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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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BCL11A is a TLX corepressor.(A) Chromatin luciferase [Luc] assay showing corepressor activity of LSD1 with TLX as previosly published [18]. 293F cells, which have a stably integrated pGL4.31 reporter gene, were transiently transfected with pM or pM TLX LBD vector (200 ng each) and with 400 ng of pcDNA empty vector, and pCMX-FLAG LSD1. (B) Chromatin luciferase [Luc] assay showing corepressor activity of BCL11A with TLX. 293F cells, which have a stably integrated pGL4.31 reporter gene, were transiently transfected with pM or pM TLX LBD vector (200 ng each) and with 400 ng of pcDNA empty vector, pEF1a-BCL11-XL or pEF1a-BCL11-L. The P values were calculated by Student’s t test (n = 3).
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pone-0037963-g004: BCL11A is a TLX corepressor.(A) Chromatin luciferase [Luc] assay showing corepressor activity of LSD1 with TLX as previosly published [18]. 293F cells, which have a stably integrated pGL4.31 reporter gene, were transiently transfected with pM or pM TLX LBD vector (200 ng each) and with 400 ng of pcDNA empty vector, and pCMX-FLAG LSD1. (B) Chromatin luciferase [Luc] assay showing corepressor activity of BCL11A with TLX. 293F cells, which have a stably integrated pGL4.31 reporter gene, were transiently transfected with pM or pM TLX LBD vector (200 ng each) and with 400 ng of pcDNA empty vector, pEF1a-BCL11-XL or pEF1a-BCL11-L. The P values were calculated by Student’s t test (n = 3).

Mentions: We next investigated the functional significance of the interaction between TLX and BCL11A in TLX transrepression activity using an in vitro luciferase reporter assay [18]. We used HEK293F cells, which have a stably integrated pGL4.31 reporter gene containing five GAL4 upstream activation sequence sites in the luciferase gene promoter, which were transiently transfected with TLX-LBD and coregulators, alone or in combination. Transfection of TLX-LBD alone repressed reporter gene activity, while co-transfection of TLX-LBD with LSD1, a known TLX coregulator, potentiated the transrepressive activity of TLX as previously described [18] (Figure 4A). When combining TLX-LBD with BCL11-XL and L isoforms a similar potentiation of TLX transrepression activity was observed (Figure 4B). Furthermore, transfection of BCL11A isoforms alone also repressed the luciferase reporter (Figure 4B). Together our data suggests that BCL11A functions as a TLX coregulator that represses transcriptional activity in TLX-dependent and TLX-independent ways.


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)

BCL11A is a TLX corepressor.(A) Chromatin luciferase [Luc] assay showing corepressor activity of LSD1 with TLX as previosly published [18]. 293F cells, which have a stably integrated pGL4.31 reporter gene, were transiently transfected with pM or pM TLX LBD vector (200 ng each) and with 400 ng of pcDNA empty vector, and pCMX-FLAG LSD1. (B) Chromatin luciferase [Luc] assay showing corepressor activity of BCL11A with TLX. 293F cells, which have a stably integrated pGL4.31 reporter gene, were transiently transfected with pM or pM TLX LBD vector (200 ng each) and with 400 ng of pcDNA empty vector, pEF1a-BCL11-XL or pEF1a-BCL11-L. The P values were calculated by Student’s t test (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0037963-g004: BCL11A is a TLX corepressor.(A) Chromatin luciferase [Luc] assay showing corepressor activity of LSD1 with TLX as previosly published [18]. 293F cells, which have a stably integrated pGL4.31 reporter gene, were transiently transfected with pM or pM TLX LBD vector (200 ng each) and with 400 ng of pcDNA empty vector, and pCMX-FLAG LSD1. (B) Chromatin luciferase [Luc] assay showing corepressor activity of BCL11A with TLX. 293F cells, which have a stably integrated pGL4.31 reporter gene, were transiently transfected with pM or pM TLX LBD vector (200 ng each) and with 400 ng of pcDNA empty vector, pEF1a-BCL11-XL or pEF1a-BCL11-L. The P values were calculated by Student’s t test (n = 3).
Mentions: We next investigated the functional significance of the interaction between TLX and BCL11A in TLX transrepression activity using an in vitro luciferase reporter assay [18]. We used HEK293F cells, which have a stably integrated pGL4.31 reporter gene containing five GAL4 upstream activation sequence sites in the luciferase gene promoter, which were transiently transfected with TLX-LBD and coregulators, alone or in combination. Transfection of TLX-LBD alone repressed reporter gene activity, while co-transfection of TLX-LBD with LSD1, a known TLX coregulator, potentiated the transrepressive activity of TLX as previously described [18] (Figure 4A). When combining TLX-LBD with BCL11-XL and L isoforms a similar potentiation of TLX transrepression activity was observed (Figure 4B). Furthermore, transfection of BCL11A isoforms alone also repressed the luciferase reporter (Figure 4B). Together our data suggests that BCL11A functions as a TLX coregulator that represses transcriptional activity in TLX-dependent and TLX-independent ways.

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