Phosphorylation by JNK switches BRD4 functions

Abstract

Bromodomain 4 (BRD4), a key regulator with pleiotropic functions, plays crucial roles in cancers and cellular stress responses. It exhibits dual functionality: chromatin-bound BRD4 regulates remodeling through its histone acetyltransferase (HAT) activity, while promoter-associated BRD4 regulates transcription through its kinase activity. Notably, chromatin-bound BRD4 lacks kinase activity, and RNA polymerase II (RNA Pol II)-bound BRD4 exhibits no HAT activity. This study unveils one mechanism underlying BRD4's functional switch. In response to diverse stimuli, c-Jun N-terminal kinase (JNK)-mediated phosphorylation of human BRD4 at Thr1186 and Thr1212 triggers its transient release from chromatin, disrupting its HAT activity and potentiating its kinase activity. Released BRD4 directly interacts with and phosphorylates RNA Pol II, PTEFb, and c-Myc, thereby promoting transcription of target genes involved in immune and inflammatory responses. JNK-mediated BRD4 functional switching induces CD8 expression in thymocytes and epithelial-to-mesenchymal transition (EMT) in prostate cancer cells. These findings elucidate the mechanism by which BRD4 transitions from a chromatin regulator to a transcriptional activator.

Keywords: BRD4; EMT; JNK; cellular stress; chromatin decompaction; histone acetyltransferase; kinase; phospho-BRD4; thymocyte stimulation; transcription activation.

Fig. 1:. JNK directly interacts with and phosphorylates BRD4

Fig 1A: BRD4 colocalizes with kinase active JNK. Proximity Ligation Assays (PLAs) with anti-BRD4 and anti-pJNK on fixed HCT116 cells. Negative control; anti-nucleolin and anti-BRD4. (Scale bars, 20 μM) Fig 1B: JNK co-immunoprecipitates with BRD4. BRD4 was immunoprecipitated from HeLa nuclear extract using anti-BRD4 and immunoblotted with anti-JNK. Fig 1C: BRD4 binds JNK directly. Recombinant JNK1 (0.1, and 0.2μg) was pulled down with 0.5μg rBRD4 immobilized on Flag-beads. Fig 1D: JNK phosphorylates BRD4. Upper: map of BRD4 and deletion mutants. Lower: autoradiograph of kinase assays with GST-JNK1 and BRD4 WT or deletion mutants. Fig 1E: JNK phosphorylation sites on BRD4. Upper: JNK consensus phosphorylation sites located on human/mouse BRD4. Lower: autoradiograph of kinase assays with His-JNK1 and BRD4 WT or the point mutants. Fig 1F: BRD4 is phosphorylated at Thr1186 and Thr1212 JNK activation. HCT116 cells weretreated with anisomycin, heat shock, LPS treatment or UV stress. BRD4 phosphorylation was assessed by immunoblotting (upper) and densitometric quantification (lower). Fig 1G: BRD4’s interaction with JNK is abrogated by phosphorylation. Co-IP of JNK with BRD4 following anisomycin treatment of WT and 3A-BRD4 expressing HCT116 cells. Also see Figs. S1 and S2

Figure 2:. JNK phosphorylation of BRD4 releases it from chromatin.

Fig. 2A: BRD4 is released from the chromatin upon JNK activation by anisomycin. Immunoblots of chromatin-free (CF) and chromatin-bound (CB) BRD4 in HCT116 cells following treatment with anisomycin. Fig 2B: Inhibition of JNK kinase blocks BRD4’s release from chromatin. Immunoblots of chromatin-free (CF) and chromatin-bound (CB) BRD4 in HCT116 cells transfected with WT JNK1 or JNK1 and dominant negative kinase mutants JNK1/JNK2 (APF) individually or in combination, followed by anisomycin treatment. Fig. 2C: BRD4 is released from the chromatin upon JNK activation by heat shock. Left: Immunoblots of chromatin-free (CF) and chromatin-bound (CB) BRD4 in HCT116 cells grown at 37°C or heat shocked at 42°C for 15 minutes in the presence or absence of JNK inhibitor SP600125. Right: Immunoblots showing pJNK levels under the above conditions. Fig 2D: JNK preferentially phosphorylates BRD4 bound to mononucleosomes. Anti-BRD4 pT1212 and anti-BRD4 immunoblots of kinase assays with recombinant JNK1 and BRD4 after preincubating BRD4 with or without assembled mononucleosomes (MN) for 10 or 20 min. Fig 2E: Mutation of BRD4 phosphorylation sites 4 prevents BRD4 release from chromatin. Left: Immunoblots of chromatin-free (CF) and chromatin-bound (CB) BRD4 in HCT116 cells transfected with WT or 3A-BRD4 and subjected to heat shock treatment. Right; Immunoblots showing pJNK levels following heat shock in WT and 3A-BRD4 expressing cells. Fig. 2F: JNK activation results in global loss of chromatin-bound BRD4. Total BRD4 peaks detected in BRD4 ChIP-seq of control untreated and anisomycin treated DLD1 BRD4-IAA7 cells expressing endogenous BRD4 or exogenous WT or 3A-BRD4 following auxin treatment. Fig 2G: Loss of JNK-phosphorylated BRD4 from chromatin is widespread. Distribution of BRD4 ChIP-seq peaks across the genomes of cells described in 2F. Fig 2H: BRD4 ChIP-seq peak distribution profiles across the gene body of representative FDPS, HMGCR and INSIG1 genes in control untreated and anisomycin treated DLD1 BRD4-IAA7 cells expressing exogenous WT or 3A-BRD4 following auxin treatment. Densitometric quantifications of CF:CB BRD4 ratio are shown below immunoblots; anti-histone H3 immunoblot monitors purity of CB and CF fractions. Also see Fig. S3

Figure 3:. JNK-mediated BRD4 release from chromatin disrupts its nucleosome clearance function.

Fig 3A: BRD4 binding to mononucleosomes is abrogated upon phosphorylation by JNK. Anti-histone H3 immunoblot of assembled mononucleosomes pulled down by recombinant WT or 3A-BRD4 that was either unphosphorylated or pre-phosphorylated (*) by JNK and immobilized on Flag-beads. Fig 3B: JNK phosphorylation of BRD4 inhibits H3K122 acetylation. Anti-histone H3K122ac immunoblot of assembled mononucleosomes subjected to an in vitro HAT assay with recombinant WT or 3A-BRD4 that was either unphosphorylated or pre-phosphorylated (*) by JNK. Fig 3C: H3K122 acetylation is reduced in vivo upon JNK activation. Immunoblots of whole cell extracts (WCEs) of HCT116 cells that were untreated (control) or treated with either DMSO (Mock) or Anisomycin. Fig 3D: In vivo H3K122 acetylation by BRD4 is regulated by JNK. Immunoblots of WCEs of HCT116 cells that were transfected with WT or 3A-BRD4 and treated with or without anisomycin. Fig. 3E: In vivo H3K122 acetylation is regulated by JNK kinase activity. Immunoblots of WCE’s of HCT116 cells that were transfected with WT JNK1, JNK2 or their respective dominant negative mutants (JNK APF), either individually or together and treated with or without anisomycin. Densitometric quantification of H3K122ac levels is shown below. Fig 3F: Nucleosome clearance activity by BRD4 is controlled by JNK phosphorylation. Autoradiograph of an in vitro nucleosome clearance assay showing dissociation of assembled mononucleosomes by unphosphorylated or JNK pre-phosphorylated (*) WT or 3A-BRD4 upon being subjected to a HAT assay in the presence or absence of AcCoA. Also see Fig. S4

Figure 4:. JNK-mediated BRD4 release from chromatin activates BRD4 kinase.

Fig. 4A: JNK activation induces phosphorylation of BRD4 kinase substrates. Immunoblots of WCE’s of HCT116 cells that were treated with DMSO (mock), anisomycin or anisomycin with JNK peptide inhibitor D-JNK1. Fig 4B: Blocking JNK activity inhibits induction of BRD4 kinase. Immunoblots of WCE’s of HCT116 cells that were transfected with Flag-tagged WT JNK1, JNK2 or respective dominant negative mutants (JNK APF), either individually or together and treated with or without anisomycin. Fig 4C: JNK phosphorylation of BRD4 is necessary for induction of BRD4 kinase. Immunoblots of WCE’s of HCT116 cells transfected with WT or 3A-BRD4 and subjected to heat shock. Fig 4D: BRD4 phosphorylation regulates MYC stability. Immunofluorescence images showing MYC levels in HCT116 cells transfected with either WT or 3A-BRD4 or empty vector (control) and subjected to heat shock. (Scale bars, 25 μM) Also see Fig. S5

Figure 5:. JNK phosphorylation toggles BRD4 enzymatic activities and is reversed by PP4 phosphatase.

Fig. 5A: BRD4 kinase and HAT activities are cross regulated by its substrates. Top: Autoradiograph of an in vitro kinase assay with BRD4 and Pol II CTD in the presence or absence of assembled mononucleosomes. Bottom: Immunoblots of an in vitro HAT assay with BRD4 and histone H3 in the presence or absence of Pol II CTD. Fig 5B: JNK activation enhances the interaction between BRD4 and its kinase substrates. Immunoblots showing co-immunoprecipitated total and T1212 phosphorylated BRD4 from HCT116 cells treated with or without anisomycin. Top: BRD4 co-immunoprecipitated with Pol II CTD. Bottom: BRD4 co-immunoprecipitated with CDK9. Fig 5C: JNK-mediated phosphorylation of BRD4 is transient. Immunoblots of WCE’s of HCT116 cells grown under normal conditions, subjected to heat shock or heat shocked and then rescued for 20 min. Fig 5D: Inhibition of phosphatases enhances phosphorylated BRD4 levels. Immunoblots of WCE’s of HCT116 cells that were treated with or without anisomycin alone or anisomycin with phosphatase inhibitor, nodularin. Fig 5E: Phosphatase PP4 dephosphorylates JNK-phosphorylated BRD4. Immunoblots of WCE’s of HCT116 cells that were transfected with either control, PP2Ac or PP4c SiRNA and treated with or without anisomycin. Fig 5F: BRD4’s interaction with Pol II CTD is modulated by PP4. Immunoblots showing total and pT1212 BRD4 co-immunoprecipitated with Pol II CTD from HCT116 cells transfected with either control or PP4c SiRNA and treated with anisomycin.

Figure 6:. JNK-mediated BRD4 release from chromatin activates transcription.

Fig. 6A: JNK activation enhances expression of BRD4 regulated genes. Volcano plots showing differential gene expression observed in RNA-seq analysis of WT and 3A-BRD4 expressing HCT116 cells after anisomycin treatment. Fig 6B: Inflammatory and immune response pathways are enriched among the BRD4 regulated genes induced by JNK activation. GO analysis of genes induced in anisomycin treated WT-BRD4 expressing cells relative to control HCT116 cells. Fig 6C: Induction of key inflammatory and immune response genes depend on JNK phosphorylation of BRD4. Quantitative RT-PCR of cDNA from anisomycin treated WT and 3A-BRD4 expressing cells relative to control cells. Error bars, s.e.m. (n=3 independent experiments; *P <0.001 by two tailed Student’s t tests) Fig 6D: JNK activation leads to increased BRD4-RNA Pol II interaction at BRD4 regulated inflammatory and immune response genes. Sequential-ChIP assays showing Pol II and Pol II- bound BRD4 at the promoter and gene body regions of CCL20, CXCL1, BIRC3 and control MYC genes. Error bars, s.e.m. (n=4 technical replicates from 2 independent experiments; *P <0.05 by two tailed Student’s t tests) Also see Fig. S6

Figure 7:. BRD4 phosphorylation and chromatin release are correlated with thymocyte activation and EMT.

Fig. 7A: Thymocyte activation correlates with JNK phosphorylation of BRD4. Left panels: Flow cytometry profiles of thymocytes activated by 0.3ng PMA/0.3μg Ionomycin or by 10ng PMA/3.75μg Ionomycin. FACS analysis of CD4/CD8 (Upper) and CD69 expression (Lower). Right panel: Immunoblots of WCE’s from unstimulated and stimulated thymocytes. Densitometric quantification of relative BRD4 phosphorylation levels is shown below. Fig. 7B: Thymocyte activation is correlated with JNK mediated release of BRD4 from chromatin. Immunoblot of chromatin-free (CF) and chromatin-bound (CB) BRD4 in thymocytes unstimulated or stimulated as described above. Densitometric quantification of CF:CB BRD4 ratio is shown below. Anti-histone H3 immunoblot monitors purity of separation. Immunoblots of WCE’s from PC3 cells at day 0 and day 5 of treatment with or without EMT-inducing media supplement. Fig. 7D: EMT induction correlates with JNK-mediated release of BRD4 from chromatin. Immunoblot of chromatin-free (CF) and chromatin-bound (CB) BRD4 in PC3 cells after five days of treatment with or without (control) EMT-inducing media. Fig. 7E: EMT induction and BRD4 phosphorylation are both dependent on JNK activity. Immunoblots of WCE’s from PC3 cells that were treated, or not, with EMT-inducing media alone or in combination with JNK peptide inhibitor D-JNK-1. Fig. 7F: EMT induction and expression of EMT regulators is dependent on BRD4 phosphorylation. Immunoblots of WCE’s from PC3 cells that were treated, or not, with EMT-inducing media and transfected with WT-BRD4, 3A-BRD4 or empty vector control on day 3 of treatment.

Figure 8:

Model of BRD4-JNK interaction and the switching of BRD4 functions. BRD4 primarily functions as a chromatin regulator by acetylating H3K122 and dissociating nucleosomes. Upon activation, JNK phosphorylates BRD4 releasing it from chromatin and activating its kinase. Chromatin-free BRD4 is then dephosphorylated by PP4, enhancing its interaction with and phosphorylation of, Pol II CTD, PTEFb and MYC, thereby activating transcription at specific genes. A portion of dephosphorylated BRD4 returns to chromatin to renew its chromatin regulatory function.