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BCL6 positively regulates AID and germinal center gene expression via repression of miR-155.

Basso K, Schneider C, Shen Q, Holmes AB, Setty M, Leslie C, Dalla-Favera R - J. Exp. Med. (2012)

Bottom Line: We have identified a core of 15 miRNAs that show binding of BCL6 in their genomic loci and are down-regulated in GC B cells.Similarly, the expression of additional genes relevant for the GC phenotype, including SPI1, IRF8, and MYB, appears to be sustained via BCL6-mediated repression of miR-155.These findings identify a novel mechanism by which BCL6, in addition to repressing protein coding genes, promotes the expression of important GC functions by repressing specific miRNAs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Cancer Genetics, Columbia University, New York, NY 10027, USA. kb451@columbia.edu

ABSTRACT
The BCL6 proto-oncogene encodes a transcriptional repressor that is required for germinal center (GC) formation and whose de-regulation is involved in lymphomagenesis. Although substantial evidence indicates that BCL6 exerts its function by repressing the transcription of hundreds of protein-coding genes, its potential role in regulating gene expression via microRNAs (miRNAs) is not known. We have identified a core of 15 miRNAs that show binding of BCL6 in their genomic loci and are down-regulated in GC B cells. Among BCL6 validated targets, miR-155 and miR-361 directly modulate AID expression, indicating that via repression of these miRNAs, BCL6 up-regulates AID. Similarly, the expression of additional genes relevant for the GC phenotype, including SPI1, IRF8, and MYB, appears to be sustained via BCL6-mediated repression of miR-155. These findings identify a novel mechanism by which BCL6, in addition to repressing protein coding genes, promotes the expression of important GC functions by repressing specific miRNAs.

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miR-155 and miR-361 target GC-relevant genes, including AICDA. (a) The effect of induction of miR-155 by doxycycline (Dox) treatment in engineered Burkitt’s lymphoma cell lines (P3HR1 and Raji) on the expression of AICDA, SPI1, IRF8, and MYB, as detected by immunoblotting (left). The right panel represents the relative fold changes obtained in three independent experiments (mean ± SD, n = 3; *, P ≤ 0.05, Student’s t test). (b) Effects of miR-361 induction on AICDA protein levels. Normalized relative fold change in protein levels are reported below each lane. The right panel represents the relative fold changes obtained in three independent experiments (mean ± SD, n = 3; *, P ≤ 0.05, Student’s t test). (c) AICDA-3′UTR reporter assay in response to increasing amount of miR-155 and miR-361. On the right, schematic representation of the regions in the AICDA-3′UTR (AICDA-3′UTR WT) that are targeted by miR-155 and miR-361 and their alignments with the miRNA sequences. The mutations introduced in the mutant UTRs (AICDA-3′UTR Mut) are displayed below (mutated nucleotides are in capital letters). The results are displayed as relative luciferase activity of the reporter construct in presence of increasing amount of miRNA compared with its basal activity, upon normalization by renilla activity (mean ± SD, n = 3). Statistically significant changes were analyzed only for the wild type compared with the mutated UTRs in the presence of the highest dose of miRNA (miR-361, P = 0.001; miR-155, P = 1 × 10−5, Student’s t test). (d) IRF8-3′UTR reporter assay in response to increasing amount of miR-155. On top, schematic representation is shown of the region in the IRF8-3′UTR (IRF8-3′UTR WT) that is targeted by miR-155 and its alignments with the miRNA sequence. The mutations introduced in the mutant UTRs (IRF8-3′UTR Mut) are displayed below (mutated nucleotides are in capital letters). The results are displayed as relative luciferase activity of the reporter construct in presence of increasing amount of miRNA compared with its basal activity, upon normalization by renilla activity (mean ± SD, n = 3). Statistically significant changes were analyzed only for the wild type compared with the mutated UTRs in the presence of the highest dose of miRNA (*, P < 0.05, Student’s t test). (e) MYB-3′UTR reporter assay in response to increasing amount of miR-155. On top, schematic representation is shown of the regions in the MYB-3′UTR (MYB-3′UTR WT) that are targeted by miR-155 and their alignments with the miRNA sequence. The mutations introduced in the mutant UTRs (MYB-3′UTR Mut) are displayed below (mutated nucleotides are in capital letters). The results are displayed as relative luciferase activity of the reporter construct in presence of increasing amount of miRNA compared with its basal activity, upon normalization by renilla activity (mean ± SD, n = 3). Statistically significant changes were analyzed only for the wild type compared with the mutated UTRs in the presence of the highest dose of miRNA (*, P < 0.05, Student’s t test).
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fig3: miR-155 and miR-361 target GC-relevant genes, including AICDA. (a) The effect of induction of miR-155 by doxycycline (Dox) treatment in engineered Burkitt’s lymphoma cell lines (P3HR1 and Raji) on the expression of AICDA, SPI1, IRF8, and MYB, as detected by immunoblotting (left). The right panel represents the relative fold changes obtained in three independent experiments (mean ± SD, n = 3; *, P ≤ 0.05, Student’s t test). (b) Effects of miR-361 induction on AICDA protein levels. Normalized relative fold change in protein levels are reported below each lane. The right panel represents the relative fold changes obtained in three independent experiments (mean ± SD, n = 3; *, P ≤ 0.05, Student’s t test). (c) AICDA-3′UTR reporter assay in response to increasing amount of miR-155 and miR-361. On the right, schematic representation of the regions in the AICDA-3′UTR (AICDA-3′UTR WT) that are targeted by miR-155 and miR-361 and their alignments with the miRNA sequences. The mutations introduced in the mutant UTRs (AICDA-3′UTR Mut) are displayed below (mutated nucleotides are in capital letters). The results are displayed as relative luciferase activity of the reporter construct in presence of increasing amount of miRNA compared with its basal activity, upon normalization by renilla activity (mean ± SD, n = 3). Statistically significant changes were analyzed only for the wild type compared with the mutated UTRs in the presence of the highest dose of miRNA (miR-361, P = 0.001; miR-155, P = 1 × 10−5, Student’s t test). (d) IRF8-3′UTR reporter assay in response to increasing amount of miR-155. On top, schematic representation is shown of the region in the IRF8-3′UTR (IRF8-3′UTR WT) that is targeted by miR-155 and its alignments with the miRNA sequence. The mutations introduced in the mutant UTRs (IRF8-3′UTR Mut) are displayed below (mutated nucleotides are in capital letters). The results are displayed as relative luciferase activity of the reporter construct in presence of increasing amount of miRNA compared with its basal activity, upon normalization by renilla activity (mean ± SD, n = 3). Statistically significant changes were analyzed only for the wild type compared with the mutated UTRs in the presence of the highest dose of miRNA (*, P < 0.05, Student’s t test). (e) MYB-3′UTR reporter assay in response to increasing amount of miR-155. On top, schematic representation is shown of the regions in the MYB-3′UTR (MYB-3′UTR WT) that are targeted by miR-155 and their alignments with the miRNA sequence. The mutations introduced in the mutant UTRs (MYB-3′UTR Mut) are displayed below (mutated nucleotides are in capital letters). The results are displayed as relative luciferase activity of the reporter construct in presence of increasing amount of miRNA compared with its basal activity, upon normalization by renilla activity (mean ± SD, n = 3). Statistically significant changes were analyzed only for the wild type compared with the mutated UTRs in the presence of the highest dose of miRNA (*, P < 0.05, Student’s t test).

Mentions: The trans-repressive activity of BCL6 on its miRNA targets becomes physiologically relevant if it is able to sustain the expression of genes that are negatively modulated by these miRNAs. To test this hypothesis, we first identified miR-155 and miR-361 candidate targets using the TargetScan (Lewis et al., 2005) and Miranda-mirSVR (John et al., 2004; Betel et al., 2010) algorithms and considered only the common predictions (Table S3). Several candidates, selected based on their relevance for GC biology, were then tested for their responsiveness to miR-155 and/or miR361 in the Burkitt lymphoma cell lines P3HR1 and Raji engineered to inducibly express miR-155 or miR-361. This approach allowed us to validate in human B cells miR-155 targets previously reported in mouse, including AID and SPI1 (Vigorito et al., 2007; Dorsett et al., 2008; Teng et al., 2008), and to identify novel GC-relevant candidates such as IRF8 and MYB (Fig. 3 a). Notably, AID was also identified as a candidate target of miR-361-5p (Fig. 3 b). As expected for miRNA targets, these candidates display a very modest down-regulation at the transcriptional level but a significant reduction in protein levels upon miRNA induction in B cells (Fig. 3, a and b). The direct inhibition of AID by both miRNAs was further demonstrated by 3′-UTR reporter assay in which the presence of the full-length AID 3′-UTR downstream of a reporter gene was associated with a dose-dependent reduction of the reporter activity in presence of miR-361 or miR-155 (Fig. 3c). Mutation of the predicted miR-155 (consistent with previous studies [Dorsett et al., 2008; Teng et al., 2008]) and/or miR361 binding sites in the AID 3′-UTR rescued the repression induced by the miRNAs (Fig. 3 c), demonstrating that both miR-155 and miR-361-5p directly target AID. The direct repression of IRF8 and MYB by miR-155 was demonstrated in an analogous assay (Fig. 4, d and e).


BCL6 positively regulates AID and germinal center gene expression via repression of miR-155.

Basso K, Schneider C, Shen Q, Holmes AB, Setty M, Leslie C, Dalla-Favera R - J. Exp. Med. (2012)

miR-155 and miR-361 target GC-relevant genes, including AICDA. (a) The effect of induction of miR-155 by doxycycline (Dox) treatment in engineered Burkitt’s lymphoma cell lines (P3HR1 and Raji) on the expression of AICDA, SPI1, IRF8, and MYB, as detected by immunoblotting (left). The right panel represents the relative fold changes obtained in three independent experiments (mean ± SD, n = 3; *, P ≤ 0.05, Student’s t test). (b) Effects of miR-361 induction on AICDA protein levels. Normalized relative fold change in protein levels are reported below each lane. The right panel represents the relative fold changes obtained in three independent experiments (mean ± SD, n = 3; *, P ≤ 0.05, Student’s t test). (c) AICDA-3′UTR reporter assay in response to increasing amount of miR-155 and miR-361. On the right, schematic representation of the regions in the AICDA-3′UTR (AICDA-3′UTR WT) that are targeted by miR-155 and miR-361 and their alignments with the miRNA sequences. The mutations introduced in the mutant UTRs (AICDA-3′UTR Mut) are displayed below (mutated nucleotides are in capital letters). The results are displayed as relative luciferase activity of the reporter construct in presence of increasing amount of miRNA compared with its basal activity, upon normalization by renilla activity (mean ± SD, n = 3). Statistically significant changes were analyzed only for the wild type compared with the mutated UTRs in the presence of the highest dose of miRNA (miR-361, P = 0.001; miR-155, P = 1 × 10−5, Student’s t test). (d) IRF8-3′UTR reporter assay in response to increasing amount of miR-155. On top, schematic representation is shown of the region in the IRF8-3′UTR (IRF8-3′UTR WT) that is targeted by miR-155 and its alignments with the miRNA sequence. The mutations introduced in the mutant UTRs (IRF8-3′UTR Mut) are displayed below (mutated nucleotides are in capital letters). The results are displayed as relative luciferase activity of the reporter construct in presence of increasing amount of miRNA compared with its basal activity, upon normalization by renilla activity (mean ± SD, n = 3). Statistically significant changes were analyzed only for the wild type compared with the mutated UTRs in the presence of the highest dose of miRNA (*, P < 0.05, Student’s t test). (e) MYB-3′UTR reporter assay in response to increasing amount of miR-155. On top, schematic representation is shown of the regions in the MYB-3′UTR (MYB-3′UTR WT) that are targeted by miR-155 and their alignments with the miRNA sequence. The mutations introduced in the mutant UTRs (MYB-3′UTR Mut) are displayed below (mutated nucleotides are in capital letters). The results are displayed as relative luciferase activity of the reporter construct in presence of increasing amount of miRNA compared with its basal activity, upon normalization by renilla activity (mean ± SD, n = 3). Statistically significant changes were analyzed only for the wild type compared with the mutated UTRs in the presence of the highest dose of miRNA (*, P < 0.05, Student’s t test).
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fig3: miR-155 and miR-361 target GC-relevant genes, including AICDA. (a) The effect of induction of miR-155 by doxycycline (Dox) treatment in engineered Burkitt’s lymphoma cell lines (P3HR1 and Raji) on the expression of AICDA, SPI1, IRF8, and MYB, as detected by immunoblotting (left). The right panel represents the relative fold changes obtained in three independent experiments (mean ± SD, n = 3; *, P ≤ 0.05, Student’s t test). (b) Effects of miR-361 induction on AICDA protein levels. Normalized relative fold change in protein levels are reported below each lane. The right panel represents the relative fold changes obtained in three independent experiments (mean ± SD, n = 3; *, P ≤ 0.05, Student’s t test). (c) AICDA-3′UTR reporter assay in response to increasing amount of miR-155 and miR-361. On the right, schematic representation of the regions in the AICDA-3′UTR (AICDA-3′UTR WT) that are targeted by miR-155 and miR-361 and their alignments with the miRNA sequences. The mutations introduced in the mutant UTRs (AICDA-3′UTR Mut) are displayed below (mutated nucleotides are in capital letters). The results are displayed as relative luciferase activity of the reporter construct in presence of increasing amount of miRNA compared with its basal activity, upon normalization by renilla activity (mean ± SD, n = 3). Statistically significant changes were analyzed only for the wild type compared with the mutated UTRs in the presence of the highest dose of miRNA (miR-361, P = 0.001; miR-155, P = 1 × 10−5, Student’s t test). (d) IRF8-3′UTR reporter assay in response to increasing amount of miR-155. On top, schematic representation is shown of the region in the IRF8-3′UTR (IRF8-3′UTR WT) that is targeted by miR-155 and its alignments with the miRNA sequence. The mutations introduced in the mutant UTRs (IRF8-3′UTR Mut) are displayed below (mutated nucleotides are in capital letters). The results are displayed as relative luciferase activity of the reporter construct in presence of increasing amount of miRNA compared with its basal activity, upon normalization by renilla activity (mean ± SD, n = 3). Statistically significant changes were analyzed only for the wild type compared with the mutated UTRs in the presence of the highest dose of miRNA (*, P < 0.05, Student’s t test). (e) MYB-3′UTR reporter assay in response to increasing amount of miR-155. On top, schematic representation is shown of the regions in the MYB-3′UTR (MYB-3′UTR WT) that are targeted by miR-155 and their alignments with the miRNA sequence. The mutations introduced in the mutant UTRs (MYB-3′UTR Mut) are displayed below (mutated nucleotides are in capital letters). The results are displayed as relative luciferase activity of the reporter construct in presence of increasing amount of miRNA compared with its basal activity, upon normalization by renilla activity (mean ± SD, n = 3). Statistically significant changes were analyzed only for the wild type compared with the mutated UTRs in the presence of the highest dose of miRNA (*, P < 0.05, Student’s t test).
Mentions: The trans-repressive activity of BCL6 on its miRNA targets becomes physiologically relevant if it is able to sustain the expression of genes that are negatively modulated by these miRNAs. To test this hypothesis, we first identified miR-155 and miR-361 candidate targets using the TargetScan (Lewis et al., 2005) and Miranda-mirSVR (John et al., 2004; Betel et al., 2010) algorithms and considered only the common predictions (Table S3). Several candidates, selected based on their relevance for GC biology, were then tested for their responsiveness to miR-155 and/or miR361 in the Burkitt lymphoma cell lines P3HR1 and Raji engineered to inducibly express miR-155 or miR-361. This approach allowed us to validate in human B cells miR-155 targets previously reported in mouse, including AID and SPI1 (Vigorito et al., 2007; Dorsett et al., 2008; Teng et al., 2008), and to identify novel GC-relevant candidates such as IRF8 and MYB (Fig. 3 a). Notably, AID was also identified as a candidate target of miR-361-5p (Fig. 3 b). As expected for miRNA targets, these candidates display a very modest down-regulation at the transcriptional level but a significant reduction in protein levels upon miRNA induction in B cells (Fig. 3, a and b). The direct inhibition of AID by both miRNAs was further demonstrated by 3′-UTR reporter assay in which the presence of the full-length AID 3′-UTR downstream of a reporter gene was associated with a dose-dependent reduction of the reporter activity in presence of miR-361 or miR-155 (Fig. 3c). Mutation of the predicted miR-155 (consistent with previous studies [Dorsett et al., 2008; Teng et al., 2008]) and/or miR361 binding sites in the AID 3′-UTR rescued the repression induced by the miRNAs (Fig. 3 c), demonstrating that both miR-155 and miR-361-5p directly target AID. The direct repression of IRF8 and MYB by miR-155 was demonstrated in an analogous assay (Fig. 4, d and e).

Bottom Line: We have identified a core of 15 miRNAs that show binding of BCL6 in their genomic loci and are down-regulated in GC B cells.Similarly, the expression of additional genes relevant for the GC phenotype, including SPI1, IRF8, and MYB, appears to be sustained via BCL6-mediated repression of miR-155.These findings identify a novel mechanism by which BCL6, in addition to repressing protein coding genes, promotes the expression of important GC functions by repressing specific miRNAs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Cancer Genetics, Columbia University, New York, NY 10027, USA. kb451@columbia.edu

ABSTRACT
The BCL6 proto-oncogene encodes a transcriptional repressor that is required for germinal center (GC) formation and whose de-regulation is involved in lymphomagenesis. Although substantial evidence indicates that BCL6 exerts its function by repressing the transcription of hundreds of protein-coding genes, its potential role in regulating gene expression via microRNAs (miRNAs) is not known. We have identified a core of 15 miRNAs that show binding of BCL6 in their genomic loci and are down-regulated in GC B cells. Among BCL6 validated targets, miR-155 and miR-361 directly modulate AID expression, indicating that via repression of these miRNAs, BCL6 up-regulates AID. Similarly, the expression of additional genes relevant for the GC phenotype, including SPI1, IRF8, and MYB, appears to be sustained via BCL6-mediated repression of miR-155. These findings identify a novel mechanism by which BCL6, in addition to repressing protein coding genes, promotes the expression of important GC functions by repressing specific miRNAs.

Show MeSH
Related in: MedlinePlus