reiterated activation domain required a second homologous or heterologous activation domain to stimulate transcription effectively when fused to the Oct-2 POU domain. These results suggest that there are different levels of synergy within and among activation domains. Analysis of reiterated activation domains containing mutated Q18III segments revealed that leucines and glutamines, but not serines or threonines, are critical for activity in vivo. Curiously, several reiterated activation domains that were inactive in vivo were active in vitro, suggesting that there are significant functional differences in our in vivo and in vitro assays. Reiteration of a second 18-amino-acid segment from the Oct-2 glutamine-rich activation domain (Q18II) was also active, but its activity was DNA-binding domain specific, because it was active when fused to the GAL4 than to the Oct-2 DNA-binding domain. The ability of separate short peptide segments derived from a single transcriptional activation domain to activate transcription after tandem reiteration emphasizes the flexible and modular nature of a transcriptional activation domain.

scholarly journals Mad proteins contain a dominant transcription repression domain.

1996 ◽  
Vol 16 (10) ◽  
pp. 5772-5781 ◽  
Author(s):  
D E Ayer ◽  
C D Laherty ◽  
Q A Lawrence ◽  
A P Armstrong ◽  
R N Eisenman
Keyword(s):  
Dna Binding ◽  
Transcriptional Repression ◽  
Binding Domain ◽  
Full Length ◽  
Dna Binding Domain ◽  
Interaction Domain ◽  
Transcription Repression ◽  
Helix Loop Helix ◽  
Repression Domain

Transcription repression by the basic region-helix-loop-helix-zipper (bHLHZip) protein Mad1 requires DNA binding as a ternary complex with Max and mSin3A or mSin3B, the mammalian orthologs of the Saccharomyces cerevisiae transcriptional corepressor SIN3. The interaction between Mad1 and mSin3 is mediated by three potential amphipathic alpha-helices: one in the N terminus of Mad (mSin interaction domain, or SID) and two within the second paired amphipathic helix domain (PAH2) of mSin3A. Mutations that alter the structure of the SID inhibit in vitro interaction between Mad and mSin3 and inactivate Mad's transcriptional repression activity. Here we show that a 35-residue region containing the SID represents a dominant repression domain whose activity can be transferred to a heterologous DNA binding region. A fusion protein comprising the Mad1 SID linked to a Ga14 DNA binding domain mediates repression of minimal as well as complex promoters dependent on Ga14 DNA binding sites. In addition, the SID represses the transcriptional activity of linked VP16 and c-Myc transactivation domains. When fused to a full-length c-Myc protein, the Mad1 SID specifically represses both c-Myc's transcriptional and transforming activities. Fusions between the GAL DNA binding domain and full-length mSin3 were also capable of repression. We show that the association between Mad1 and mSin3 is not only dependent on the helical SID but is also dependent on both putative helices of the mSin3 PAH2 region, suggesting that stable interaction requires all three helices. Our results indicate that the SID is necessary and sufficient for transcriptional repression mediated by the Mad protein family and that SID repression is dominant over several distinct transcriptional activators.

scholarly journals Phosphorylation of BATF regulates DNA binding: a novel mechanism for AP-1 (activator protein-1) regulation

2003 ◽  
Vol 374 (2) ◽  
pp. 423-431 ◽  
Author(s):  
Christopher D. DEPPMANN ◽  
Tina M. THORNTON ◽  
Fransiscus E. UTAMA ◽  
Elizabeth J. TAPAROWSKY
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Leucine Zipper ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activator Protein 1 ◽  
Basic Leucine Zipper ◽  
Activator Protein

BATF is a member of the AP-1 (activator protein-1) family of bZIP (basic leucine zipper) transcription factors that form transcriptionally inhibitory, DNA binding heterodimers with Jun proteins. In the present study, we demonstrate that BATF is phosphorylated in vivo on multiple serine and threonine residues and at least one tyrosine residue. Reverse-polarity PAGE revealed that serine-43 and threonine-48 within the DNA binding domain of BATF are phosphorylated. To model phosphorylation of the BATF DNA binding domain, serine-43 was replaced by an aspartate residue. BATF(S43D) retains the ability to dimerize with Jun proteins in vitro and in vivo, and the BATF(S43D):Jun heterodimer localizes properly to the nucleus of cells. Interestingly, BATF(S43D) functions like wild-type BATF to reduce AP-1-mediated gene transcription, despite the observed inability of the BATF(S43D):Jun heterodimer to bind DNA. These data demonstrate that phosphorylation of serine-43 converts BATF from a DNA binding into a non-DNA binding inhibitor of AP-1 activity. Given that 40% of mammalian bZIP transcription factors contain a residue analogous to serine-43 of BATF in their DNA binding domains, the phosphorylation event described here represents a mechanism that is potentially applicable to the regulation of many bZIP proteins.

scholarly journals Accurate and sensitive quantification of protein-DNA binding affinity

2018 ◽  
Vol 115 (16) ◽  
pp. E3692-E3701 ◽  
Author(s):  
Chaitanya Rastogi ◽  
H. Tomas Rube ◽  
Judith F. Kribelbauer ◽  
Justin Crocker ◽  
Ryan E. Loker ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Sites ◽  
Biophysical Model ◽  
Regulatory Sequences ◽  
Binding Modes ◽  
Perfect Agreement ◽  
Dna Binding Sites

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes.

Structure of the DNA-binding domain of the response regulator SaeR fromStaphylococcus aureus

2015 ◽  
Vol 71 (8) ◽  
pp. 1768-1776 ◽  
Author(s):  
Xiaojiao Fan ◽  
Xu Zhang ◽  
Yuwei Zhu ◽  
Liwen Niu ◽  
Maikun Teng ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Response Regulator ◽  
Regulatory System ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Promoter Regions ◽  
Winged Helix

The SaeR/S two-component regulatory system is essential for controlling the expression of many virulence factors inStaphylococcus aureus. SaeR, a member of the OmpR/PhoB family, is a response regulator with an N-terminal regulatory domain and a C-terminal DNA-binding domain. In order to elucidate how SaeR binds to the promoter regions of target genes, the crystal structure of the DNA-binding domain of SaeR (SaeRDBD) was solved at 2.5 Å resolution. The structure reveals that SaeRDBDexists as a monomer and has the canonical winged helix–turn–helix module. EMSA experiments suggested that full-length SaeR can bind to the P1 promoter and that the binding affinity is higher than that of its C-terminal DNA-binding domain. Five key residues on the winged helix–turn–helix module were verified to be important for binding to the P1 promoterin vitroand for the physiological function of SaeRin vivo.

scholarly journals Regulation of the Myxococcus xanthus C-Signal-Dependent Ω4400 Promoter by the Essential Developmental Protein FruA

2006 ◽  
Vol 188 (14) ◽  
pp. 5167-5176 ◽  
Author(s):  
Deborah R. Yoder-Himes ◽  
Lee Kroos
Keyword(s):  
Dna Binding ◽  
Response Regulator ◽  
Myxococcus Xanthus ◽  
Cell Movement ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Consensus Binding Site ◽  
Multicellular Development

ABSTRACT The bacterium Myxococcus xanthus employs extracellular signals to coordinate aggregation and sporulation during multicellular development. Extracellular, contact-dependent signaling that involves the CsgA protein (called C-signaling) activates FruA, a putative response regulator that governs a branched signaling pathway inside cells. One branch regulates cell movement, leading to aggregation. The other branch regulates gene expression, leading to sporulation. C-signaling is required for full expression of most genes induced after 6 h into development, including the gene identified by Tn5 lac insertion Ω4400. To determine if FruA is a direct regulator of Ω4400 transcription, a combination of in vivo and in vitro experiments was performed. Ω4400 expression was abolished in a fruA mutant. The DNA-binding domain of FruA bound specifically to DNA upstream of the promoter −35 region in vitro. Mutations between bp −86 and −77 greatly reduced binding. One of these mutations had been shown previously to reduce Ω4400 expression in vivo and make it independent of C-signaling. For the first time, chromatin immunoprecipitation (ChIP) experiments were performed on M. xanthus. The ChIP experiments demonstrated that FruA is associated with the Ω4400 promoter region late in development, even in the absence of C-signaling. Based on these results, we propose that FruA directly activates Ω4400 transcription to a moderate level prior to C-signaling and, in response to C-signaling, binds near bp −80 and activates transcription to a higher level. Also, the highly localized effects of mutations between bp −86 and −77 on DNA binding in vitro, together with recently published footprints, allow us to predict a consensus binding site of GTCG/CGA/G for the FruA DNA-binding domain.

scholarly journals Unbiased Mutagenesis of MHV68 LANA Reveals a DNA-Binding Domain Required for LANA Function In Vitro and In Vivo

2012 ◽  
Vol 8 (9) ◽  
pp. e1002906 ◽  
Author(s):  
Clinton R. Paden ◽  
J. Craig Forrest ◽  
Scott A. Tibbetts ◽  
Samuel H. Speck
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Dna Binding Domain

scholarly journals Estrogen Receptors Recruit SMRT and N-CoR Corepressors through Newly Recognized Contacts between the Corepressor N Terminus and the Receptor DNA Binding Domain

2010 ◽  
Vol 30 (6) ◽  
pp. 1434-1445 ◽  
Author(s):  
Natalia Varlakhanova ◽  
Chelsea Snyder ◽  
Soumia Jose ◽  
Johnnie B. Hahm ◽  
Martin L. Privalsky
Keyword(s):  
Dna Binding ◽  
Estrogen Receptors ◽  
Nuclear Receptors ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Dna Binding Sites ◽  
Hormone Antagonists ◽  
Key Aspects ◽  
Hormone Binding Domain

ABSTRACT Estrogen receptors (ERs) are hormone-regulated transcription factors that regulate key aspects of reproduction and development. ERs are unusual in that they do not typically repress transcription in the absence of hormone but instead possess otherwise cryptic repressive functions that are revealed upon binding to certain hormone antagonists. The roles of corepressors in the control of these aspects of ER function are complex and incompletely understood. We report here that ERs recruit SMRT through an unusual mode of interaction involving multiple contact surfaces. Two surfaces of SMRT, located at the N- and C-terminal domains, contribute to the recruitment of the corepressor to ERs in vitro and are crucial for the corepressor modulation of ER transcriptional activity in cells. These corepressor surfaces contact the DNA binding domain of the receptor, rather than the hormone binding domain previously elucidated for other corepressor/nuclear receptor interactions, and are modulated by the ER's recognition of cognate DNA binding sites. Several additional nuclear receptors, and at least one other corepressor, N-CoR, share aspects of this novel mode of corepressor recruitment. Our results highlight a molecular mechanism that helps explain several previously paradoxical aspects of ER-mediated transcriptional antagonism, which may have a broader significance for an understanding of target gene repression by other nuclear receptors.

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