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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-p65, P-TEFb and TNFα influence MYB elongation.(A) 293 cells were treated with the P-TEFb inhibitor DRB for 6 h and endogenous MYB expression assessed by QPCR. (B) Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT reporter and 0.125 μg of pcDNA NFκBp50-p65. At 12 h post transfection cells were treated with DRB and incubated for a further 24 h. (C) NFκBp50-p65 induces endogenous MYB. Total RNA was isolated from 293 cells transfected with; 1 μg of pcDNA NFκB p50-p65, 1 μg of pcDNA NFκBp50 K148A-p65 or 4 μg of pcDNA NFκBp50 K146-148A-p65 and analyzed by Q-PCR to measure MYB expression levels and (D) 1 μg of pcDNA NFκB p50-p65 and analyzed by Q-PCR to measure intronic pre-mRNA MYB transcript upstream (preSLR) and downstream (post SLR) of the MYB SLR polyU. Data are expressed as a ratio “post/pre”, a measure of the amount of transcription through the SLR. (E) ChIP analysis of RNA polymerase II levels at the MYB SLR polyU. 293 cells were transfected with pcDNA, NFκB p50-p65 or pcDNA NFκBp50 K146-148A-p65. Cross-linked chromatin extracts were prepared at 48h post transfection and RNA polymerase II was detected by anti-pol II followed by Q-PCR. (F) SI organoids cultures and 293 cells were exposed to TNFα (20–100 ng/ml) for the times indicated and total RNA was isolated and analyzed by Q-PCR to measure MYB expression levels. (G) 293 cells were transfected with 2 μg of the MYB SLR polyU CAT or MYB ΔSLR polyU CAT reporter. At 24 h post transfection cells were exposed to 100 ng/ml TNFα for 10 h and CAT reporter activity assessed. (H) 293 cells were exposed to BAY inhibitor for the times indicated and total RNA was isolated and analyzed by Q-PCR to measure MYB expression levels. Error bars represent mean ± SEM, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.
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pone.0122919.g005: NFκBp50-p65, P-TEFb and TNFα influence MYB elongation.(A) 293 cells were treated with the P-TEFb inhibitor DRB for 6 h and endogenous MYB expression assessed by QPCR. (B) Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT reporter and 0.125 μg of pcDNA NFκBp50-p65. At 12 h post transfection cells were treated with DRB and incubated for a further 24 h. (C) NFκBp50-p65 induces endogenous MYB. Total RNA was isolated from 293 cells transfected with; 1 μg of pcDNA NFκB p50-p65, 1 μg of pcDNA NFκBp50 K148A-p65 or 4 μg of pcDNA NFκBp50 K146-148A-p65 and analyzed by Q-PCR to measure MYB expression levels and (D) 1 μg of pcDNA NFκB p50-p65 and analyzed by Q-PCR to measure intronic pre-mRNA MYB transcript upstream (preSLR) and downstream (post SLR) of the MYB SLR polyU. Data are expressed as a ratio “post/pre”, a measure of the amount of transcription through the SLR. (E) ChIP analysis of RNA polymerase II levels at the MYB SLR polyU. 293 cells were transfected with pcDNA, NFκB p50-p65 or pcDNA NFκBp50 K146-148A-p65. Cross-linked chromatin extracts were prepared at 48h post transfection and RNA polymerase II was detected by anti-pol II followed by Q-PCR. (F) SI organoids cultures and 293 cells were exposed to TNFα (20–100 ng/ml) for the times indicated and total RNA was isolated and analyzed by Q-PCR to measure MYB expression levels. (G) 293 cells were transfected with 2 μg of the MYB SLR polyU CAT or MYB ΔSLR polyU CAT reporter. At 24 h post transfection cells were exposed to 100 ng/ml TNFα for 10 h and CAT reporter activity assessed. (H) 293 cells were exposed to BAY inhibitor for the times indicated and total RNA was isolated and analyzed by Q-PCR to measure MYB expression levels. Error bars represent mean ± SEM, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Mentions: We next examined whether the elongation factor P-TEFb influenced MYB expression. Previous studies have established that the P-TEFb inhibitory drug DRB inhibits elongation that leads to the synthesis of longer MYB transcripts [22]. We therefore treated 293 cells with DRB for 6 h and assessed the expression of MYB by QPCR. MYB expression was significantly down regulated in drug treated cells (Fig 5A). Furthermore, DRB treatment of 293 cells inhibited the NFκBp50-65 mediated induction of MYB elongation from the MYB SLR polyU CAT reporter (Fig 5B). Collectively these data suggest that P-TEFb could influence NFκBp50-65 mediated induction of MYB elongation.


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-p65, P-TEFb and TNFα influence MYB elongation.(A) 293 cells were treated with the P-TEFb inhibitor DRB for 6 h and endogenous MYB expression assessed by QPCR. (B) Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT reporter and 0.125 μg of pcDNA NFκBp50-p65. At 12 h post transfection cells were treated with DRB and incubated for a further 24 h. (C) NFκBp50-p65 induces endogenous MYB. Total RNA was isolated from 293 cells transfected with; 1 μg of pcDNA NFκB p50-p65, 1 μg of pcDNA NFκBp50 K148A-p65 or 4 μg of pcDNA NFκBp50 K146-148A-p65 and analyzed by Q-PCR to measure MYB expression levels and (D) 1 μg of pcDNA NFκB p50-p65 and analyzed by Q-PCR to measure intronic pre-mRNA MYB transcript upstream (preSLR) and downstream (post SLR) of the MYB SLR polyU. Data are expressed as a ratio “post/pre”, a measure of the amount of transcription through the SLR. (E) ChIP analysis of RNA polymerase II levels at the MYB SLR polyU. 293 cells were transfected with pcDNA, NFκB p50-p65 or pcDNA NFκBp50 K146-148A-p65. Cross-linked chromatin extracts were prepared at 48h post transfection and RNA polymerase II was detected by anti-pol II followed by Q-PCR. (F) SI organoids cultures and 293 cells were exposed to TNFα (20–100 ng/ml) for the times indicated and total RNA was isolated and analyzed by Q-PCR to measure MYB expression levels. (G) 293 cells were transfected with 2 μg of the MYB SLR polyU CAT or MYB ΔSLR polyU CAT reporter. At 24 h post transfection cells were exposed to 100 ng/ml TNFα for 10 h and CAT reporter activity assessed. (H) 293 cells were exposed to BAY inhibitor for the times indicated and total RNA was isolated and analyzed by Q-PCR to measure MYB expression levels. Error bars represent mean ± SEM, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.
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pone.0122919.g005: NFκBp50-p65, P-TEFb and TNFα influence MYB elongation.(A) 293 cells were treated with the P-TEFb inhibitor DRB for 6 h and endogenous MYB expression assessed by QPCR. (B) Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT reporter and 0.125 μg of pcDNA NFκBp50-p65. At 12 h post transfection cells were treated with DRB and incubated for a further 24 h. (C) NFκBp50-p65 induces endogenous MYB. Total RNA was isolated from 293 cells transfected with; 1 μg of pcDNA NFκB p50-p65, 1 μg of pcDNA NFκBp50 K148A-p65 or 4 μg of pcDNA NFκBp50 K146-148A-p65 and analyzed by Q-PCR to measure MYB expression levels and (D) 1 μg of pcDNA NFκB p50-p65 and analyzed by Q-PCR to measure intronic pre-mRNA MYB transcript upstream (preSLR) and downstream (post SLR) of the MYB SLR polyU. Data are expressed as a ratio “post/pre”, a measure of the amount of transcription through the SLR. (E) ChIP analysis of RNA polymerase II levels at the MYB SLR polyU. 293 cells were transfected with pcDNA, NFκB p50-p65 or pcDNA NFκBp50 K146-148A-p65. Cross-linked chromatin extracts were prepared at 48h post transfection and RNA polymerase II was detected by anti-pol II followed by Q-PCR. (F) SI organoids cultures and 293 cells were exposed to TNFα (20–100 ng/ml) for the times indicated and total RNA was isolated and analyzed by Q-PCR to measure MYB expression levels. (G) 293 cells were transfected with 2 μg of the MYB SLR polyU CAT or MYB ΔSLR polyU CAT reporter. At 24 h post transfection cells were exposed to 100 ng/ml TNFα for 10 h and CAT reporter activity assessed. (H) 293 cells were exposed to BAY inhibitor for the times indicated and total RNA was isolated and analyzed by Q-PCR to measure MYB expression levels. Error bars represent mean ± SEM, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.
Mentions: We next examined whether the elongation factor P-TEFb influenced MYB expression. Previous studies have established that the P-TEFb inhibitory drug DRB inhibits elongation that leads to the synthesis of longer MYB transcripts [22]. We therefore treated 293 cells with DRB for 6 h and assessed the expression of MYB by QPCR. MYB expression was significantly down regulated in drug treated cells (Fig 5A). Furthermore, DRB treatment of 293 cells inhibited the NFκBp50-65 mediated induction of MYB elongation from the MYB SLR polyU CAT reporter (Fig 5B). Collectively these data suggest that P-TEFb could influence NFκBp50-65 mediated induction of MYB elongation.

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