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MYB elongation is regulated by the nucleic acid binding of NFκB p50 to the intronic stem-loop region.

Pereira LA, Hugo HJ, Malaterre J, Huiling X, Sonza S, Cures A, Purcell DF, Ramsland PA, Gerondakis S, Gonda TJ, Ramsay RG - PLoS ONE (2015)

Bottom Line: We identified a conserved lysine-rich motif within the Rel homology domain (RHD) of NFκBp50, mutation of which abrogated the interaction of NFκBp50 with the SLR polyU and impaired NFκBp50 mediated MYB elongation.Furthermore, we identify the DNA binding activity of NFκBp50 as a key component required for the SLR polyU mediated regulation of MYB.Collectively these results suggest that the MYB SLR polyU provides a platform for proteins to regulate MYB and reveals novel nucleic acid binding properties of NFκBp50 required for MYB regulation.

View Article: PubMed Central - PubMed

Affiliation: Differentiation and Transcription Laboratory, Peter MacCallum Cancer Centre, Locked Bag #1, Melbourne, Victoria, 8006, Australia; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, 3010, Australia.

ABSTRACT
MYB transcriptional elongation is regulated by an attenuator sequence within intron 1 that has been proposed to encode a RNA stem loop (SLR) followed by a polyU tract. We report that NFκBp50 can bind the SLR polyU RNA and promote MYB transcriptional elongation together with NFκBp65. We identified a conserved lysine-rich motif within the Rel homology domain (RHD) of NFκBp50, mutation of which abrogated the interaction of NFκBp50 with the SLR polyU and impaired NFκBp50 mediated MYB elongation. We observed that the TAR RNA-binding region of Tat is homologous to the NFκBp50 RHD lysine-rich motif, a finding consistent with HIV Tat acting as an effector of MYB transcriptional elongation in an SLR dependent manner. Furthermore, we identify the DNA binding activity of NFκBp50 as a key component required for the SLR polyU mediated regulation of MYB. Collectively these results suggest that the MYB SLR polyU provides a platform for proteins to regulate MYB and reveals novel nucleic acid binding properties of NFκBp50 required for MYB regulation.

No MeSH data available.


Related in: MedlinePlus

NFκBp50 and NFκBp65 induce MYB elongation via the MYB SLR polyU.(A) Top panel: The 5´ genomic structure of MYB and the CAT reporter constructs is depicted. MYB ΔSLR polyU CAT has a 76 bp deletion of the SLR sequence up to the 19 nucleotide polyU stretch. MYB SLR ΔpolyU CAT contains a deletion of the 19 nucleotide polyU stretch. Bottom left panel: Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT reporter and 0.5 μg of pcDNA NFκBp50. Bottom right panel: Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT, MYB ΔSLR polyU CAT or MYB SLR ΔpolyU CAT reporters and 0.5 μg of pcDNA NFκBp65. (B) Left panel: Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT, MYB ΔSLR polyU CAT or MYB SLR ΔpolyU CAT reporters and 0.25 μg of pcDNA NFκBp50-p65; Right panel: Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT or MYB Promoter CAT reporters and 0.25 μg of pcDNA NFκBp50-p65. (C) Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT, MYB SLR 3L mutation polyU CAT, MYB SLR 5C mutation polyU CAT or MYB SLR 23C mutation polyU CAT reporters with 0.25 μg of pcDNA NFκBp50-p65. (D) Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT reporter with 0.25 μg of pcDNA NFκBp50-p65, 0.25 μg of pcDNA NFκBp50 K148A-p65 or 1 μg of pcDNA NFκBp50 K146-148A-p65. Error bars represent mean ± SEM, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.
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pone.0122919.g004: NFκBp50 and NFκBp65 induce MYB elongation via the MYB SLR polyU.(A) Top panel: The 5´ genomic structure of MYB and the CAT reporter constructs is depicted. MYB ΔSLR polyU CAT has a 76 bp deletion of the SLR sequence up to the 19 nucleotide polyU stretch. MYB SLR ΔpolyU CAT contains a deletion of the 19 nucleotide polyU stretch. Bottom left panel: Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT reporter and 0.5 μg of pcDNA NFκBp50. Bottom right panel: Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT, MYB ΔSLR polyU CAT or MYB SLR ΔpolyU CAT reporters and 0.5 μg of pcDNA NFκBp65. (B) Left panel: Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT, MYB ΔSLR polyU CAT or MYB SLR ΔpolyU CAT reporters and 0.25 μg of pcDNA NFκBp50-p65; Right panel: Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT or MYB Promoter CAT reporters and 0.25 μg of pcDNA NFκBp50-p65. (C) Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT, MYB SLR 3L mutation polyU CAT, MYB SLR 5C mutation polyU CAT or MYB SLR 23C mutation polyU CAT reporters with 0.25 μg of pcDNA NFκBp50-p65. (D) Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT reporter with 0.25 μg of pcDNA NFκBp50-p65, 0.25 μg of pcDNA NFκBp50 K148A-p65 or 1 μg of pcDNA NFκBp50 K146-148A-p65. Error bars represent mean ± SEM, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Mentions: The above experiments suggested that NFκBp50 might engage the MYB SLR polyU to regulate MYB elongation. To examine this possibility, the potential of NFκBp50 to regulate elongation in gene reporter assays was assessed using a series of CAT reporter constructs containing the promoter, exon 1 and intron 1 with or without the MYB SLR or polyU [21] (Fig 4A).


MYB elongation is regulated by the nucleic acid binding of NFκB p50 to the intronic stem-loop region.

Pereira LA, Hugo HJ, Malaterre J, Huiling X, Sonza S, Cures A, Purcell DF, Ramsland PA, Gerondakis S, Gonda TJ, Ramsay RG - PLoS ONE (2015)

NFκBp50 and NFκBp65 induce MYB elongation via the MYB SLR polyU.(A) Top panel: The 5´ genomic structure of MYB and the CAT reporter constructs is depicted. MYB ΔSLR polyU CAT has a 76 bp deletion of the SLR sequence up to the 19 nucleotide polyU stretch. MYB SLR ΔpolyU CAT contains a deletion of the 19 nucleotide polyU stretch. Bottom left panel: Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT reporter and 0.5 μg of pcDNA NFκBp50. Bottom right panel: Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT, MYB ΔSLR polyU CAT or MYB SLR ΔpolyU CAT reporters and 0.5 μg of pcDNA NFκBp65. (B) Left panel: Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT, MYB ΔSLR polyU CAT or MYB SLR ΔpolyU CAT reporters and 0.25 μg of pcDNA NFκBp50-p65; Right panel: Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT or MYB Promoter CAT reporters and 0.25 μg of pcDNA NFκBp50-p65. (C) Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT, MYB SLR 3L mutation polyU CAT, MYB SLR 5C mutation polyU CAT or MYB SLR 23C mutation polyU CAT reporters with 0.25 μg of pcDNA NFκBp50-p65. (D) Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT reporter with 0.25 μg of pcDNA NFκBp50-p65, 0.25 μg of pcDNA NFκBp50 K148A-p65 or 1 μg of pcDNA NFκBp50 K146-148A-p65. Error bars represent mean ± SEM, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.
© Copyright Policy
Related In: Results  -  Collection

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pone.0122919.g004: NFκBp50 and NFκBp65 induce MYB elongation via the MYB SLR polyU.(A) Top panel: The 5´ genomic structure of MYB and the CAT reporter constructs is depicted. MYB ΔSLR polyU CAT has a 76 bp deletion of the SLR sequence up to the 19 nucleotide polyU stretch. MYB SLR ΔpolyU CAT contains a deletion of the 19 nucleotide polyU stretch. Bottom left panel: Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT reporter and 0.5 μg of pcDNA NFκBp50. Bottom right panel: Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT, MYB ΔSLR polyU CAT or MYB SLR ΔpolyU CAT reporters and 0.5 μg of pcDNA NFκBp65. (B) Left panel: Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT, MYB ΔSLR polyU CAT or MYB SLR ΔpolyU CAT reporters and 0.25 μg of pcDNA NFκBp50-p65; Right panel: Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT or MYB Promoter CAT reporters and 0.25 μg of pcDNA NFκBp50-p65. (C) Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT, MYB SLR 3L mutation polyU CAT, MYB SLR 5C mutation polyU CAT or MYB SLR 23C mutation polyU CAT reporters with 0.25 μg of pcDNA NFκBp50-p65. (D) Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT reporter with 0.25 μg of pcDNA NFκBp50-p65, 0.25 μg of pcDNA NFκBp50 K148A-p65 or 1 μg of pcDNA NFκBp50 K146-148A-p65. Error bars represent mean ± SEM, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.
Mentions: The above experiments suggested that NFκBp50 might engage the MYB SLR polyU to regulate MYB elongation. To examine this possibility, the potential of NFκBp50 to regulate elongation in gene reporter assays was assessed using a series of CAT reporter constructs containing the promoter, exon 1 and intron 1 with or without the MYB SLR or polyU [21] (Fig 4A).

Bottom Line: We identified a conserved lysine-rich motif within the Rel homology domain (RHD) of NFκBp50, mutation of which abrogated the interaction of NFκBp50 with the SLR polyU and impaired NFκBp50 mediated MYB elongation.Furthermore, we identify the DNA binding activity of NFκBp50 as a key component required for the SLR polyU mediated regulation of MYB.Collectively these results suggest that the MYB SLR polyU provides a platform for proteins to regulate MYB and reveals novel nucleic acid binding properties of NFκBp50 required for MYB regulation.

View Article: PubMed Central - PubMed

Affiliation: Differentiation and Transcription Laboratory, Peter MacCallum Cancer Centre, Locked Bag #1, Melbourne, Victoria, 8006, Australia; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, 3010, Australia.

ABSTRACT
MYB transcriptional elongation is regulated by an attenuator sequence within intron 1 that has been proposed to encode a RNA stem loop (SLR) followed by a polyU tract. We report that NFκBp50 can bind the SLR polyU RNA and promote MYB transcriptional elongation together with NFκBp65. We identified a conserved lysine-rich motif within the Rel homology domain (RHD) of NFκBp50, mutation of which abrogated the interaction of NFκBp50 with the SLR polyU and impaired NFκBp50 mediated MYB elongation. We observed that the TAR RNA-binding region of Tat is homologous to the NFκBp50 RHD lysine-rich motif, a finding consistent with HIV Tat acting as an effector of MYB transcriptional elongation in an SLR dependent manner. Furthermore, we identify the DNA binding activity of NFκBp50 as a key component required for the SLR polyU mediated regulation of MYB. Collectively these results suggest that the MYB SLR polyU provides a platform for proteins to regulate MYB and reveals novel nucleic acid binding properties of NFκBp50 required for MYB regulation.

No MeSH data available.


Related in: MedlinePlus