scholarly journals Enhancer-binding activity of the tal-1 oncoprotein in association with the E47/E12 helix-loop-helix proteins.

1991 ◽  
Vol 11 (6) ◽  
pp. 3037-3042 ◽  
Author(s):  
H L Hsu ◽  
J T Cheng ◽  
Q Chen ◽  
R Baer
Keyword(s):  
Protein Interactions ◽  
Lymphoblastic Leukemia ◽  
Control Cell ◽  
Biochemical Properties ◽  
Binding Activity ◽  
Sequence Motif ◽  
Cell Type ◽  
Dimerization Domain ◽  
Helix Loop Helix

Almost 30% of patients with T-cell acute lymphoblastic leukemia (T-ALL) bear structural alterations of tal-1, a presumptive proto-oncogene that encodes sequences homologous to the helix-loop-helix (HLH) DNA-binding and dimerization domain. Analysis of the tal-1 gene product reveals that its HLH domain mediates protein-protein interactions with either of the ubiquitously expressed HLH proteins E47 and E12. The resultant tal-1/E47 and tal-1/E12 heterodimers specifically recognize the E-box DNA sequence motif found in eucaryotic transcriptional enhancers. Hence, the tal-1 protein shares biochemical properties with other tissue-specific HLH proteins that control cell type determination during myogenesis (e.g., MyoD1) and neurogenesis (e.g., achaete-scute). The data suggest that HLH heterodimers involving tal-1 may function in vivo as transcriptional regulatory factors that influence cell type determination during hematopoietic development.

Enhancer-binding activity of the tal-1 oncoprotein in association with the E47/E12 helix-loop-helix proteins

1991 ◽  
Vol 11 (6) ◽  
pp. 3037-3042 ◽  
Author(s):  
H L Hsu ◽  
J T Cheng ◽  
Q Chen ◽  
R Baer
Keyword(s):  
Protein Interactions ◽  
Lymphoblastic Leukemia ◽  
Control Cell ◽  
Biochemical Properties ◽  
Binding Activity ◽  
Sequence Motif ◽  
Cell Type ◽  
Dimerization Domain ◽  
Helix Loop Helix

Almost 30% of patients with T-cell acute lymphoblastic leukemia (T-ALL) bear structural alterations of tal-1, a presumptive proto-oncogene that encodes sequences homologous to the helix-loop-helix (HLH) DNA-binding and dimerization domain. Analysis of the tal-1 gene product reveals that its HLH domain mediates protein-protein interactions with either of the ubiquitously expressed HLH proteins E47 and E12. The resultant tal-1/E47 and tal-1/E12 heterodimers specifically recognize the E-box DNA sequence motif found in eucaryotic transcriptional enhancers. Hence, the tal-1 protein shares biochemical properties with other tissue-specific HLH proteins that control cell type determination during myogenesis (e.g., MyoD1) and neurogenesis (e.g., achaete-scute). The data suggest that HLH heterodimers involving tal-1 may function in vivo as transcriptional regulatory factors that influence cell type determination during hematopoietic development.

Differential use of SCL/TAL-1 DNA-binding domain in developmental hematopoiesis

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 1056-1067 ◽  
Author(s):  
Mira T. Kassouf ◽  
Hedia Chagraoui ◽  
Paresh Vyas ◽  
Catherine Porcher
Keyword(s):  
Dna Binding ◽  
Molecular Mechanisms ◽  
Binding Activity ◽  
Hematopoietic Stem ◽  
Helix Loop Helix ◽  
Experimental Systems ◽  
Dna Binding Activity ◽  
Independent Functions

Abstract Dissecting the molecular mechanisms used by developmental regulators is essential to understand tissue specification/differentiation. SCL/TAL-1 is a basic helix-loop-helix transcription factor absolutely critical for hematopoietic stem/progenitor cell specification and lineage maturation. Using in vitro and forced expression experimental systems, we previously suggested that SCL might have DNA-binding–independent functions. Here, to assess the requirements for SCL DNA-binding activity in vivo, we examined hematopoietic development in mice carrying a germline DNA-binding mutation. Remarkably, in contrast to complete absence of hematopoiesis and early lethality in scl-null embryos, specification of hematopoietic cells occurred in homozygous mutant embryos, indicating that direct DNA binding is dispensable for this process. Lethality was forestalled to later in development, although some mice survived to adulthood. Anemia was documented throughout development and in adulthood. Cellular and molecular studies showed requirements for SCL direct DNA binding in red cell maturation and indicated that scl expression is positively autoregulated in terminally differentiating erythroid cells. Thus, different mechanisms of SCL's action predominate depending on the developmental/cellular context: indirect DNA binding activities and/or sequestration of other nuclear regulators are sufficient in specification processes, whereas direct DNA binding functions with transcriptional autoregulation are critically required in terminal maturation processes.

scholarly journals Fused protein domains inhibit DNA binding by LexA.

1992 ◽  
Vol 12 (7) ◽  
pp. 3006-3014 ◽  
Author(s):  
E A Golemis ◽  
R Brent
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
In Vitro Assays ◽  
Dimerization Domain ◽  
Activation Domains ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Operator Binding

Many studies of transcription activation employ fusions of activation domains to DNA binding domains derived from the bacterial repressor LexA and the yeast activator GAL4. Such studies often implicitly assume that DNA binding by the chimeric proteins is equivalent to that of the protein donating the DNA binding moiety. To directly investigate this issue, we compared operator binding by a series of LexA-derivative proteins to operator binding by native LexA, by using both in vivo and in vitro assays. We show that operator binding by many proteins such as LexA-Myc, LexA-Fos, and LexA-Bicoid is severely impaired, while binding of other LexA-derivative proteins, such as those that carry bacterially encoded acidic sequences ("acid blobs"), is not. Our results also show that DNA binding by LexA derivatives that contain the LexA carboxy-terminal dimerization domain (amino acids 88 to 202) is considerably stronger than binding by fusions that lack it and that heterologous dimerization motifs cannot substitute for the LexA88-202 function. These results suggest the need to reevaluate some previous studies of activation that employed LexA derivatives and modifications to recent experimental approaches that use LexA and GAL4 derivatives to detect and study protein-protein interactions.

scholarly journals Preferred sequences for DNA recognition by the TAL1 helix-loop-helix proteins.

1994 ◽  
Vol 14 (2) ◽  
pp. 1256-1265 ◽  
Author(s):  
H L Hsu ◽  
L Huang ◽  
J T Tsan ◽  
W Funk ◽  
W E Wright ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Dna Binding ◽  
Lymphoblastic Leukemia ◽  
Dna Recognition ◽  
Binding Activity ◽  
Class A ◽  
Helix Loop Helix ◽  
Dna Binding Activity ◽  
Cell Acute Lymphoblastic Leukemia

Tumor-specific activation of the TAL1 gene is the most common genetic alteration seen in patients with T-cell acute lymphoblastic leukemia. The TAL1 gene products contain the basic helix-loop-helix (bHLH) domain, a protein dimerization and DNA-binding motif common to several known transcription factors. A binding-site selection procedure has now been used to evaluate the DNA recognition properties of TAL1. These studies demonstrate that TAL1 polypeptides do not have intrinsic DNA-binding activity, presumably because of their inability to form bHLH homodimers. However, TAL1 readily interacts with any of the known class A bHLH proteins (E12, E47, E2-2, and HEB) to form heterodimers that bind DNA in a sequence-specific manner. The TAL1 heterodimers preferentially recognize a subset of E-box elements (CANNTG) that can be represented by the consensus sequence AACAGATGGT. This consensus is composed of half-sites for recognition by the participating class A bHLH polypeptide (AACAG) and the TAL1 polypeptide (ATGGT). TAL1 heterodimers with DNA-binding activity are readily detected in nuclear extracts of Jurkat, a leukemic cell line derived from a patient with T-cell acute lymphoblastic leukemia. Hence, TAL1 is likely to bind and regulate the transcription of a unique subset of subordinate target genes, some of which may mediate the malignant function of TAL1 during T-cell leukemogenesis.

LYL1, Class II Basic Helix-Loop-Helix Transcription Factor, Induces the Differentiation of Common Marmoset Embryonic Stem Cells to Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2348-2348
Author(s):  
Hirotaka Kawano ◽  
Tomotoshi Marumoto ◽  
Michiyo Okada ◽  
Tomoko Inoue ◽  
Takenobu Nii ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Lymphoblastic Leukemia ◽  
Embryonic Stem ◽  
Common Marmoset ◽  
Hematopoietic Stem ◽  
Helix Loop Helix ◽  
Cd34 Cells

Abstract Abstract 2348 Since the successful establishment of human embryonic stem cells (ESCs) in 1998, transplantation of functional cells differentiated from ESCs to the specific impaired organ has been expected to cure its defective function [Thomson JA et al., Science 282:1145–47, 1998]. For the establishment of the regenerative medicine using ESCs, the preclinical studies utilizing animal model systems including non-human primates are essential. We have demonstrated that non-human primate of common marmoset (CM) is a suitable experimental animal for the preclinical studies of hematopoietic stem cells (HSCs) therapy [Hibino H et al., Blood 93:2839–48, 1999]. Since then we have continuously investigated the in vitro and in vivo differentiation of CM ESCs to hematopoietic cells by the exogenous hematopoietic gene transfer. In earlier study, we showed that the induction of CD34+ cells having a blood colony forming capacity from CM ESCs is promoted by lentiviral transduction of TAL1 cDNA [Kurita R et al., Stem Cells 24:2014-22,2006]. However those CD34+ cells did not have a bone marrow reconstituting ability in irradiated NOG (NOD/Shi-scid/IL-2Rγnull) mice, suggesting that transduction of TAL1 gene is not enough to induce functional HSCs which have self-renewal capability and multipotency. Thus we tried to find other hematopoietic genes being able to promote hematopoietic differetiation more efficiently than TAL1. We selected 6 genes (LYL1, HOXB4, BMI1, GATA2, c-MYB and LMO2) as candidates for factors that induce the differentiation from ESCs to HSCs, based on the comparison of gene expression level between human ESCs and HSCs by Digital Differential Display from the Uni-Gene database at the NCBI web site (http://www.ncbi.nlm.nih.gov/UniGene/). Then, we transduced the respective candidate gene in CM ESCs (Cj11), and performed embryoid body (EB) formation assay to induce their differentiation to HSCs for 9 days. We found that lentiviral transduction of LYL1, a basic helix-loop-helix transcription factor, in EBs derived from Cj11, one of CM ESC lines, markedly increased the number of cells positive for CD34, a marker for hematopoietic stem/progenitors. The lymphoblastic leukemia 1 (LYL1) was originally identified as the factor of a chromosomal translocation, resulting in T cell acute lymphoblastic leukemia [Mellentin JD et al., Cell 58:77-83.1989]. These class II bHLH transcription factors regulate gene expression by binding to target gene sequences as heterodimers with E-proteins, in association with Gata1 and Gata2 [Goldfarb AN et al., Blood 85:465-71.1995][Hofmann T et al., Oncogene 13:617-24.1996][Hsu HL et al., Proc Natl Acad Sci USA 91:5947-51.1994]. The Lyl1-deficient mice display the reduction of B cells and impaired long-term hematopoietic reconstitution capacity [Capron C et al., Blood 107:4678-4686. 2006]. And, overexpression of Lyl1 in mouse bone marrow cells induced the increase of HSCs, HPCs and lymphocytes in vitro and in vivo [Lukov GL et al., Leuk Res 35:405-12. 2011]. These information indicate that LYL1 plays important roles in hematopoietic differentiation in primate animals including human and common marmoset. To examine whether overexpression of LYL1 in EBs can promote hematopoietic differentiation in vitro we performed colony-forming unit (CFU) assay, and found that LYL1-overexpressing EBs showed the formation of multi-lineage blood cells consisting of erythroid cells, granulocytes and macrophages. Next, we analyzed gene expression level by RT-PCR, and found that the transduction of LYL1 induced the expression of various hematopoietic genes. These results suggested that the overexpression of LYL1 can promote the differentiation of CM ESCs to HSCs in vitro. Furthermore we found that the combined overexpression of TAL1 and LYL1 could enhance the differentiation of CD34+ cells from CM ESCs than the respective overexrpession of TAL1 or LYL1. Collectively, our novel technology to differentiate hematopoietic cells from ESCs by the transduction of specific transcription factors is novel, and might be applicable to expand human hematopoietic stem/progenitor cells in vitro for future regenerative medicine to cure human hematopoietic cell dyscrasias. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Novel Methyltransferase Methylates Cucumber Mosaic Virus 1a Protein and Promotes Systemic Spread

2008 ◽  
Vol 82 (10) ◽  
pp. 4823-4833 ◽  
Author(s):  
Min Jung Kim ◽  
Sung Un Huh ◽  
Byung-Kook Ham ◽  
Kyung-Hee Paek
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Protein Interactions ◽  
Protein Localization ◽  
Binding Activity ◽  
Virus Infectivity ◽  
Gene Encoding ◽  
Systemic Spread

ABSTRACT In mammalian and yeast systems, methyltransferases have been implicated in the regulation of diverse processes, such as protein-protein interactions, protein localization, signal transduction, RNA processing, and transcription. The Cucumber mosaic virus (CMV) 1a protein is essential not only for virus replication but also for movement. Using a yeast two-hybrid system with tobacco plants, we have identified a novel gene encoding a methyltransferase that interacts with the CMV 1a protein and have designated this gene Tcoi1 (tobacco CMV 1a-interacting protein 1). Tcoi1 specifically interacted with the methyltransferase domain of CMV 1a, and the expression of Tcoi1 was increased by CMV inoculation. Biochemical studies revealed that the interaction of Tcoi1 with CMV 1a protein was direct and that Tcoi1 methylated CMV 1a protein both in vitro and in vivo. The CMV 1a binding activity of Tcoi1 is in the C-terminal domain, which shows the methyltransferase activity. The overexpression of Tcoi1 enhanced the CMV infection, while the reduced expression of Tcoi1 decreased virus infectivity. These results suggest that Tcoi1 controls the propagation of CMV through an interaction with the CMV 1a protein.

scholarly journals Specificity and Regulation of DNA Binding by the Yeast Glucose Transporter Gene Repressor Rgt1

2003 ◽  
Vol 23 (15) ◽  
pp. 5208-5216 ◽  
Author(s):  
Jeong-Ho Kim ◽  
Jeffrey Polish ◽  
Mark Johnston
Keyword(s):  
Dna Binding ◽  
Glucose Transporter ◽  
Binding Activity ◽  
Sequence Motif ◽  
Consensus Binding Site ◽  
Binding Ability ◽  
Dna Binding Activity ◽  
Genes Encoding

ABSTRACT Rgt1 is a glucose-responsive transcription factor that binds to the promoters of several HXT genes encoding glucose transporters in Saccharomyces cerevisiae and regulates their expression in response to glucose. Rgt1 contains a Zn2Cys6 binuclear cluster responsible for DNA binding. Most proteins that contain this sequence motif bind as dimers to regularly spaced pairs of the sequence CGG. However, there are no CGG pairs with regular spacing in promoters of genes regulated by Rgt1, suggesting that Rgt1 binds as a monomer to CGG or to another sequence. We identified the Rgt1 consensus binding site sequence 5′-CGGANNA-3′, multiple copies of which are present in all HXT promoters regulated by Rgt1. Rgt1 binds in vivo to multiple sites in the HXT3 promoter in a nonadditive, synergistic manner, leading to synergistic repression of HXT3 transcription. We show that glucose inhibits the DNA-binding ability of Rgt1, thereby relieving repression of HXT gene expression. This regulation of Rgt1 DNA-binding activity is caused by its glucose-induced phosphorylation: the hyperphosphorylated Rgt1 present in cells growing on high levels of glucose does not bind DNA in vivo or in vitro; dephosphorylation of this form of Rgt1 in vitro restores its DNA-binding ability. Furthermore, an altered Rgt1 that functions as a constitutive repressor remains hypophosphorylated when glucose is added to cells and binds DNA under these conditions. These results suggest that glucose regulates the DNA-binding ability of Rgt1 by inducing its phosphorylation.

scholarly journals The adenovirus E4-6/7 protein transactivates the E2 promoter by inducing dimerization of a heteromeric E2F complex.

1994 ◽  
Vol 14 (2) ◽  
pp. 1333-1346 ◽  
Author(s):  
S Obert ◽  
R J O'Connor ◽  
S Schmid ◽  
P Hearing
Keyword(s):  
Binding Activity ◽  
Mutant Protein ◽  
Stable Complex ◽  
Physical Interaction ◽  
Dimerization Domain ◽  
Fab Fragment ◽  
Dna Binding Activity ◽  
Transcription Factor E2f

Binding of the mammalian transcription factor E2F to the adenovirus E2a early promoter is modulated through interaction with the viral E4-6/7 protein. E4-6/7 induces the cooperative and stable binding of E2F in vitro to two correctly spaced and inverted E2F binding sites in the E2a promoter (E2F induction) by physical interaction in the protein-DNA complex. The E2a promoter is transactivated in vivo by the E4-6/7 product. The C-terminal 70 amino acids of E4-6/7 are necessary and sufficient for induction of E2F binding and for transactivation. To assess the mechanism(s) of E2a transactivation and the induction of cooperative E2F binding by the E4-6/7 protein, we have analyzed a series of point mutants in the functional C-terminal domain of E4-6/7. Two distinct segments of E4-6/7 are required for interaction with E2F. Additionally, and E4-6/7 mutant with a phenylalanine-to-proline substitution at amino acid 125 (F-125-P) efficiently interacts with E2F but does not induce E2F binding to the E2a promoter and is defective for transactivation. Induction of E2F stable complex formation at the E2a promoter by the F-125-P mutant protein is restored by divalent E4-6/7-specific monoclonal antibodies, but not a monovalent Fab fragment, or by appending a heterologous dimerization domain to the N terminus of the mutant protein. These and other data support the involvement of E4-6/7 dimerization in the induction of cooperative and stable E2F binding and transactivation of the E2a promoter. We present evidence that at least two cellular components are involved in E2F DNA binding activity and that both are required for E2F induction by the E4-6/7 product. The recently cloned E2F-related activities E2F-1 and DP-1 individually bind to an E2F binding site weakly, but when combined generate an activity that is indistinguishable from endogenous cellular E2F. Recombinant E2F-1, DP-1, and E4-6/7 are sufficient to form the induced E2F complex at the E2a promoter.

scholarly journals DeepG4: A deep learning approach to predict cell-type specific active G-quadruplex regions

2021 ◽  
Vol 17 (8) ◽  
pp. e1009308
Author(s):  
Vincent Rocher ◽  
Matthieu Genais ◽  
Elissar Nassereddine ◽  
Raphael Mourad
Keyword(s):  
Deep Learning ◽  
Double Helix ◽  
Complex Molecule ◽  
Cell Types ◽  
Sequence Motif ◽  
Cell Type ◽  
G Quadruplex ◽  
Cell Type Specific

DNA is a complex molecule carrying the instructions an organism needs to develop, live and reproduce. In 1953, Watson and Crick discovered that DNA is composed of two chains forming a double-helix. Later on, other structures of DNA were discovered and shown to play important roles in the cell, in particular G-quadruplex (G4). Following genome sequencing, several bioinformatic algorithms were developed to map G4s in vitro based on a canonical sequence motif, G-richness and G-skewness or alternatively sequence features including k-mers, and more recently machine/deep learning. Recently, new sequencing techniques were developed to map G4s in vitro (G4-seq) and G4s in vivo (G4 ChIP-seq) at few hundred base resolution. Here, we propose a novel convolutional neural network (DeepG4) to map cell-type specific active G4 regions (e.g. regions within which G4s form both in vitro and in vivo). DeepG4 is very accurate to predict active G4 regions in different cell types. Moreover, DeepG4 identifies key DNA motifs that are predictive of G4 region activity. We found that such motifs do not follow a very flexible sequence pattern as current algorithms seek for. Instead, active G4 regions are determined by numerous specific motifs. Moreover, among those motifs, we identified known transcription factors (TFs) which could play important roles in G4 activity by contributing either directly to G4 structures themselves or indirectly by participating in G4 formation in the vicinity. In addition, we used DeepG4 to predict active G4 regions in a large number of tissues and cancers, thereby providing a comprehensive resource for researchers. Availability: https://github.com/morphos30/DeepG4.

scholarly journals Proximity labeling of protein complexes and cell-type-specific organellar proteomes in Arabidopsis enabled by TurboID

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Andrea Mair ◽  
Shou-Ling Xu ◽  
Tess C Branon ◽  
Alice Y Ting ◽  
Dominique C Bergmann
Keyword(s):  
Transcription Factor ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Cell Types ◽  
Specific Protein ◽  
Cell Type ◽  
Subcellular Compartment ◽  
Cellular Processes ◽  
Highly Active

Defining specific protein interactions and spatially or temporally restricted local proteomes improves our understanding of all cellular processes, but obtaining such data is challenging, especially for rare proteins, cell types, or events. Proximity labeling enables discovery of protein neighborhoods defining functional complexes and/or organellar protein compositions. Recent technological improvements, namely two highly active biotin ligase variants (TurboID and miniTurbo), allowed us to address two challenging questions in plants: (1) what are in vivo partners of a low abundant key developmental transcription factor and (2) what is the nuclear proteome of a rare cell type? Proteins identified with FAMA-TurboID include known interactors of this stomatal transcription factor and novel proteins that could facilitate its activator and repressor functions. Directing TurboID to stomatal nuclei enabled purification of cell type- and subcellular compartment-specific proteins. Broad tests of TurboID and miniTurbo in Arabidopsis and Nicotiana benthamiana and versatile vectors enable customization by plant researchers.

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