scholarly journals MBD3L1 Is a Transcriptional Repressor That Interacts with Methyl-CpG-binding Protein 2 (MBD2) and Components of the NuRD Complex

2004 ◽  
Vol 279 (50) ◽  
pp. 52456-52464 ◽  
Author(s):  
Chun-Ling Jiang ◽  
Seung-Gi Jin ◽  
Gerd P. Pfeifer
Keyword(s):  
Transcriptional Repression ◽  
Binding Protein ◽  
Transcriptional Repressor ◽  
Binding Domain ◽  
Nurd Complex ◽  
Methylated Dna ◽  
Binding Domains ◽  
Mouse Cells

Methyl-CpG-binding domain proteins 2 and 3 (MBD2 and MBD3) are transcriptional repressors that contain methyl-CpG binding domains and are components of a CpG-methylated DNA binding complex named MeCP1. Methyl-CpG-binding protein 3-like 1 (MBD3L1) is a protein with substantial homology to MBD2 and MBD3, but it lacks the methyl-CpG binding domain. MBD3L1 interacts with MBD2 and MBD3in vitroand in yeast two-hybrid assays. Gel shift experiments with a CpG-methylated DNA probe indicate that recombinant MBD3L1 can supershift an MBD2-methylated DNA complex.In vivo, MBD3L1 associates with and colocalizes with MBD2 but not with MBD3 and is recruited to 5-methylcytosine-rich pericentromeric heterochromatin in mouse cells. In glutathioneS-transferase pull-down assays MBD3L1 is found associated with several known components of the MeCP1·NuRD complex, including HDAC1, HDAC2, MTA2, MBD2, RbAp46, and RbAp48, but MBD3 is not found in the MBD3L1-bound fraction. MBD3L1 enhances transcriptional repression of methylated DNA by MBD2. The data are consistent with a role of MBD3L1 as a methylation-dependent transcriptional repressor that may interchange with MBD3 as an MBD2-interacting component of the NuRD complex. MBD3L1 knockout mice were created and were found to be viable and fertile, indicating that MBD3L1 may not be essential or there is functional redundancy (through MBD3) in this pathway. Overall, this study reveals additional complexities in the mechanisms of transcriptional repression by the MBD family proteins.

scholarly journals The transcriptional repressor even-skipped interacts directly with TATA-binding protein.

1995 ◽  
Vol 15 (9) ◽  
pp. 5007-5016 ◽  
Author(s):  
M Um ◽  
C Li ◽  
J L Manley
Keyword(s):  
Protein Interactions ◽  
Transcriptional Repression ◽  
Binding Protein ◽  
Transcriptional Repressor ◽  
Direct Interaction ◽  
Tata Binding Protein ◽  
Homeodomain Protein ◽  
Repression Domain

The Drosophila homeodomain protein Even-skipped (Eve) has previously been shown to function as a sequence-specific transcriptional repressor, and in vitro and in vivo experiments have shown that the protein can actively block basal transcription. However, the mechanism of repression is not known. Here, we present evidence establishing a direct interaction between Eve and the TATA-binding protein (TBP). Using cotransfection assays with minimal basal promoters whose activity can be enhanced by coexpression of TBP, we found that Eve could efficiently block, or squelch, this enhancement. Squelching did not require Eve DNA-binding sites on the reporter plasmids but was dependent on the presence of the Eve repression domain. Further support for an in vivo interaction between the Eve repression domain and TBP was derived from a two-hybrid-type assay with transfected cells. Evidence that Eve and TBP interact directly was provided by in vitro binding assays, which revealed a specific protein-protein interaction that required an intact Eve repression domain and the conserved C terminus of TBP. The Eve homeodomain was also required for these associations, suggesting that it may function in protein-protein interactions. We also show that a previously characterized artificial repression region behaves in a manner similar to that of the Eve repression domain, including its ability to squelch TBP-enhanced expression in vivo and to bind TBP specifically in vitro. Our results suggest a model for transcriptional repression that involves an interaction between Eve and TBP.

scholarly journals A Human TATA Binding Protein-Related Protein with Altered DNA Binding Specificity Inhibits Transcription from Multiple Promoters and Activators

1999 ◽  
Vol 19 (11) ◽  
pp. 7610-7620 ◽  
Author(s):  
Paul A. Moore ◽  
Josef Ozer ◽  
Moreh Salunek ◽  
Gwenael Jan ◽  
Dennis Zerby ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcriptional Repression ◽  
Transcription Initiation ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Related Protein ◽  
Multiple Promoters ◽  
Binding Surface

ABSTRACT The TATA binding protein (TBP) plays a central role in eukaryotic and archael transcription initiation. We describe the isolation of a novel 23-kDa human protein that displays 41% identity to TBP and is expressed in most human tissue. Recombinant TBP-related protein (TRP) displayed barely detectable binding to consensus TATA box sequences but bound with slightly higher affinities to nonconsensus TATA sequences. TRP did not substitute for TBP in transcription reactions in vitro. However, addition of TRP potently inhibited basal and activated transcription from multiple promoters in vitro and in vivo. General transcription factors TFIIA and TFIIB bound glutathioneS-transferase–TRP in solution but failed to stimulate TRP binding to DNA. Preincubation of TRP with TFIIA inhibited TBP-TFIIA-DNA complex formation and addition of TFIIA overcame TRP-mediated transcription repression. TRP transcriptional repression activity was specifically reduced by mutations in TRP that disrupt the TFIIA binding surface but not by mutations that disrupt the TFIIB or DNA binding surface of TRP. These results suggest that TFIIA is a primary target of TRP transcription inhibition and that TRP may modulate transcription by a novel mechanism involving the partial mimicry of TBP functions.

scholarly journals Histone H3 N-terminal mimicry drives a novel network of methyl-effector interactions

2021 ◽  
Author(s):  
Jianji Chen ◽  
John Horton ◽  
Cari Sagum ◽  
Jujun Zhou ◽  
Xiaodong Cheng ◽  
...  
Keyword(s):  
Histone H3 ◽  
Transcriptional Repressor ◽  
Protein Domain ◽  
Nurd Complex ◽  
Phd Finger ◽  
Effector Domains ◽  
Methionine Residue ◽  
Repressor Complex

The reader ability of PHD fingers is largely limited to the recognition of the histone H3 N-terminal tail. Distinct subsets of PHDs bind either H3K4me3 (a transcriptional activator mark) or H3K4me0 (a transcriptional repressor state). Structural studies have identified common features among the different H3K4me3 effector PHDs, including 1) removal of the initiator methionine residue of H3 to prevent steric interference, 2) a groove where arginine-2 binds, and 3) an aromatic cage that engages methylated lysine-4. We hypothesize that  PHDs  have the ability to engage with non-histone ligands, as long as they adhere to these three rules. A search of the human proteome revealed an enrichment of chromatin-binding proteins that met these criteria, which we termed H3 N-terminal mimicry proteins (H3TMs). Seven H3TMs were selected, and used to screen a protein domain microarray for potential effector domains, and they all had the ability to bind H3K4me3-interacting effector domains. Furthermore, the binding affinity between the VRK1 peptide and the PHD domain of PHF2 is ~3-fold stronger than that of PHF2 and H3K4me3 interaction. The crystal structure of PHF2 PHD finger bound with VRK1 K4me3 peptide provides a molecular basis for stronger binding of VRK1 peptide. In addition, a number of the H3TMs peptides, in their unmethylated form, interact with NuRD transcriptional repressor complex. Our findings provide in vitro evidence that methylation of H3TMs can promote interactions with PHD and Tudor domain-containing proteins and potentially block interactions with the NuRD complex. We propose that these interactions can occur in vivo as well.

scholarly journals Multivalent interactions with RNA drive RNA binding protein recruitment and dynamics in biomolecular condensates in Xenopus oocytes

2021 ◽  
Author(s):  
Sarah E Cabral ◽  
Kimberly Mowry
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Dependent Manner ◽  
Binding Domains ◽  
Multivalent Interactions

RNA localization and biomolecular condensate formation are key biological strategies for organizing the cytoplasm and generating cellular and developmental polarity. While enrichment of RNAs and RNA-binding proteins (RBPs) is a hallmark of both processes, the functional and structural roles of RNA-RNA and RNA-protein interactions within condensates remain unclear. Recent work from our laboratory has shown that RNAs required for germ layer patterning in Xenopus oocytes localize in novel biomolecular condensates, termed Localization bodies (L-bodies). L-bodies are composed of a non-dynamic RNA phase enmeshed in a more dynamic protein-containing phase. However, the interactions that drive the biophysical characteristics of L-bodies are not known. Here, we test the role of RNA-protein interactions using an L-body RNA-binding protein, PTBP3, which contains four RNA-binding domains (RBDs). We find that binding of RNA to PTB is required for both RNA and PTBP3 to be enriched in L-bodies in vivo. Importantly, while RNA binding to a single RBD is sufficient to drive PTBP3 localization to L-bodies, interactions between multiple RRMs and RNA tunes the dynamics of PTBP3 within L-bodies. In vitro, recombinant PTBP3 phase separates into non-dynamic structures in an RNA-dependent manner, supporting a role for RNA-protein interactions as a driver of both recruitment of components to L-bodies and the dynamics of the components after enrichment. Our results point to a model where RNA serves as a concentration-dependent, non-dynamic substructure and multivalent interactions with RNA are a key driver of protein dynamics.

scholarly journals Importance of the Conserved Carboxyl-Terminal CNOT1 Binding Domain to Tristetraprolin ActivityIn Vivo

2019 ◽  
Vol 39 (13) ◽  
Author(s):  
Wi S. Lai ◽  
Deborah J. Stumpo ◽  
Melissa L. Wells ◽  
Artiom Gruzdev ◽  
Stephanie N. Hicks ◽  
...  
Keyword(s):  
Binding Domain ◽  
Content Type ◽  
Target Mrna ◽  
Binding Domains ◽  
Inflammatory Protein ◽  
Carboxyl Terminal ◽  
The Stability ◽  
Inflammatory Syndrome

ABSTRACTTristetraprolin (TTP) is an anti-inflammatory protein that modulates the stability of certain cytokine/chemokine mRNAs. After initial high-affinity binding to AU-rich elements in 3′ untranslated regions of target mRNAs, mediated through its tandem zinc finger (TZF) domain, TTP promotes the deadenylation and ultimate decay of target transcripts. These transcripts and their encoded proteins accumulate abnormally in TTP knockout (KO) mice, leading to a severe inflammatory syndrome. To assess the importance of the highly conserved C-terminal CNOT1 binding domain (CNBD) of TTP to the TTP deficiency phenotype in mice, we created a mouse model in which TTP lacked its CNBD. CNBD deletion mice exhibited a less severe phenotype than the complete TTP KO mice. In macrophages, the stabilization of target transcripts seen in KO mice was partially normalized in the CNBD deletion mice. In cell-free experiments, recombinant TTP lacking its CNBD could still activate target mRNA deadenylation by purified recombinantSchizosaccharomyces pombeCCR4/NOT complexes, although to a lesser extent than full-length TTP. Thus, TTP lacking its CNBD can still act to promote target mRNA instabilityin vitroandin vivo. These data have implications for TTP family members throughout the eukarya, since species from all four kingdoms contain proteins with linked TZF and CNOT1 binding domains.

scholarly journals A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis

2002 ◽  
Vol 13 (10) ◽  
pp. 3683-3695 ◽  
Author(s):  
Petra Björk ◽  
Göran Baurén ◽  
ShaoBo Jin ◽  
Yong-Guang Tong ◽  
Thomas R. Bürglin ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Ribosomal Subunit ◽  
Binding Domain ◽  
Dependent Manner ◽  
Binding Domains ◽  
40S Ribosomal Subunit ◽  
Rna Binding Domain

Synthesis of the ribosomal subunits from pre-rRNA requires a large number of trans-acting proteins and small nucleolar ribonucleoprotein particles to execute base modifications, RNA cleavages, and structural rearrangements. We have characterized a novel protein, RNA-binding domain-1 (RBD-1), that is involved in ribosome biogenesis. This protein contains six consensus RNA-binding domains and is conserved as to sequence, domain organization, and cellular location from yeast to human. RBD-1 is essential in Caenorhabditis elegans. In the dipteran Chironomus tentans, RBD-1 (Ct-RBD-1) binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In cytoplasmic extracts, 20–30% of Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes.

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 Rhodopsin targeted transcriptional silencing by DNA-binding

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Salvatore Botta ◽  
Elena Marrocco ◽  
Nicola de Prisco ◽  
Fabiola Curion ◽  
Mario Renda ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcriptional Repression ◽  
Binding Protein ◽  
Regulatory Element ◽  
Transcriptional Silencing ◽  
Dna Binding Protein ◽  
Gain Of Function ◽  
Dna Binding Domains ◽  
Binding Domains

Transcription factors (TFs) operate by the combined activity of their DNA-binding domains (DBDs) and effector domains (EDs) enabling the coordination of gene expression on a genomic scale. Here we show that in vivo delivery of an engineered DNA-binding protein uncoupled from the repressor domain can produce efficient and gene-specific transcriptional silencing. To interfere with RHODOPSIN (RHO) gain-of-function mutations we engineered the ZF6-DNA-binding protein (ZF6-DB) that targets 20 base pairs (bp) of a RHOcis-regulatory element (CRE) and demonstrate Rho specific transcriptional silencing upon adeno-associated viral (AAV) vector-mediated expression in photoreceptors. The data show that the 20 bp-long genomic DNA sequence is necessary for RHO expression and that photoreceptor delivery of the corresponding cognate synthetic trans-acting factor ZF6-DB without the intrinsic transcriptional repression properties of the canonical ED blocks Rho expression with negligible genome-wide transcript perturbations. The data support DNA-binding-mediated silencing as a novel mode to treat gain-of-function mutations.

DNMT1 Cxxc Domain Can Functionally Substitute In An MLL Fusion Protein

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4193-4193
Author(s):  
Laurie E. Risner ◽  
Aravinda Kuntimaddi ◽  
John H. Bushweller ◽  
Nancy J. Zeleznik-Le
Keyword(s):  
Dna Binding ◽  
Binding Affinity ◽  
Fusion Proteins ◽  
Binding Domain ◽  
Cpg Dna ◽  
Domain Swap ◽  
Binding Domains ◽  
Dna Binding Affinity

Abstract Abstract 4193 The MLL gene encodes a multi-domain protein that is involved in the maintenance of Hox gene expression during development and hematopoiesis, and was first identified through its involvement in chromosome translocations that cause leukemia. The CXXC domain of MLL, which is retained in leukemic MLL fusion proteins, is a cysteine rich DNA binding domain, with specificity for binding nonmethylated CpG-containing DNA, and is essential for MLL fusion proteins' oncogenic properties. We performed domain swap experiments in which CXXC domains from other proteins were swapped in to replace MLL's CXXC domain in the context of an oncogenic MLL fusion. CXXC domains from DNA methyltransferase 1 (DNMT1), CpG binding protein (CGBP), and methyl-CpG binding domain protein 1 (MBD1), as well as a methyl binding domain (MBD) from MBD1 were swapped into the MLL-AF9 fusion. These particular domains were chosen because their described CpG DNA binding capacity is either similar or different from that described for MLL. In vitro colony assays on isolated murine bone marrow progenitor cells infected with domain swapped or wild type MLL-AF9 fusion genes were performed in order to determine whether CpG binding domains from other proteins would affect the ability of MLL-AF9 to give an enhanced proliferative capacity to bone marrow progenitor cells. In vivo murine studies determined whether the different CpG binding domains alter the ability of MLL fusion proteins to cause leukemia. We predicted that the different CpG binding domains would change the strength or specificity of MLL binding to DNA, which would affect the ability of MLL-AF9 to cause leukemia. The results of both in vitro replating assays and in vivo leukemogenesis experiments have shown significant differences between the ability of various CpG DNA binding domains to function in the context of an MLL-AF9 fusion protein. MLL-AF9 containing the DNMT1 CXXC domain shows robust in vitro colony forming activity and in vivo leukemogenesis activity, similar to the oncogenic MLL-AF9 fusion. However, MLL-AF9 containing either the CXXC domain from CGBP or MBD1, or the MBD domain of MBD1 all show reduced colony forming ability and leukemogenicity in vivo. In vitro DNA binding experiments are currently being performed to directly measure and compare the DNA binding affinity of the CXXC domain from MLL to the other domain swap proteins. Preliminary data suggests that MLL CXXC has a stronger DNA binding affinity to non-methylated DNA compared to the other CXXC domains. Furthermore, the DNMT and CGBP CXXC domains both show lower affinity DNA binding compared to MLL CXXC, but they have different effects in MLL-AF9. This suggests that CXXC domain properties in addition to DNA binding affinity, perhaps including protein recruitment, also contribute to an MLL fusion protein's leukemogenic properties. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Identification and Characterization of a Family of Mammalian Methyl-CpG Binding Proteins

1998 ◽  
Vol 18 (11) ◽  
pp. 6538-6547 ◽  
Author(s):  
Brian Hendrich ◽  
Adrian Bird
Keyword(s):  
Fluorescent Protein ◽  
Embryonic Stem ◽  
Mouse Development ◽  
Methylated Dna ◽  
Somatic Tissues ◽  
Mouse Cells ◽  
Green Fluorescent

ABSTRACT Methylation at the DNA sequence 5′-CpG is required for mouse development. MeCP2 and MBD1 (formerly PCM1) are two known proteins that bind specifically to methylated DNA via a related amino acid motif and that can repress transcription. We describe here three novel human and mouse proteins (MBD2, MBD3, and MBD4) that contain the methyl-CpG binding domain. MBD2 and MBD4 bind specifically to methylated DNA in vitro. Expression of MBD2 and MBD4 tagged with green fluorescent protein in mouse cells shows that both proteins colocalize with foci of heavily methylated satellite DNA. Localization is disrupted in cells that have greatly reduced levels of CpG methylation. MBD3 does not bind methylated DNA in vivo or in vitro. MBD1, MBD2, MBD3, and MBD4 are expressed in somatic tissues, but MBD1 and MBD2 expression is reduced or absent in embryonic stem cells which are known to be deficient in MeCP1 activity. The data demonstrate that MBD2 and MBD4 bind specifically to methyl-CpG in vitro and in vivo and are therefore likely to be mediators of the biological consequences of the methylation signal.

Sign in / Sign up

Export Citation Format

Share Document