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. 2017 Mar 16;65(6):1068-1080.e5.
doi: 10.1016/j.molcel.2016.12.022. Epub 2017 Mar 3.

Distinct Roles of Brd2 and Brd4 in Potentiating the Transcriptional Program for Th17 Cell Differentiation

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Free PMC article

Distinct Roles of Brd2 and Brd4 in Potentiating the Transcriptional Program for Th17 Cell Differentiation

Ka Lung Cheung et al. Mol Cell. .
Free PMC article

Abstract

The BET proteins are major transcriptional regulators and have emerged as new drug targets, but their functional distinction has remained elusive. In this study, we report that the BET family members Brd2 and Brd4 exert distinct genomic functions at genes whose transcription they co-regulate during mouse T helper 17 (Th17) cell differentiation. Brd2 is associated with the chromatin insulator CTCF and the cohesin complex to support cis-regulatory enhancer assembly for gene transcriptional activation. In this context, Brd2 binds the transcription factor Stat3 in an acetylation-sensitive manner and facilitates Stat3 recruitment to active enhancers occupied with transcription factors Irf4 and Batf. In parallel, Brd4 temporally controls RNA polymerase II (Pol II) processivity during transcription elongation through cyclin T1 and Cdk9 recruitment and Pol II Ser2 phosphorylation. Collectively, our study uncovers both separate and interdependent Brd2 and Brd4 functions in potentiating the genetic program required for Th17 cell development and adaptive immunity.

Keywords: BRD2; BRD4; CTCF; Th17 cell differentiation; gene transcription; the cohesin complex.

Conflict of interest statement

Conflict of interest statement: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Genomic analysis of Brd2 and Brd4 in Th17 cells
(A) ChIP-seq analysis revealing Brd2 and Brd4 genome-wide binding sites in Th17 cells. The Brd2 and Brd4 peaks are grouped according to their location in promoter, exon, intron, or intergenic regions. (B) Venn diagrams showing the number of overlapping peaks of Brd2 and Brd4 (left) and genes co-bound by Brd2 and Brd4 (right) in Th17 cells. (C) ChIP-seq tracks of Brd2, Brd4 and transcription factors revealing co-localization on Il17 and Rorc gene loci in Th17 cells. (D) Brd2, Stat3, PolII, Brd4, H4Ac, PolII-S2P occupancy at gene loci of Il17a, Il17f, Rorc and Il21 after 24 and 48 hours of Th17 cell differentiation from murine primary naïve CD4+ T cells isolated from mouse spleen and lymph nodes, as determined by ChIP-qPCR. The primer target site is indicated as Stat3-bs1 in C. Data are represented as mean ± SEM of n=3. *P<0.05. (E) mRNA expression levels of Il17a, Rorc, and Il21 during 72-hour lineage-specific differentiation of murine Th17 cells as described in D, normalized to their corresponding expression levels in mouse primary naïve CD4+ T cells. See also Figure S1 and Tables S1 and S2.
Figure 2
Figure 2. Brd2, but not Brd4, is associated with CTCF/Cohesin complex in Th17 cells
(A) DNA binding motifs identified for Brd2 and Brd4 with their ChIP-seq data from Th17 cells. (B) Venn diagrams showing the number of overlapping peaks of Brd2, Brd4 and CTCF (left); and normalized Brd2 and Brd4 ChIP-signal centered around CTCF peak regions (right). (C) ChIP-seq tracks of CTCF, Nipbl, Smc1, Smc3, Brd2, Brd4 and Stat3 on Il17a and Rorc gene loci in Th17 cells. The ChIP-seq data for CTCF and Stat3 were reported previously (Ciofani et al., 2012), whereas the others were generated in this study. (D) Immunoprecipitation of Brd2 and Brd4, and immunoblotting with various specific antibodies to assess Brd2 or Brd4 interactions with CTCF and cohesin components (Nipbl, Rad21 and Stag1) in Th17 cells differentiated for 48 hours. (E) Occupancy of Brd2, Brd4 and cohesin components (Nipbl, Smc1 and Smc3) at the CTCF and Stat3 binding sites in the Il17a and Rorc gene loci in Th17 cells differentiated for 24 and 48 hours, as determined ChIP. Data are represented as mean ± SEM of n=3. The primer target sites are indicated in C. (F) Immunoprecipitation of Stat3 and Irf4, and immunoblotting with specific antibodies to examine Stat3 and Irf4 interactions with cohesin components (Nipbl, Smc1, Smc3, Rad21 and Stag1) in Th17 cells. See also Figure S2.
Figure 3
Figure 3. Brd2/Brd4 co-bound genes mark super-enhancers with highest transcriptional expression levels in Th17 cells
(A) Heatmap for ChIP-seq signals of Brd2 and Brd4 marked by the indicated antibodies ±1.5kb from the center of Smc1 peaks. (B) Boxplots of normalized counts of Brd2 and Brd4 signals illustrated at the four clusters of peaks. (C) Boxplot indicating transcriptional expression levels of genes associated with the clustered peaks in Th17 cells. (D) Heatmap of ChIP-seq signals of Stat3 located ±1.5kb from the center of Smc1 peaks (left), and boxplots of normalized counts of these signals at the four clusters of peaks (right). (E) Heatmap of ChIP-seq signals of H3K27Ac located ±1.5kb from the center of Smc1 peaks (left), and boxplots of normalized counts of these signals at the four clusters of peaks (right). (F) Heatmap of ChIP-seq signals of H3K4me1 located ±1.5kb from the center of Smc1 peaks (left), and boxplots of normalized counts of these signals at the four clusters of peaks (right). (G) ChIP-seq tracks representing examples of Brd2-Brd4 co-bound genes (such as Il21) and Brd2-bound only genes (such as Rock2). See also Figure S3.
Figure 4
Figure 4. Endogenous Stat3 interaction with Brd2 is dependent upon its acetylation by p300
(A) Immunoprecipitation of Stat3 and immunoblotting with specific antibodies to assess Stat3 interactions with Brd2, Brd4, Irf4, or p300 in Th17 cell lysates treated with or without TSA. (B) Immunoprecipitation of Brd2 or Brd4, and immunoblotting with specific antibodies to examine Brd2 or Brd4 interactions with Stat3, p300 or Irf4 in Th17 cell lysates treated with TSA. (C) Th17 cells lysates treated with TSA immunoprecipitated with Stat3, and then treated with or without eithidium bromide (EtBr) and followed by western blot with antibodies against Brd2 and Stat3. (D) Dose-dependent effects of BET BrD inhibition by MS417 on Brd2/Stat3 association in Th17 cell lysates treated with TSA, as assessed immunoprecipitated with Stat3, and then treated with MS417, and followed by western blot with antibodies against Brd2 and Stat3. (E) Assessing the role of lysine acetylation in Brd2/Stat3 association. Left, schematic representations of various Brd2 plasmids used in the study. Middle, HEK293 cells overexpressed with Flag-Stat3, GFP-Brd2 and myc-p300 were lysed and immunoprecipitated with antibody against flag to detect Brd2/Stat3 interactions with or without p300. Right, HEK293 cells overexpressed with Flag-Stat3, GFP-Brd2-BD and GFP-Brd2-BDMut1+2 were lysed and immunoprecipitated. The acetyl-lysine binding deficient mutations in BD1 and BD2 of Brd2 are Y154F and Y427F, respectively. (F) Immunoprecipitation of Brd2 or Brd4 in Th17 cell lysates, and immunoblotting with specific antibodies to assess their interactions with p300, Stat3, Irf4, Batf, PolII or Cdk9. (G) Venn diagrams showing the number of overlapping peaks of Brd2, Stat3 and Irf4 identified from ChIP-seq datasets collected in Th17 cells. (H) Normalized Irf4 and Stat3 ChIP-signal centered around Irf4-Stat3 co-bound peak regions stratified by the presence of Brd2. (I) Assessing effects of BET BrD inhibition by MS417 on Brd2/Cohesin/Stat3 association, as determined by immunoprecipitation of Brd2 from Th17 cell lysates, and immunoblotting with specific antibodies to Smc1, Smc3 and Stat3 with or without MS417 treatment. See also Figure S4.
Figure 5
Figure 5. Structural analysis of Brd2/Stat3 interaction
(A) 2D 15N-HSQC spectra of Brd2-BD1 or BD2 illustrating changes of the protein backbone amide resonances in the free form (black), and in the presence of Stat3-K87ac peptide (red). Upper panel, the three main lysine acetylation sites (K49ac, K87ac and K685ac) in Stat3 are indicated in the protein domain organization diagram. (B) 3D NMR structure of the Brd2-BD2 bound to Stat3-K87ac peptide (yellow), illustrating Stat3-K87ac recognition by the key residues at the acetyl-lysine binding pocket as indicated in green. Lower panel, electrostatic potential representation of the protein depicts the acetyl-lysine binding pocket for Stat3-K87ac recognition. (C) Assessing the site-specific lysine acetylation in Stat3/Brd2 association. Immunoprecipitation of Flag-tagged Stat3 wild-type, or point mutants of the three known lysine acetylation sites in HEK293 cells co-transfected with myc-300, and immunoblotting with specific antibodies to examine Brd2 interactions with Stat3. See also Figure S5 and Table S3.
Figure 6
Figure 6. Brd2 and Brd4 functionally cooperate to regulate gene transcription in Th17 cells
(A) mRNA expression levels of Brd2, Brd4, Il17a, Il17f, Rorc and Il21 determined in mouse CD4+ T cells transfected with siControl, siBrd2 or siBrd4 RNA and after 24 or 48 hours of Th17 cell differentiation. All results are statistically significant (P<0.05), and represented as mean ± SEM of more than two independent experiments. (B) Th17 cell lysates transfected with siControl, siBrd2 or siBrd4 RNA immunoprecipitated with PolII and western blot with antibodies against Irf4, Stat3, PolII-S2P and Cdk9. (C) Th17 cell lysates transfected with siControl, siBrd2 or siBrd4 RNA immunoprecipitated with Stat3 and western blot with antibodies against PolII and Irf4. (D) Th17 cell lysates transfected with siControl, siBrd2 (left) or siNipbl (right) RNA immunoprecipitated with Stat3 and western blot with antibodies against Nipbl, Brd2, Stat3 and Irf4. (E) Brd2, Brd4, Stat3, PolII, PolII-S2P, Irf4, MED1 and p300 occupancy at gene loci of IL17a, IL21 and Rorc in Th17 cell lysates transfected with siControl, siBrd2 or siBrd4 RNA, as determined by ChIP-qPCR. Data are represented as mean ± SEM of n=3. See also Figure S6.
Figure 7
Figure 7. Distinct roles of Brd2 and Brd4 in potentiating the gene transcriptional program for Th17 cell differentiation
Schematic diagram illustrating Brd2 and Brd4 functionally cooperate with each other to regulate gene transcription in chromatin in Th17 cells.

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