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The oncoprotein BCL11A binds to orphan nuclear receptor TLX and potentiates its transrepressive function.

Estruch SB, Buzón V, Carbó LR, Schorova L, Lüders J, Estébanez-Perpiñá E - PLoS ONE (2012)

Bottom Line: This interaction was validated by expression and coimmunoprecipitation in human cells.BCL11A potentiated the transrepressive function of TLX in an in vitro reporter gene assay.Our work suggests that BCL11A is a novel TLX coregulator that might be involved in TLX-dependent gene regulation in the brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology and Institute of Biomedicine from the University of Barcelona, University of Barcelona, Barcelona, Spain.

ABSTRACT
Nuclear orphan receptor TLX (NR2E1) functions primarily as a transcriptional repressor and its pivotal role in brain development, glioblastoma, mental retardation and retinopathologies make it an attractive drug target. TLX is expressed in the neural stem cells (NSCs) of the subventricular zone and the hippocampus subgranular zone, regions with persistent neurogenesis in the adult brain, and functions as an essential regulator of NSCs maintenance and self-renewal. Little is known about the TLX social network of interactors and only few TLX coregulators are described. To identify and characterize novel TLX-binders and possible coregulators, we performed yeast-two-hybrid (Y2H) screens of a human adult brain cDNA library using different TLX constructs as baits. Our screens identified multiple clones of Atrophin-1 (ATN1), a previously described TLX interactor. In addition, we identified an interaction with the oncoprotein and zinc finger transcription factor BCL11A (CTIP1/Evi9), a key player in the hematopoietic system and in major blood-related malignancies. This interaction was validated by expression and coimmunoprecipitation in human cells. BCL11A potentiated the transrepressive function of TLX in an in vitro reporter gene assay. Our work suggests that BCL11A is a novel TLX coregulator that might be involved in TLX-dependent gene regulation in the brain.

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Human TLX recruits ATN1.Y2H screens using TLX-FL and TLX-LBD domain as baits against an adult brain cDNA library identified ATN1 as a TLX-interactor. (A) Schematic diagram of the identified ATN1 clones. All clones share a 378 amino acid overlapping region (residues 813–1190) featuring the ATRO-BOX region. (B) Validation of the interaction between TLX and the identified ATN1 clones by forward one-to-one Y2H assay. TLX constructs FL-TLX (1–385), TLX-LBD (172–385), TLX-H-LBD (94–385) and TLX-DBD (1–95) (baits) were tested for interaction with the ATN1 clones identified (preys). Yeast transformants were plated on a control plate (lacking Trp and Leu) and plated on a selective plate (lacking Trp, Leu, His supplemented with 50 mM 3AT).
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pone-0037963-g001: Human TLX recruits ATN1.Y2H screens using TLX-FL and TLX-LBD domain as baits against an adult brain cDNA library identified ATN1 as a TLX-interactor. (A) Schematic diagram of the identified ATN1 clones. All clones share a 378 amino acid overlapping region (residues 813–1190) featuring the ATRO-BOX region. (B) Validation of the interaction between TLX and the identified ATN1 clones by forward one-to-one Y2H assay. TLX constructs FL-TLX (1–385), TLX-LBD (172–385), TLX-H-LBD (94–385) and TLX-DBD (1–95) (baits) were tested for interaction with the ATN1 clones identified (preys). Yeast transformants were plated on a control plate (lacking Trp and Leu) and plated on a selective plate (lacking Trp, Leu, His supplemented with 50 mM 3AT).

Mentions: Our screenings with the two different baits (FL-TLX and TLX-LBD) yielded a large number of clones that were identified as overlapping sequences of ATN1 (Figure 1A and Text S1-A). The region shared by all ATN1 clones comprises residues 813 to 1190, which includes the so-called ATRO-Box and is in good agreement with the previously defined TLX-binding region in ATN1 (residues 800–1000) [16][18][23]–[24] (Figure 1A). The identification of ATN1 as a TLX interactor confirmed the functionality of our bait constructs and Y2H screens. All identified ATN1 clones were also validated in one-to-one Y2H assays against all TLX constructs. The interaction ATN1-TLX only occurred in the TLX constructs featuring a LBD domain and was lost when TLX-NT-DBD construct was tested (Figure 1B).


The oncoprotein BCL11A binds to orphan nuclear receptor TLX and potentiates its transrepressive function.

Estruch SB, Buzón V, Carbó LR, Schorova L, Lüders J, Estébanez-Perpiñá E - PLoS ONE (2012)

Human TLX recruits ATN1.Y2H screens using TLX-FL and TLX-LBD domain as baits against an adult brain cDNA library identified ATN1 as a TLX-interactor. (A) Schematic diagram of the identified ATN1 clones. All clones share a 378 amino acid overlapping region (residues 813–1190) featuring the ATRO-BOX region. (B) Validation of the interaction between TLX and the identified ATN1 clones by forward one-to-one Y2H assay. TLX constructs FL-TLX (1–385), TLX-LBD (172–385), TLX-H-LBD (94–385) and TLX-DBD (1–95) (baits) were tested for interaction with the ATN1 clones identified (preys). Yeast transformants were plated on a control plate (lacking Trp and Leu) and plated on a selective plate (lacking Trp, Leu, His supplemented with 50 mM 3AT).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366998&req=5

pone-0037963-g001: Human TLX recruits ATN1.Y2H screens using TLX-FL and TLX-LBD domain as baits against an adult brain cDNA library identified ATN1 as a TLX-interactor. (A) Schematic diagram of the identified ATN1 clones. All clones share a 378 amino acid overlapping region (residues 813–1190) featuring the ATRO-BOX region. (B) Validation of the interaction between TLX and the identified ATN1 clones by forward one-to-one Y2H assay. TLX constructs FL-TLX (1–385), TLX-LBD (172–385), TLX-H-LBD (94–385) and TLX-DBD (1–95) (baits) were tested for interaction with the ATN1 clones identified (preys). Yeast transformants were plated on a control plate (lacking Trp and Leu) and plated on a selective plate (lacking Trp, Leu, His supplemented with 50 mM 3AT).
Mentions: Our screenings with the two different baits (FL-TLX and TLX-LBD) yielded a large number of clones that were identified as overlapping sequences of ATN1 (Figure 1A and Text S1-A). The region shared by all ATN1 clones comprises residues 813 to 1190, which includes the so-called ATRO-Box and is in good agreement with the previously defined TLX-binding region in ATN1 (residues 800–1000) [16][18][23]–[24] (Figure 1A). The identification of ATN1 as a TLX interactor confirmed the functionality of our bait constructs and Y2H screens. All identified ATN1 clones were also validated in one-to-one Y2H assays against all TLX constructs. The interaction ATN1-TLX only occurred in the TLX constructs featuring a LBD domain and was lost when TLX-NT-DBD construct was tested (Figure 1B).

Bottom Line: This interaction was validated by expression and coimmunoprecipitation in human cells.BCL11A potentiated the transrepressive function of TLX in an in vitro reporter gene assay.Our work suggests that BCL11A is a novel TLX coregulator that might be involved in TLX-dependent gene regulation in the brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology and Institute of Biomedicine from the University of Barcelona, University of Barcelona, Barcelona, Spain.

ABSTRACT
Nuclear orphan receptor TLX (NR2E1) functions primarily as a transcriptional repressor and its pivotal role in brain development, glioblastoma, mental retardation and retinopathologies make it an attractive drug target. TLX is expressed in the neural stem cells (NSCs) of the subventricular zone and the hippocampus subgranular zone, regions with persistent neurogenesis in the adult brain, and functions as an essential regulator of NSCs maintenance and self-renewal. Little is known about the TLX social network of interactors and only few TLX coregulators are described. To identify and characterize novel TLX-binders and possible coregulators, we performed yeast-two-hybrid (Y2H) screens of a human adult brain cDNA library using different TLX constructs as baits. Our screens identified multiple clones of Atrophin-1 (ATN1), a previously described TLX interactor. In addition, we identified an interaction with the oncoprotein and zinc finger transcription factor BCL11A (CTIP1/Evi9), a key player in the hematopoietic system and in major blood-related malignancies. This interaction was validated by expression and coimmunoprecipitation in human cells. BCL11A potentiated the transrepressive function of TLX in an in vitro reporter gene assay. Our work suggests that BCL11A is a novel TLX coregulator that might be involved in TLX-dependent gene regulation in the brain.

Show MeSH
Related in: MedlinePlus