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A human B-cell interactome identifies MYB and FOXM1 as master regulators of proliferation in germinal centers.

Lefebvre C, Rajbhandari P, Alvarez MJ, Bandaru P, Lim WK, Sato M, Wang K, Sumazin P, Kustagi M, Bisikirska BC, Basso K, Beltrao P, Krogan N, Gautier J, Dalla-Favera R, Califano A - Mol. Syst. Biol. (2010)

Bottom Line: Assembly of a transcriptional and post-translational molecular interaction network in B cells, the human B-cell interactome (HBCI), reveals a hierarchical, transcriptional control module, where MYB and FOXM1 act as synergistic master regulators of proliferation in the germinal center (GC).Eighty percent of genes jointly regulated by these transcription factors are activated in the GC, including those encoding proteins in a complex regulating DNA pre-replication, replication, and mitosis.These results indicate that the HBCI analysis can be used for the identification of determinants of major human cell phenotypes and provides a paradigm of general applicability to normal and pathologic tissues.

View Article: PubMed Central - PubMed

Affiliation: Center for Computational Biology and Bioinformatics, Columbia University, New York, NY 10032, USA.

ABSTRACT
Assembly of a transcriptional and post-translational molecular interaction network in B cells, the human B-cell interactome (HBCI), reveals a hierarchical, transcriptional control module, where MYB and FOXM1 act as synergistic master regulators of proliferation in the germinal center (GC). Eighty percent of genes jointly regulated by these transcription factors are activated in the GC, including those encoding proteins in a complex regulating DNA pre-replication, replication, and mitosis. These results indicate that the HBCI analysis can be used for the identification of determinants of major human cell phenotypes and provides a paradigm of general applicability to normal and pathologic tissues.

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MYB directly regulates the transcription of FOXM1. (A) Western blot analysis of ST486 total cell lysates obtained at 24, 48, and 72 h after lentiviral-mediated transduction of control, FOXM1, or MYB shRNA. Viral load was set for complete silencing of FOXM1 at 24 h but only partial (>50%) silencing of MYB, as complete silencing induced >80% apoptosis at 48 h (data from previous experiment). GAPDH was used as a loading control. (B) Percentage of FOXM1 protein level on MYB silencing were calculated by densitometric analysis (in ImageJ) and normalized to GAPDH protein level. (C) qPCR analysis showing the mRNA expression level of FOXM1 and MYB at 24 h. Similar results were observed at 48 and 72 h (data not shown). (D) qChIP with control IgG or anti-MYB antibody at FOXM1-binding site. Data shown in (A, C) are representative of one of three independent experiments. Error bars were calculated (C) from samples run in duplicate or (D) from two sets of three independent real-time PCR reactions. (E) qChIP with control IgG, anti-FOXM1, and anti-MYB antibodies performed in the nuclear extract of ST486 cells. Samples from two independent experiments were pooled together and real-time PCR reactions were run in duplicate. Error bars represent the standard error.
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f3: MYB directly regulates the transcription of FOXM1. (A) Western blot analysis of ST486 total cell lysates obtained at 24, 48, and 72 h after lentiviral-mediated transduction of control, FOXM1, or MYB shRNA. Viral load was set for complete silencing of FOXM1 at 24 h but only partial (>50%) silencing of MYB, as complete silencing induced >80% apoptosis at 48 h (data from previous experiment). GAPDH was used as a loading control. (B) Percentage of FOXM1 protein level on MYB silencing were calculated by densitometric analysis (in ImageJ) and normalized to GAPDH protein level. (C) qPCR analysis showing the mRNA expression level of FOXM1 and MYB at 24 h. Similar results were observed at 48 and 72 h (data not shown). (D) qChIP with control IgG or anti-MYB antibody at FOXM1-binding site. Data shown in (A, C) are representative of one of three independent experiments. Error bars were calculated (C) from samples run in duplicate or (D) from two sets of three independent real-time PCR reactions. (E) qChIP with control IgG, anti-FOXM1, and anti-MYB antibodies performed in the nuclear extract of ST486 cells. Samples from two independent experiments were pooled together and real-time PCR reactions were run in duplicate. Error bars represent the standard error.

Mentions: We first tested whether MYB and FOXM1 may regulate each other as predicted in the HBCI. Synergistic TFs often participate in feed-forward loops (Palomero et al, 2006; Carro et al, 2010), increasing the resilience of regulatory networks to transient perturbations by the maintenance of the cell in a phenotype that could be affected by the fluctuation in the levels of either TF alone. MYB and FOXM1 were silenced in the ST486 Burkitt's lymphoma line, representative of transformed GC B cells, by lentiviral-mediated transduction of MYB/FOXM1-specific and control shRNAs (Figure 3). Decrease of endogenous MYB and FOXM1 protein levels in specific versus control shRNA-transduced samples was confirmed by western blot (Figure 3A and B). qPCR assays confirmed mRNA-level reduction of both TFs at 24 h (Figure 3C), persisting at 48 and 72 h (data not shown). MYB mRNA and protein levels were not affected by FOXM1 silencing, whereas FOXM1 mRNA and protein levels were both reduced in a time-dependent manner after MYB silencing (Figure 3A–C). These results suggest that MYB is a transcriptional activator of FOXM1. Quantitative chromatin IP (qChIP) assays showed that MYB binds in vivo to the FOXM1 promoter (Figure 3D). Together, these findings suggest that MYB and FOXM1 form a feed-forward loop, involved in the synergistic activation of a large subset of GC-specific genes.


A human B-cell interactome identifies MYB and FOXM1 as master regulators of proliferation in germinal centers.

Lefebvre C, Rajbhandari P, Alvarez MJ, Bandaru P, Lim WK, Sato M, Wang K, Sumazin P, Kustagi M, Bisikirska BC, Basso K, Beltrao P, Krogan N, Gautier J, Dalla-Favera R, Califano A - Mol. Syst. Biol. (2010)

MYB directly regulates the transcription of FOXM1. (A) Western blot analysis of ST486 total cell lysates obtained at 24, 48, and 72 h after lentiviral-mediated transduction of control, FOXM1, or MYB shRNA. Viral load was set for complete silencing of FOXM1 at 24 h but only partial (>50%) silencing of MYB, as complete silencing induced >80% apoptosis at 48 h (data from previous experiment). GAPDH was used as a loading control. (B) Percentage of FOXM1 protein level on MYB silencing were calculated by densitometric analysis (in ImageJ) and normalized to GAPDH protein level. (C) qPCR analysis showing the mRNA expression level of FOXM1 and MYB at 24 h. Similar results were observed at 48 and 72 h (data not shown). (D) qChIP with control IgG or anti-MYB antibody at FOXM1-binding site. Data shown in (A, C) are representative of one of three independent experiments. Error bars were calculated (C) from samples run in duplicate or (D) from two sets of three independent real-time PCR reactions. (E) qChIP with control IgG, anti-FOXM1, and anti-MYB antibodies performed in the nuclear extract of ST486 cells. Samples from two independent experiments were pooled together and real-time PCR reactions were run in duplicate. Error bars represent the standard error.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f3: MYB directly regulates the transcription of FOXM1. (A) Western blot analysis of ST486 total cell lysates obtained at 24, 48, and 72 h after lentiviral-mediated transduction of control, FOXM1, or MYB shRNA. Viral load was set for complete silencing of FOXM1 at 24 h but only partial (>50%) silencing of MYB, as complete silencing induced >80% apoptosis at 48 h (data from previous experiment). GAPDH was used as a loading control. (B) Percentage of FOXM1 protein level on MYB silencing were calculated by densitometric analysis (in ImageJ) and normalized to GAPDH protein level. (C) qPCR analysis showing the mRNA expression level of FOXM1 and MYB at 24 h. Similar results were observed at 48 and 72 h (data not shown). (D) qChIP with control IgG or anti-MYB antibody at FOXM1-binding site. Data shown in (A, C) are representative of one of three independent experiments. Error bars were calculated (C) from samples run in duplicate or (D) from two sets of three independent real-time PCR reactions. (E) qChIP with control IgG, anti-FOXM1, and anti-MYB antibodies performed in the nuclear extract of ST486 cells. Samples from two independent experiments were pooled together and real-time PCR reactions were run in duplicate. Error bars represent the standard error.
Mentions: We first tested whether MYB and FOXM1 may regulate each other as predicted in the HBCI. Synergistic TFs often participate in feed-forward loops (Palomero et al, 2006; Carro et al, 2010), increasing the resilience of regulatory networks to transient perturbations by the maintenance of the cell in a phenotype that could be affected by the fluctuation in the levels of either TF alone. MYB and FOXM1 were silenced in the ST486 Burkitt's lymphoma line, representative of transformed GC B cells, by lentiviral-mediated transduction of MYB/FOXM1-specific and control shRNAs (Figure 3). Decrease of endogenous MYB and FOXM1 protein levels in specific versus control shRNA-transduced samples was confirmed by western blot (Figure 3A and B). qPCR assays confirmed mRNA-level reduction of both TFs at 24 h (Figure 3C), persisting at 48 and 72 h (data not shown). MYB mRNA and protein levels were not affected by FOXM1 silencing, whereas FOXM1 mRNA and protein levels were both reduced in a time-dependent manner after MYB silencing (Figure 3A–C). These results suggest that MYB is a transcriptional activator of FOXM1. Quantitative chromatin IP (qChIP) assays showed that MYB binds in vivo to the FOXM1 promoter (Figure 3D). Together, these findings suggest that MYB and FOXM1 form a feed-forward loop, involved in the synergistic activation of a large subset of GC-specific genes.

Bottom Line: Assembly of a transcriptional and post-translational molecular interaction network in B cells, the human B-cell interactome (HBCI), reveals a hierarchical, transcriptional control module, where MYB and FOXM1 act as synergistic master regulators of proliferation in the germinal center (GC).Eighty percent of genes jointly regulated by these transcription factors are activated in the GC, including those encoding proteins in a complex regulating DNA pre-replication, replication, and mitosis.These results indicate that the HBCI analysis can be used for the identification of determinants of major human cell phenotypes and provides a paradigm of general applicability to normal and pathologic tissues.

View Article: PubMed Central - PubMed

Affiliation: Center for Computational Biology and Bioinformatics, Columbia University, New York, NY 10032, USA.

ABSTRACT
Assembly of a transcriptional and post-translational molecular interaction network in B cells, the human B-cell interactome (HBCI), reveals a hierarchical, transcriptional control module, where MYB and FOXM1 act as synergistic master regulators of proliferation in the germinal center (GC). Eighty percent of genes jointly regulated by these transcription factors are activated in the GC, including those encoding proteins in a complex regulating DNA pre-replication, replication, and mitosis. These results indicate that the HBCI analysis can be used for the identification of determinants of major human cell phenotypes and provides a paradigm of general applicability to normal and pathologic tissues.

Show MeSH