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. 2019 Jul 11;8:e43235. doi: 10.7554/eLife.43235

Acetylation of BMAL1 by TIP60 controls BRD4-P-TEFb recruitment to circadian promoters

Nikolai Petkau 1, Harun Budak 1,†, Xunlei Zhou 1,‡, Henrik Oster 1,§, Gregor Eichele 1,✉
Editors: Asifa Akhtar2, K VijayRaghavan3
PMCID: PMC6650244  PMID: 31294688

Abstract

Many physiological processes exhibit circadian rhythms driven by cellular clocks composed of interlinked activating and repressing elements. To investigate temporal regulation in this molecular oscillator, we combined mouse genetic approaches and analyses of interactions of key circadian proteins with each other and with clock gene promoters. We show that transcriptional activators control BRD4-PTEFb recruitment to E-box-containing circadian promoters. During the activating phase of the circadian cycle, the lysine acetyltransferase TIP60 acetylates the transcriptional activator BMAL1 leading to recruitment of BRD4 and the pause release factor P-TEFb, followed by productive elongation of circadian transcripts. We propose that the control of BRD4-P-TEFb recruitment is a novel temporal checkpoint in the circadian clock cycle.

Research organism: Mouse

Introduction

Circadian clocks are genetically encoded, self-sustained molecular oscillators that drive biological rhythms with a period close to 24 hr (Partch et al., 2014). They are found in most organisms and even in cultured tissues and cells (Balsalobre et al., 1998; Brown et al., 2012; Yoo et al., 2004). In mammals, these clocks are organized in a network controlled by a master pacemaker in the hypothalamic suprachiasmatic nucleus (SCN) (Dibner et al., 2010). Physiological processes such as energy metabolism, locomotion, hormone secretion, and cognitive functions are subject to circadian regulation (Bass and Takahashi, 2010; Gerstner and Yin, 2010; Panda, 2016). The influence of circadian rhythmicity is highlighted by the fact that in each tissue ~10% of the transcriptome is under the control of the circadian clock (Zhang et al., 2014) and that disruption of circadian rhythms is tied to the development of various diseases including metabolic disorders and cancer (Bedrosian et al., 2016; Musiek and Holtzman, 2016).

The molecular circadian oscillator consists of interlinked positive and negative feedback elements (Reppert and Weaver, 2002). Positive components are the heterodimers of CLOCK (circadian locomotor output cycles kaput) and BMAL1 (brain and muscle ARNT-like protein 1) that bind to E-box promoter elements of Per1-3 (Period), Cry1/2 (Cryptochome) and Rev-ErbAα/β (Nr1d1/2; nuclear receptor subfamily 1, group D, members 1 and 2) that encode repressor proteins PER, CRY and NR1D (Takahashi, 2017).

Genome-wide analysis of core circadian regulators revealed temporally restricted chromatin associations of circadian activators and repressors in the course of the circadian day (Koike et al., 2012; Menet et al., 2012; Rey et al., 2011). It is proposed that at the onset of the circadian cycle repressor CRY1 is part of the chromatin-bound CLOCK-BMAL1-complex located near transcriptionally silent RNA polymerase II (Pol II). Upon degradation of CRY1, coactivators such as CBP/p300 are recruited followed by Per and Cry transcription. During the repression phase of the circadian cycle, newly synthesized CRY2 and PER proteins associate with CLOCK-BMAL1, thereby shutting down their own transcription (Koike et al., 2012). Recruitment of CRY to the CLOCK-BMAL1 heterodimer is facilitated by acetylation of BMAL1 at lysine 538 by the acetyltransferase activity attributed to CLOCK (Doi et al., 2006; Hirayama et al., 2007). BMAL1 acetylation peaks during the repression phase (Hirayama et al., 2007), although data by Nakakata and co-workers (Nakahata et al., 2008; Nakahata et al., 2009) suggest that the presence of acetylated BMAL1 extends into the activation phase.

An important issue that the above feedback mechanisms leave unresolved concerns the binding of CLOCK-BMAL1 heterodimers to E-box promoter elements that initiates Pol II-mediated transcription (Koike et al., 2012; Stratmann et al., 2012). Well-recognized, rate-limiting steps that regulate transcription are the recruitment of Pol II to promoters and Pol II release from promoter-proximal pause sites (Jonkers and Lis, 2015; Liu et al., 2015). Pause release of Pol II and the transition to productive elongation requires the activity of the P-TEFb (positive transcription elongation factor b) complex, composed of T-type cyclins and CDK9 (cyclin-dependent kinase 9) (Jonkers and Lis, 2015; Liu et al., 2015). P-TEFb is recruited to promoters through interaction with specific transcription factors and other proteins like BRD4 (bromodomain-containing protein 4) and SEC (super elongation complex) (Jonkers and Lis, 2015; Liu et al., 2015; Luo et al., 2012; Shi and Vakoc, 2014). After such recruitment, P-TEFb phosphorylates the carboxy-terminal domain (CTD) of Pol II at Ser2 and additionally phosphorylates the pausing factors NELF (negative elongation factor) and DSIF (DRB sensitivity inducing factor). Upon phosphorylation, NELF is evicted from Pol II and DSIF becomes a positive elongation factor. Pol II pausing serves as a checkpoint allowing for rapid and synchronous expression of genes (Boettiger and Levine, 2009; Gilchrist et al., 2012; Lagha et al., 2013; Lin et al., 2011; Liu et al., 2015).

BRD4 is a member of the bromo-domain and extra terminal domain (BET) protein family which bind through their bromo-domains to acetylated lysines of histones and of transcription factors (Shi and Vakoc, 2014). Bound BRD4 recruits P-TEFb to promoter proximal paused Pol II (Jonkers and Lis, 2015; Liu et al., 2015). Small molecule inhibitors that block BRD4 (e.g. JQ1) from binding to acetylated lysines prevent productive elongation (Shi and Vakoc, 2014). Thus BRD4 protein is a key player in the regulation of productive transcription elongation. Therefore, acetyltransferases that acetylate BRD4 binding partners would play an important role in regulating Pol II pause release. The lysine acetyltransferase TIP60 (60 kDa Tat-interactive protein) is recruited to active promoters (Ravens et al., 2015) and plays a role in early steps of transcription elongation in mammals and flies (Kusch et al., 2014; Ravens et al., 2015; Shi et al., 2014). TIP60 is an essential protein (Hu et al., 2009).

Here we provide evidence that TIP60 is a critical component of the circadian clock. During the activation phase of the circadian cycle, TIP60 acetylates BMAL1, which triggers recruitment of the BRD4-P-TEFb complex to E-box-containing circadian promoters leading to Pol II pause release and productive elongation of circadian transcripts. These findings suggest that pause release is a critical regulatory step in the transcription of circadian clock regulated genes and that TIP60 plays a central role in this process.

Results

Inhibition of CDK9 and BRD4 abolishes circadian oscillations

Synchronized fibroblasts stably expressing the clock-driven luciferase reporter Bmal1-LUC (Nagoshi et al., 2004) show sustained reporter expression with a period of ~26 hr. In the presence of flavopiridol (FP), a potent inhibitor of the CDK9 subunit of P-TEFb (Rahl et al., 2010), the amplitude of luciferase activity rhythms was suppressed in a dose dependent manner concomitant with an increased period length until, at high doses, the rhythm was fully lost (Figure 1A; Figure 1—figure supplement 1A). Treatment of fibroblasts with FP also strongly reduced endogenous mRNA expression levels of Dbp (D-site albumin promoter binding protein), Per1, and Nr1d1 (Figure 1—figure supplement 1C) that are direct CLOCK-BMAL1 targets (Stratmann et al., 2012). These findings suggest that circadian clock gene regulation not only occurs at the initiation of transcription (Le Martelot et al., 2012) but also at the level of Pol II pause release and productive elongation. Triazolothienodiazepine (JQ1) is a potent and selective inhibitor of BRD4 (Shi and Vakoc, 2014). JQ1 treatment of Bmal1-LUC reporter fibroblasts dampened luciferase activity rhythms in a dose-dependent manner and evoked a lengthening of the period (Figure 1B; Figure 1—figure supplement 1B). JQ1 also resulted in a strong dampening of the rhythm of endogenous Dbp expression in these cells (Figure 1C). In the presence of JQ1, enrichment of BRD4 and CDK9 at the transcription start site (TSS) of Dbp was markedly reduced (Figure 1D). Ser2 phosphorylation of Pol II, at the 3’-end of Dbp, was strongly diminished (Figure 1E; Figure 1—figure supplement 1D) suggesting that Pol II pause release had been suppressed (Harlen and Churchman, 2017). Reduced Dbp expression could be due to lower levels of BMAL1. To show that this is not the case, we constitutively expressed ectopic CMV promoter-driven Bmal1-myc. BRD4 inhibition by JQ1 strongly reduced endogenous expression levels of Dbp mRNA but had no effect on mRNA or protein levels of Bmal1-myc (Figure 1—figure supplement 1E). Furthermore, BRD4 inhibition neither affected endogenous mRNA nor protein levels of Bmal1 (Figure 1—figure supplement 1F) in Bmal1-LUC fibroblasts at 24 hr after Dex synchronization. Finally, the binding of BMAL1 and of acetylated BMAL1 (AcBMAL1) to the Dbp promoter was not affected by BRD4 inhibition in these cells (Figure 1—figure supplement 1G). Together, our JQ1 inhibitor studies suggest that BRD4-P-TEFb is recruited to the E-box-containing clock-controlled gene Dbp which subsequently allows Pol II pause release and transcription elongation. Thus, for this circadian clock-controlled gene, Pol II pause release may serve as an important regulatory step of its transcription.

Figure 1. BRD4 controls clock gene expression.

(A and B) Bioluminescence recordings of dexamethasone (Dex) synchronized Bmal1-LUC fibroblasts treated with increasing concentrations of flavoperidol (A) or JQ1 (B) (n = 4). (C) Dbp mRNA dampening in Dex-synchronized fibroblasts by 500 nM JQ1 (n = 3; two-way ANOVA, see Supplementary file 1). (D and E) ChIP analysis of BRD4 and CDK9 (D) and Ser2-phosphorylated Pol II (normalized to total Pol II) (E) for the Dbp gene carried out 24 hr after Dex synchronization of the cells (n = 3). Schematic diagrams show the genomic structure of the Dbp locus. Horizontal lines represent PCR-amplified genomic regions. All data are shown as mean ± SD. Note that Dbp has several additional intronic Pol II pausing sites (Sobel et al., 2017). For validation of ChIP-grade quality of antibodies see Figure 1—figure supplement 2.

Figure 1.

Figure 1—figure supplement 1. BRD4 controls expression of E-box-controlled clock genes.

Figure 1—figure supplement 1.

(A - B) Change of period, luminescence intensity, and damping rate in flavopiridol (FP) (A) and JQ1 (B) treated dexamethasone (Dex) synchronized Bmal1-LUC fibroblasts (n = 4). LR: loss of rhythm. (C) mRNA expression analysis of fibroblasts treated with either DMSO or flavopiridol (300 nM) carried out 24 hr after Dex synchronization (n = 3). (D) Pol II and Ser2P-Pol II ChIP analysis of Dex synchronized DMSO or JQ1 treated fibroblasts (n = 3). (E) Immunoblot and mRNA expression analysis of Dex synchronized (24 hr after) Bmal1-/- MEFs transfected with empty vector or Bmal1-myc treated with either DMSO or JQ1 (500 nM) (n = 3). (F) BMAL1 protein abundance and Bmal1 mRNA expression in DMSO or JQ1 (500 nM) treated cells isolated 24 hr after Dex synchronization (n = 3). (G) ChIP analysis of BMAL1 and acetylated BMAL1 for the Dbp gene carried out 24 hr after Dex synchronization of MEFs (n = 3). (H) Interaction of BMAL1 with BRD4 in fibroblasts treated with either DMSO or JQ1 (500 nM). Immunoblots show results with increasing equivalents of IPs (two-fold dilutions) from nuclear extracts isolated from cells 24 hr after Dex synchronization. All data are shown as mean ± SD; ns > 0.05 and ***p<0.001 Student’s t-test).
Figure 1—figure supplement 2. Validation of antibodies used for ChIP studies either deficient cells or antibody isotype controls were used to validate the ChIP-grade quality of the antisera.

Figure 1—figure supplement 2.

The test gene used was Dbp. Data are shown as mean ± SD (n = 3; **p<0.01, and ***p<0.001 Student’s t-test).
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Acetylation of BMAL1 at lysine 538 is required to initiate circadian productive transcription elongation

BRD4 binds to acetylated lysines of transcription factors/histones at the promoter of paused genes (Shi and Vakoc, 2014). The observed strong effect of JQ1 on the cellular circadian rhythm raises the possibility that among BRD4 targets there are core clock components, such as BMAL1, which is acetylated at Lys538 (Hirayama et al., 2007) and could therefore provide a binding site for BRD4. We mutated this particular lysine to an arginine (BMAL1K538R) using the CRISPR/Cas9 technique (Figure 2—figure supplement 1A–1C). BMAL1K538R protein was as stable as wildtype BMAL1 (Figure 5—figure supplement 1A), and subcellular localization and phosphorylation status of both forms were similar (Hirayama et al., 2007). We also found an unchanged enrichment of both BMAL1 variants at the promoters of the Dbp, Per1, and Nr1d1 genes (Figure 2—figure supplement 1C). Next, the interaction of BRD4 and CDK9 with BMAL1 was examined in control fibroblasts and fibroblasts containing engineered BMAL1K538R. Immunoprecipitation experiments showed that in BMAL1K538R expressing cells this interaction was markedly reduced (Figure 2A). When wildtype cells were JQ1-treated, this interaction was substantially dampened (Figure 1—figure supplement 1H). ChIP experiments demonstrated that BMAL1K538R mutant cells showed a diminished recruitment of BRD4 and of the P-TEFb subunit CDK9 to the TSS of Dbp (Figure 2B), Per1, and Nr1d1 (Figure 2—figure supplement 1E). In addition, enrichment of Ser2-phosphorylated Pol II was markedly reduced (Figure 2B; Figure 2—figure supplement 1E; Figure 2—figure supplement 2). Since both, control fibroblasts and BMAL1K538R mutant cells stably expressed a clock-driven luciferase reporter, we assessed the effect of the K538R mutation on luminescence rhythms. In BMAL1K538R cells virtually no rhythmic luciferase reporter expression was recorded for any of the three clones (Figure 2C). In addition, in these cells there was a pronounced reduction of peak expression of endogenous Dbp (Figure 2D), Per1, and Nr1d1 mRNAs (Figure 2—figure supplement 1F). It is possible that BMAL1 acetylation could additionally promote transcription initiation. However, the occupancy of the general transcription factor TFIIEα (part of the Pol II transcription initiation complex; Sainsbury et al., 2015) at the TSS of Dbp, Per1, and Nr1d1 genes was not affected in mutant cells (Figure 2—figure supplement 1G). This favors the idea that transcription elongation, and not initiation, is the process that is primarily regulated by acetylation of BMAL1. Our data support a mechanism in which acetylated BMAL1 recruits BRD4-P and TEFb to E-box-containing circadian clock genes which then results in a release of Pol II from its paused state, thereby allowing productive elongation.

Figure 2. Lys538 acetylation of BMAL1 Is essential for transcription elongation.

(A) Interaction of BMAL1 or BMAL1K538R with BRD4 and CDK9 as shown by immunoblotting of nuclear extracts from synchronized fibroblast 24 hr after Dex treatment (two-fold dilutions). TBP (TATA-binding protein) was used as loading control. Numerical values represent intensities of chemiluminescence signals of individual bands, normalized to wildtype and loading control for input samples. (B) ChIP analysis of BRD4, CDK9 and Ser2P-Pol II (normalized to total Pol II) binding to the Dbp gene in wildtype and BMAL1K538R synchronized fibroblasts as shown in (A) (n = 3). (C) Bmal1-LUC bioluminescence tracings of synchronized wildtype fibroblasts and of three independent BMAL1K538R clones (n = 4). (D) Dbp mRNA expression analysis of wildtype or BMAL1K538R fibroblasts taken from the experiment shown in (C) (n = 3, two-way ANOVA, see Supplementary file 1). All data are shown as mean ± SD.

Figure 2.

Figure 2—figure supplement 1. CRISPR/Cas9-mediated generation and characterization of BMAL1K538Rmutant cells.

Figure 2—figure supplement 1.

(A) Schematic representation of the gene targeting strategy for generating BMAL1K538R mutant cells using the CRISPR/Cas9 system. The gRNA-targeting sequence is underlined and the PAM sequence is indicated in red. The oligonucleotide donor (120 bp) is shown below the targeted site, with the Lys-to-Arg substitution indicated in red. Silent point mutations were introduced for the two adjacent Gly to prevent cutting of the donor DNA or re-cutting of the genome after homology-directed recombination (shown in red). (B) Representative sequencing results from PCR amplicons of the Lys538 region from three individual clones that were subsequently used for experimental work. Primer sequences are specified in Supplementary file 2. (C) ChIP analysis of BMAL1 binding to Dbp, Per1, and Nr1d1 genes in wildtype and BMAL1K538R synchronized fibroblasts 24 hr after Dex synchronization (n = 3). (D) BMAL1 IPs from wildtype or BMAL1K538R fibroblast nuclear extracts carried out 24 hr after Dex synchronization of fibroblasts were immunoblotted with anti-AcBMAL1 and anti-BMAL1 antibodies. (E) BRD4, CDK9, and Ser2P-Pol II (normalized to total Pol II) ChIP analysis of Dex synchronized wildtype or BMAL1K538R fibroblasts (n = 3). (F) Per1 and Nr1d1 mRNA expression analysis of wildtype or BMAL1K538R fibroblasts (n = 3, two-way ANOVA, see Supplementary file 1). (G) ChIP analysis of TFIIEα binding to Dbp, Per1, and Nr1d1 genes in wildtype and BMAL1K538R synchronized fibroblasts (n = 3). All data are shown as mean ± SD.
Figure 2—figure supplement 2. Characterization of BMAL1K538Rmutant cells Pol II and Ser2P-Pol II ChIP analysis of Dex synchronized wildtype or BMAL1K538R fibroblasts (n = 3).

Figure 2—figure supplement 2.

All data are shown as mean ± SD.
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TIP60 is essential for a functional circadian clock in the mouse

BMAL1 acetylation is carried out by CLOCK (Hirayama et al., 2007). However, we found that in CLOCK-deficient fibroblasts BMAL1 was acetylated at Lys538 to a similar extent as observed in wildtype cells (Figure 3A). CLOCKmutA fails to acetylate histones H3 and H4 (Doi et al., 2006) and shows greatly diminished activity in the acetylation of BMAL1 (Hirayama et al., 2007). We found that both CLOCK variants showed a minimal increase of BMAL1 acetylation compared to a control (Figure 3B). Importantly, BMAL1 acetylation levels in the presence of CLOCK or CLOCKmutA were indistinguishable. Together, these data suggest the existence of alternative acetyltransferases that catalyze BMAL1 acetylation. A candidate is TIP60 that co-purifies with CLOCK-BMAL1 (Doi et al., 2006) and acetylates a broad range of transcription factors (Judes et al., 2015). Addition of TIP60, but not enzymatically inactive TIP60C369A;E403Q (Yang et al., 2012), strongly enhanced BMAL1 acetylation (Figure 3B).

Figure 3. Acetylation of BMAL1 in CLOCK-deficient cells.

Figure 3.

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(A) BMAL1 IP analysis of control and Clock-deficient MEF nuclear extracts over a 24 hr time course. For both genotypes, equivalent amounts of BMAL1 were included as shown in the anti-BMAL1 probed western blot. Lys538 acetylation of BMAL1 was detected using an anti-AcBMAL1 antibody (left). Wildtype and mutant cells were PCR-genotyped to confirm their genotype (right) (Debruyne et al., 2006). (B) BMAL1FLAG, CLOCKmyc, CLOCKmutA;myc, TIP60V5, and TIP60C369A;E403Q;V5 were transiently overexpressed in HEK293T cells in the combinations indicated. Lysates were subjected to IPs and immunoblotted with antibodies indicated (n = 3). Data are shown as mean ± SD.

This unexpected discovery of a new potential regulator of the circadian clock prompted us to generate conditional TIP60 mice to assess the in-vivo function of this enzyme and provide fibroblasts that we then used to study the molecular clock in vitro. Tip60fl/- mice carrying one conditional and one deleted allele of Tip60 (Figure 4—figure supplement 1A and B) were mated to Syt10-CRE driver mice. This driver is strongly active in the postmitotic neurons of the SCN (Husse et al., 2011) circumventing cell lethality associated with TIP60-deficiency (Hu et al., 2009). All offspring carrying two copies of the knock-in Cre allele (Syt10Cre/Cre Tip60fl/-) and half of the mice carrying one copy of the knock-in Cre allele (Syt10Cre/+Tip60 fl/-) became immediately arrhythmic upon release into constant darkness (DD) (Figure 4A and B). The rest of the Syt10Cre/+Tip60 fl/- mice showed either longer (29%) or normal (21%) free-running periods. Such a Cre dosage dependence was also observed with Syt10-CRE-mediated deletion of Bmal1fl/fl and, therefore, characterizes this particular driver (Husse et al., 2011). Immunohistochemical staining for TIP60 in the SCN showed nuclear signal, which was absent in arrhythmic Syt10Cre/+Tip60 fl/- mice (Figure 4C). Moreover, circadian expression patterns of Per1, Dbp, and Bmal1 in the SCN were severely dampened (Figure 4D) in line with the behavioral arrhythmicity observed in these animals. Nuclear staining of SCN sections from Syt10Cre/+Tip60+/+ and arrhythmic Syt10Cre/+Tip60 fl/- mice showed no obvious changes in SCN morphology or in the number of SCN cells (control: 827 ± 60 cells per section; mutant: 850 ± 68 cells per section). In addition, no TUNEL signal, indicating apoptotic cells, could be detected in the SCN of TIP60-deficient animals (Figure 4—figure supplement 1C). Together, these experiments indicate that SCN cells were fully viable after deletion of Tip60.

Figure 4. TIP60-deficiency evokes a circadian phenotype in mice and disrupts rhythmic clock gene expression in the SCN and in MEFs.

(A) Double-plotted actograms of control (Syt10Cre/+Tip60+/+) and mutant (Syt10Cre/+Tip60 fl/-, Syt10Cre/Cre Tip60fl/-) mice under 12 hr:12 hr light-dark and constant darkness conditions. Gray shadings indicate dark phases. (B) Free-running periods determined by χ2 periodogram analysis. AR: arrhythmic (***p<0.001, one-way ANOVA with Bonferroni post-test). (C) TIP60-immunoreactivity (ir) in the SCN of control (Syt10Cre/+Tip60+/+) and arrhythmic mutant (Syt10Cre/+Tip60 fl/-) mice. (D) Densitometric quantification of clock gene mRNA expression from radioactive in situ hybridization analysis of control (Syt10Cre/+Tip60+/+; black) and mutant (Syt10Cre/+Tip60 fl/-; gray) SCN sections (n = 3; two-way ANOVA, see Supplementary file 1). (E) Bioluminescence recordings of synchronized Tip60fl/-; Bmal1-LUC MEFs transduced with AdGFP (black) or AdCre (gray) (n = 4). (F) mRNA expression analysis of Dex synchronized Tip60fl/- MEFs transduced with either AdGFP (black) or AdCre (gray) (n = 3; two-way ANOVA, see Supplementary file 1). All data are shown as mean ± SD.

Figure 4.

Figure 4—figure supplement 1. Generation and validation of the Tip60-deficient mice and MEFs derived from such animals.

Figure 4—figure supplement 1.

(A) Targeting strategy for the Tip60 locus. Tip60 wildtype allele, targeting vector, targeted allele, floxed allele after removal of the PGK-Neo cassette, and the mutant Tip60 allele created by Cre-mediated recombination are shown. LoxP (black triangle) and FRT (open triangle) sites are marked. The arrows indicate the position of diagnostic primers P1-P3 (see Supplementary file 1). (B) PCR genotyping of Tip60 alleles. The wildtype allele produces a 331 bp amplicon (primers P1, (P2) while the floxed allele produces a 429 bp amplicon (P1, P2). Deletion of the LoxP-flanked region of the Tip60 locus leads to a 487 bp fragment (P1, P3). (C) TUNEL immunoreactivity on brain sections of Syt10Cre/+Tip60+/+ (control), arrhythmic Syt10Cre/+Tip60 fl/- animals, and Syt10Cre/+Tip60 fl/- sections treated in situ with DNase I. (D) mRNA expression of housekeeping genes in Tip60fl/- MEFs transduced with either AdGFP or AdCre, 24 hr after Dex synchronization. Data are mean ± SD (n = 3; ns > 0.05 and ***p<0.001, Student’s t-test). (E) Flow cytometric analysis of AdGFP, AdCre transduced, and heat-treated (3 min, 95°C) confluent Tip60fl/- MEFs. (F) Bioluminescence tracing of Dex-synchronized Bmal1-LUC MEFs transduced with AdGFP (black) or AdCre (red) (n = 4).
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The essential circadian function of TIP60 was also seen in MEFs. Immortalized MEFs were prepared from Tip60fl/- mouse embryos and were transduced with a Bmal1-LUC reporter. Confluent, that is non-dividing, MEFs were infected with AdCre to delete TIP60. Cell confluency is sufficient to induce cell cycle quiescence (Hayes et al., 2005), thus bypassing the role of TIP60 in cell cycle regulation (Sykes et al., 2006; Tang et al., 2006). Deletion resulted in a major dampening of the rhythmic expression of the luciferase reporter and a significantly reduced expression of endogenous clock genes (Figure 4E and F), but not of several housekeeping genes (Figure 4F; Figure 4—figure supplement 1D). The deletion of TIP60 in confluent MEFs did not affect cell viability (Figure 4—figure supplement 1E) nor did adenoviral transduction itself alter the circadian rhythm of a luciferase reporter (Figure 4—figure supplement 1F). Thus, abrogation of TIP60 function has major effects on locomotor activity and the rhythmic expression of clock genes in the SCN in vivo and in postmitotic MEFs.

TIP60 acetylates BMAL1 at lysine 538

The severe circadian phenotype in the mouse, the strong changes of clock gene expression in TIP60-deficient SCN and MEFs, and co-transfection experiments (Figure 3B) all suggest that BMAL1 could be a direct substrate of TIP60. Co-transfection experiments showed that Lys538 of BMAL1 was acetylated by TIP60 in a dose-dependent manner and that acetyltransferase-deficient TIP60C369A;E403Q (Yang et al., 2012) led to baseline acetylation levels of BMAL1 similar to mock transfected cells (Figure 3B; Figure 5A). Lys538 appeared to be the predominant lysine acetylated by TIP60 as the signal obtained with an anti-pan-AcK antibody was strongly diminished in cells co-transfected with BMAL1K538R and TIP60 (Figure 5A). TIP60 co-precipitated with wildtype and BMAL1K538R suggesting an interaction between the two proteins (Figure 5A; Figure 5—figure supplement 1A). Acetylation of BMAL1 did not influence this interaction. Both, BMAL1 and TIP60V5 proteins bound to the E-boxes of the Dbp gene (Figure 5—figure supplement 1B). Recombinant TIP60GST acetylated recombinant BMAL1GST at Lys538 (Figure 5B) and, in TIP60-deficient MEFs, Lys538 acetylation of endogenous BMAL1 was markedly reduced (Figure 5C). When endogenous TIP60 was replaced by TIP60C369A;E403Q in the Bmal1-LUC reporter cell line, luciferase rhythms and mRNA rhythms of endogenous Dbp were abolished (Figure 5D and E). Collectively, these data provide strong evidence that BMAL1 is a substrate of TIP60.

Figure 5. TIP60 acetylates BMAL1.

(A) BMAL1myc, BMAL1K538R, TIP60V5, and TIP60C369A;E403Q;V5 were transiently overexpressed in HEK293T cells in the combinations indicated on top. Lysates were subjected to IPs using the antibodies indicated. (B) Recombinant BMAL1GST, TIP60GST and acetyl-CoA were incubated and the presence of acetylated BMAL1 was detected by immunoblotting using either a pan-acetyl or a Lys538-specific AcBMAL1 antibody. (C) IPs (top) and nuclear extracts (bottom) from AdGFP (control) and AdCre (mutant) transduced unsynchronized Tip60fl/- MEF (two-fold dilutions) were immunoblotted with antibodies indicated and signal intensities were quantified and normalized. Data are shown as mean of all relative values ± SD (n = 3). (D) Tip60fl/-; Bmal1-LUC MEFs stably expressing TIP60V5 (red), TIP60C369A;E403Q;V5 (blue) or empty vector (black, coinciding with the blue tracing) were transduced with AdCre and bioluminescence was recorded (n = 4). (E) Dbp expression profiles in TIP60V5 and TIP60C369A;E403Q;V5 cells shown in (D). (n = 3; two-way ANOVA, see Supplementary file 1).

Figure 5.

Figure 5—figure supplement 1. TIP60 interacts with BMAL1 on chromatin.

Figure 5—figure supplement 1.

(A) Immunoblot analysis of the interaction between endogenous TIP60 with BMAL1 or BMAL1K538R. IPs from Dex synchronized MEF (24 hr after Dex) nuclear extracts were immunoblotted with the antibodies indicated. IgG and buffer (mock IP) served as negative controls. (B) ChIP analysis of Tip60-/-; Tip60V5 MEFs using anti-V5 and anti-BMAL1 antibodies. The diagram shows the 5’ region of the Dbp gene. E-boxes that bind CLOCK-BMAL1 at exons 1 (E1), exon 2 (E2), and transcription start sites are shown. Horizontal lines represent PCR-amplified genomic regions. The 3’ UTR region was used as a control. Data are shown as mean ± SD (n = 3).
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TIP60 controls productive elongation of circadian transcripts

So far, we have shown that TIP60 acetylates BMAL1 at Lys538 and that abrogation of TIP60 function has major effects on the rhythmic expression of clock genes (Figure 4). BMAL1K538R failed to interact with BRD4 and P-TEFb (Figure 2A and B). Furthermore, Pol II Ser2 phosphorylation, an indicator of productive elongation, was markedly reduced in BMAL1K538R cells (Figure 2B, Figure 2—figure supplement 1E; Figure 2—figure supplement 2). It follows that deletion of TIP60 should abrogate BRD4-P-TEFb recruitment and Pol II pause release. Indeed, in confluent TIP60-deficient Dex synchronized fibroblasts, BMAL1 was hypoacetylated (Figures 5C and 6A) and there was a major loss of interaction between BMAL1 and BRD4 (Figure 6A and B) as well as between BMAL1 and CDK9 (Figure 6C). ChIP experiments showed that enrichment of BRD4 and CDK9 at the TSS of Dbp, Per1, and Nr1d1 genes was strongly reduced and Ser2-phosphorylated Pol II occupancy was diminished (Figure 6D; Figure 6—figure supplement 1A and B).

Figure 6. TIP60 controls productive elongation.

(A) Interaction of BMAL1 with BRD4 in Tip60fl/- MEFs transduced with either AdGFP (control) or AdCre (mutant). Immunoblots show results with increasing equivalents of IPs (two-fold dilutions) from nuclear extracts isolated from cells 24 hr after Dex synchronization. (B) BRD4 or (C) CDK9 IPs from AdGFP or AdCre transduced Tip60fl/- MEF nuclear extracts were immunoblotted with antibodies indicated. Analyses were carried out 24 hr after Dex synchronization. (D) ChIP analysis for BRD4, CDK9 and Ser2P-Pol II (normalized to total Pol II) in the promoter or the 3’-end of Dbp for the cells shown in (A). Data are shown as mean ± SD (n = 3). Numerical values represent intensities of chemiluminescence signals of individual bands, normalized to wildtype and loading control for input samples.

Figure 6.

Figure 6—figure supplement 1. TIP60 controls productive elongation of circadian genes.

Figure 6—figure supplement 1.

(A, B and C) BRD4, CDK9, and Ser2P-Pol II (normalized to total Pol II) (A), Pol two and Ser2P-Pol II (B) or TFIIEα (C) ChIP analysis of Dex synchronized Tip60fl/- MEFs transduced with either AdGFP or AdCre. All data are shown as mean ± SD (n = 3).
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Above we provided evidence that acetylation of BMAL1 plays a key role in transcription elongation rather than initiation (Figure 2—figure supplement 1G). Since TIP60 acetylates BMAL1, TIP60 deletion should not affect transcription initiation, which was indeed the case. Deletion of Tip60 did not affect TFIIEα recruitment to the TSS of Dbp, Per1, and Nr1d1 genes (Figure 6—figure supplement 1C).

Rhythmicity of productive elongation of circadian transcripts

Collectively, our experiments provide evidence that TIP60-mediated acetylation of BMAL1 is an essential element of the positive limb of the circadian clock oscillator. TIP60-mediated BMAL1 acetylation leads to the recruitment of BRD4 and of the pause release factor P-TEFb to clock gene TSSs. This, in turn, allows productive elongation of CLOCK-BMAL1-controlled genes. This positive limb of the circadian cycle is counteracted by a negative feedback. This balance between positive and negative regulation, characteristic for the circadian cycle, should also be seen in temporal occupancy profiles of pause release factors at the promoters of E-box-controlled clock genes. Approximately 24 hr after synchronization of the cells, we observed the strongest enrichment of BMAL1 and of acetylated BMAL1 at the promoters of Dbp, Per1, and Nr1d1 genes (Figure 7A, green). The concentration of acetylated BMAL1 in nuclear extracts also showed its maximum by that time (Figure 7B). In parallel, the occupancy of BRD4 peaked at target gene promoters (Figure 7A) and Ser2 phosphorylation of Pol II showed a maximum (Figure 7A; Figure 7—figure supplement 1). Consequently, the abundance of Dbp, Per1, and Nr1d1 mRNA peaked between 24 and 28 hr (Figure 7A). Taken together, our data show that TIP60-dependent BRD4 recruitment, Pol II pause release, and productive elongation are precisely timed over the circadian cycle and in this way exert a temporal control over the circadian clock oscillator.

Figure 7. Rhythmic profile of productive elongation.

(A) ChIP profiles of Dex-synchronized MEFs over a course of 24 hr. ChIP shows time-of-day-dependent enrichment of BMAL1, acetylated BMAL1, BRD4, and Ser2P-Pol II (normalized to total Pol II) at the promoter or 3’-end (Ser2P-Pol II) of Dbp, Per1, and Nr1d1 genes. The rightmost column reveals time-dependence of mRNA accumulation for these genes. Data are shown as mean ± SD (n = 3). (B) Immunoblot analysis with the indicated antibodies of Dex-synchronized fibroblast nuclear extracts and BMAL1 IPs. The color bars represent the activation (green) and repression (red) phases of the circadian cycle.

Figure 7.

Figure 7—figure supplement 1. Rhythmic profile of Pol II and Ser2P-Pol II abundance ChIP profiles of Dex-synchronized MEFs over a course of 24 hr.

Figure 7—figure supplement 1.

ChIP shows time-of-day-dependent enrichment of Pol II and Ser2P-Pol II at the 3’-end of Dbp, Per1, and Nr1d1 genes. Data are shown as mean ± SD (n = 3).
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Discussion

Genome-wide ChIP studies (Koike et al., 2012; Menet et al., 2012; Rey et al., 2011) show that at the onset of the circadian cycle, the circadian repressor protein CRY1 is associated with chromatin-bound CLOCK-BMAL1 located near Pol II. In this complex Pol II is bound to the clock gene promoters but is not competent to drive RNA synthesis. At this time in the circadian cycle Pol II is phosphorylated at Ser5 (Koike et al., 2012), which is indicative for promoter-proximal pausing and recruitment of the mRNA capping complex to Pol II (Harlen and Churchman, 2017). Evidence for Pol II pausing to occur at this time at circadian promoters can also be found in global run-on experiments measuring transcriptionally engaged Pol II during the circadian cycle (Fang et al., 2014). Strong enrichment of Pol II at the TSS of the Nr1d1 gene was observed at the onset of the circadian cycle, suggesting that Pol II is in a paused state. Genome-wide and circadian time-resolved quantitative analysis of Pol II abundance at TSSs and gene bodies show a peak of Pol II binding at TSSs, but not the gene bodies of clock genes, at the onset of the circadian cycle (Zhu et al., 2018). A circadian control of promoter proximal pausing and also of Pol II pause release would provide mechanisms capable to accurately time circadian transcription (Jonkers and Lis, 2015; Liu et al., 2015).

In the present work, we have addressed the mechanism of the circadian regulation of Pol II pause release with a focus on proteins that are known to mediate this process. We propose that pause release involves a TIP60-mediated on-chromatin acetylation of Lys538 of BMAL1. BMAL1 acetylation allows the recruitment of the co-activator BRD4 to AcBMAL1. BRD4, in turn, recruits the pause release factor P-TEFb, whose kinase subunit CDK9 then phosphorylates Ser2 of Pol II leading to a release of Pol II from the paused state enabling productive elongation of E-box-containing circadian genes (Figure 7A). In TIP60-deficient cells, Lys538 acetylation of BMAL1 was strongly diminished, BRD4-P-TEFb recruitment was reduced, and the expression of Dbp, Per1, and Nr1d1 genes was dampened. If acetylation of BMAL at Lys538 was prevented by a Lys to Arg substitution (BMAL1K538R), this mutant protein still interacted with TIP60, but recruitment of BRD4 and P-TEFb to the complex was reduced and Pol II pause release and productive elongation were diminished. The dysregulation of the circadian rhythm of locomotor activity in the mouse and perturbation of circadian gene expression in the SCN and in MEFs provide strong genetic evidence for a key role of TIP60 in the circadian clock. Our studies of TIP60-deficient MEFs suggest that rhythmic control of Pol II pause release may be a main process in which TIP60 is involved. Our studies thus implicate Pol II pause release as an additional regulatory step that contributes to the temporal control of the mammalian circadian clock. However, we cannot completely rule out the possibility that the loss of TIP60 could affect post-initiation event other than Pol II pause release and thereby control RNA processing or RNA stability (Core and Adelman, 2019). Kinetic analysis of all steps of the transcription cycle could address this issue.

TIP60 is evolutionarily highly conserved and is involved in the regulation of a series of cellular processes (see Introduction). Thereby, TIP60 has the role of an enzymatically active cofactor in larger multiprotein complexes (Jacquet et al., 2016; Judes et al., 2015). Our studies suggest a similar function of TIP60 within the circadian clock.

Our studies are based on three robustly rhythmic and clock phase-specific genes that harbor multiple CLOCK-BMAL1 binding sites such as the tandem E-box motif (E1-E2) (Paquet et al., 2008; Rey et al., 2011). There are many other strongly rhythmic, phase specific CLOCK-BMAL1-target genes that also harbor multiple E-boxes (Rey et al., 2011; Shimomura et al., 2013). This suggests that regulation by Pol II pause release could extend to the entire class of E-box controlled clock genes. These genes are synchronously expressed and would benefit from a precisely timed Pol II pause release process. It is not clear, however, whether BRD4-P-TEFb mediated Pol II pause release applies for other types of clock-controlled genes. Temporal analyses of Pol II pause release at the transcriptome-wide scale can address these issues.

We found that in fibroblasts, but also in the SCN, deletion of TIP60 resulted in a nearly complete loss of rhythmic expression of numerous circadian genes suggesting that in these tissues and for these genes TIP60 function is non-redundant with CBP/p300 acetyltransferase that also binds to circadian promoters. The strongest argument for BMAL1 being a substrate of TIP60 is that recombinant TIP60 directly acetylates recombinant BMAL1. Although p300 can acetylate BMAL1 it does so at lysine 500 (Weinert et al., 2018). The abundance of BRD4 at clock gene promoters was drastically reduced in BMAL1K538R cells, but it is likely that there are other acetylated substrates, such as histones H3 and H4 (Etchegaray et al., 2003; Koike et al., 2012; Ripperger and Schibler, 2006; Vollmers et al., 2012), to which BRD4 could also be recruited and affect the circadian clock.

It is proposed that CLOCK acetylates its heterodimeric partner BMAL1 at lysine 538. This leads to recruitment of CRY protein and, thus, initiates the repression phase of the circadian cycle (Hirayama et al., 2007). We found that TIP60-mediated acetylation of BMAL1 occurs during the activation phase of the cycle, which is consistent with the data of Nakahata and co-workers (Nakahata et al., 2008; Nakahata et al., 2009). Our finding of strong acetylation of Lys538 of BMAL1 in CLOCK-deficient fibroblasts and the absence of an effect on the BMAL1 acetylation status in a reaction with a catalytically inactive CLOCK mutant protein raises some doubt about the efficiency of CLOCK as an acetyltransferase for BMAL1. It is noteworthy that CLOCK acetylates argininosuccinate synthase in the cytosol of U2OS cells (Lin et al., 2017). Since our experiments aimed at transcriptional regulation of the clock, they were carried out with nuclear extracts, they do not address cytosolic acetyltransferase activity of CLOCK.

TIP60 acetylates BMAL1 during the activation phase, but it is not clear by which mechanism BMAL1 is deacetylated during the repression phase. One possibility is a SIRT1-dependent deacetylation of BMAL1 (Nakahata et al., 2008). Alternatively, recruitment of histone deacetylases by PER to the CLOCK-BMAL1 heterodimer (Duong et al., 2011; Kim et al., 2014) would lead to deacetylation of BMAL1. Another possibility is that promoter-associated acetylated BMAL1 could be subject to rapid proteolytic turnover (Stratmann et al., 2012) obviating the need for a specific deacetylase.

Taken together, our study provides evidence that the positive limb of the circadian clock requires TIP60-mediated acetylation of BMAL1, at least for the genes studied here. This could allow a precise and synchronous expression of E-box-containing clock genes and in this way also contribute to robustness and precision of the circadian clock. Robust and synchronous expression are the hallmarks of genes involved in stimulus-controlled pathways, whose transcription is also often regulated through Pol II pause release (Jonkers and Lis, 2015; Liu et al., 2015; Sawarkar et al., 2012; Siegal and Rushlow, 2012).

Materials and methods

Tip60 targeting vector and generation of experimental animals

Bacteria (Strain EL350) and plasmids (pL451 and pL452) used for recombineering were obtained from the NCI (Biological Resources Branch). A 15.7 kb fragment of Tip60 genomic DNA was cloned from a BAC clone (bMQ-331N14, Sanger Institute) into a targeting vector carrying a Pol2-DTA cassette for negative selection. The first loxP site was inserted 637 bp upstream of the Tip60 ATG. The second loxP site together with a FRT-flanked PGK-neo cassette was inserted 3,065 bp downstream of the ATG. The 5’ end of the homologous arm of the targeting vector was 4.5 kb and the 3’ end of the homologous arm was 7.6 kb. ES cell targeting and generation of Tip60fl/+ founder mice was commissioned to PolyGene Transgenics (Switzerland). The FRT-flanked PGK-neo selection cassette was removed by crossing mice with a Flippase-expressing deleter line. Primers P1-P3 used for PCR genotyping of the Tip60 mice are listed in Supplementary file 2.

To generate animals for locomotor activity recording, Tip60fl/fl mice were crossed to a Syt10Cre driver mouse line resulting in Cre-mediated deletion of TIP60 predominately in the SCN (Husse et al., 2011). To generate Tip60fl/- mice for subsequent MEF isolation, Tip60fl/fl mice were crossed to a ubiquitous CMVCre driver mouse line to generate Tip60+/- ± which were then used for breeding with Tip60fl/fl mice to obtain Tip60fl/- offspring. For both types of studies, mice were previously backcrossed to a C57BL/6 background for at least 10 generations. Mouse handling was carried out in accordance with the German Law on Animal Welfare and was ethically approved and licensed by the Office of Consumer Protection and Food Safety of the State of Lower Saxony (license numbers 33.11.42502-04/072/07 and 33.9-42502-04-12/0719).

Generation of Bmal1K538R mutant cells

Bmal1K538 mutant cell lines were generated using CRISPR/Cas9-based genome editing. Fibroblasts stably expressing a clock-driven luciferase reporter (Bmal1-LUC) (Nagoshi et al., 2004) were co-transfected with an all-in-one plasmid pSpCas9(BB)−2A-Puro (PX459) harboring CAS9, puromycin-resistance gene, and guide RNA (sgRNA), and a single-stranded oligodeoxynucleotide (ssODNs) template comprising the point mutation. The sgRNA was designed using the online tool (http://crispr.mit.edu/). The target sequence of sgRNA was 5’-GGTCCTCCGTTCTTCCATTCGTA-3’ (PAM sequence is underlined). Transfected cells were selected with puromycin (1 µg/ml) for two days and clonal cell lines were isolated by limiting dilution. Genomic DNA of these clones was extracted and subjected to sequencing. Primers used for screening are listed in Supplementary file 2.

Plasmids

Bmal1, Tip60 ORFs were amplified by PCR and cloned into pcDNA3.1 vector containing either C-terminal myc- or V5-tags. Single site mutations were introduced by using the QuikChange II Site-directed Mutagenesis Kit (Agilent Technologies). Tip60V5 and Tip60C369A;E403Q;V5 were cloned into pLenti PGK Hygro DEST vector. Bmal1-FLAG was from M.J. Rossner (LMU Munich). Myc-Clock and myc-ClockmutA were from P. Sassone-Corsi. Lentiviral Bmal1-dLuc reporters were from S.A. Kay (USC Los Angeles). pBABE-puro SV40 LT (#13970), pCL-Eco (#12371), pMD2.G (#12259), psPAX2 (# 12260), pLenti PGK Hygro DEST (#19066), and pSpCas9(BB)−2A-Puro (# 48139) plasmids were from Addgene.

RNA analysis by quantitative Real-Time PCR (qPCR)

Total RNA was extracted using RNeasy Plus Mini Kit (Qiagen). cDNA was synthesized using Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Real-time PCR reactions were performed on a CFX96 Real-Time PCR Detection System (Bio-Rad) with IQ SYBR Green Supermix (Bio-Rad). Eef1a or Gapdh expression was used for normalization. Primers were as described (Oster et al., 2006) except for Gapdh, TIP60, 18S, Actb, and 36b4 (Supplementary file 2).

Radioactive in situ Hybridization

Mice were entrained for about 2 weeks to a 12 hr:12 hr light dark cycle and subsequently released into constant darkness. On the second day after lights-off mice were sacrificed at four time points and brains were collected and frozen in O.C.T. medium (Tissue-Tek). Templates for Dbp, Per1, and Bmal1 were as described (Oster et al., 2003; Oster et al., 2006). In situ hybridization with 35S-labeled antisense RNA probes was carried out on frozen sections through the SCN. Relative quantification of expression strength was performed by densitometric analysis of autoradiograph films.

Immunohistochemistry

Cryo-sections through the SCN were fixed with 4% paraformaldehyde for 15 min at 4°C, boiled several times in 10 mM sodium citrate (pH 6.4) for 5 min, and permeabilized for 15 min with 0.5% Triton-X-100. Sections were blocked with 10% FBS in PBS followed by incubation with anti-TIP60 antibody and a fluorescent secondary antibody. Nuclei were stained with DAPI.

Cell culture

Tip60fl/- MEFs were isolated following standard procedures. MEFs, fibroblasts (NIH-3T3 Bmal1-LUC; Nagoshi et al., 2004), and HEK293T cells were cultured and passaged in standard medium (DMEM, high glucose, GlutaMAX supplement, pyruvate; 10% fetal bovine serum; 100 U/ml penicillin and 100 µg/ml streptomycin [all Thermo Fisher Scientific]) at 37°C in a humidified incubator with 5% CO2. MEFs and fibroblasts were synchronized by treatment with 100 nM dexamethasone, a synthetic glucocorticoid that induces expression of Per1 (Reddy et al., 2009; Reddy et al., 2012). JQ1 (500 nM), Flavopiridol (300 nM) or vehicle (final 0.05% DMSO) were added to standard medium after synchronization. All cells were tested using morphology and PCR-based approaches to confirm their identity. Cell lines were tested negative for mycoplasma (Sigma-Aldrich).

Transfection and viral transduction

HEK293T cells were transiently transfected using 1 mg/ml polyethylenimine solution (Sigma-Aldrich). After two days, cells were used for western blotting and immunoprecipitations (IPs). Tip60fl/- MEFs were immortalized with SV40 LT retrovirus. Immortalized MEFs were grown to confluence and transduced with AdGFP or AdCre adenoviruses (Vector Development Laboratory, Baylor College of Medicine, Houston, TX, USA) in standard medium containing 3% fetal bovine serum (low-serum) and 8 µg/ml polybrene (Sigma-Aldrich). After two days, medium was changed to low-serum medium and cells were cultured for another 4–6 days. For lentiviral transduction (luciferase experiments) viruses were produced (Ramezani and Hawley, 2002) and concentrated using Lenti-X Concentrator (Clontech).

Preparation of cell and nuclear extracts

Transiently transfected HEK293T cells were lysed in 50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 5 mM EDTA; 15 mM MgCl2; 1% NP40; 1 mM DTT; 10 mM NaF; 10 mM NAM; Complete EDTA-free (Roche) for 30 min on ice and lysate was cleared by centrifugation (20,000 x g, 4°C, 10 min). Samples were boiled in SDS sample buffer or used for IP. Anti-Myc agarose beads (Sigma-Aldrich) were used to precipitate myc-tagged proteins. The precipitates were washed three times with lysis buffer and bound proteins eluted by denaturation in SDS sample buffer.

Nuclear extracts were prepared as described (Dimauro et al., 2012). Nuclei were lysed with 15 passages through an 18-gauge needle, sonicated using Bioruptor (30 s ON/30 s OFF) and cleared by centrifugation (9,000 g, 4°C, 30 min). For western blotting samples were denatured in SDS sample buffer. For IPs samples were diluted 3-fold in dilution buffer (20 mM HEPES pH 7.9; 1.5 mM MgCl2; 0.2 mM EDTA; 20% glycerol; 10 mM NAM; Complete EDTA-free [Roche]) and incubated for 2 hr at 4°C on a rotating wheel. Antibody was added and samples were put back on the rotating wheel. The next day, antibody-protein complexes were collected by Dynabead Protein G Beads (Thermo Fisher Scientific) and washed three times with wash buffer (20 mM HEPES pH 7.9; 1.5 mM MgCl2; 166 mM NaCl; 0.2 mM EDTA; 20% glycerol; 0,33% Triton-X-100; 10 mM NAM; Complete EDTA-free [Roche]). Proteins were eluted by boiling in SDS sample buffer and analyzed by western blotting. For acetylated BMAL1, western blotting was first performed with AcBMAL1 antibody followed by a harsh stripping (Abcam) step and subsequent reaction with the pan BMAL1 antibody. Signal intensities were determined on an ImageQuant LAS4010 imager (General Electric) and quantified using ImageQuant software.

Chromatin immunoprecipitation (ChIP)

For BMAL1 (NB100-2288), acetylated BMAL1 (AB15396), BRD4 (A301-985A), CDK9 (sc-484 or sc-13130), TFIIEα (ab28177), Pol II (MABI0601), and Ser2 phosphorylated Pol II (04–1571) ChIP analysis were performed as described (Lin et al., 2012) with minor modifications. Fibroblasts were crosslinked with 1% formaldehyde for 10 min at room temperature. The reaction was stopped by adding 125 mM glycine and incubating for 5 min. Cells were washed twice with PBS, scraped and frozen in liquid nitrogen. Dynabead Protein G Beads (Thermo Fisher Scientific) were blocked with 0.5% BSA (w/v) in PBS and bound to the indicated antibodies. Crosslinked cells were lysed in lysis buffer one and washed with lysis buffer 2. Pellets were resuspended and sonicated in lysis buffer 3 (Bioruptor; 30 s ON/30 s OFF) to obtain chromatin fragments of 150–600 bp length for promoter analysis and 100–300 bp for TSS analysis to allow a higher resolution. Sonicated lysates were cleared and incubated overnight at 4°C with beads bound with antibody for indicated factors. Beads were washed twice (CDK9, TFIIE, and Pol II Ser2P) or three times (BMAL1, AcBMAL1, Pol II, and BRD4) with lysis buffer 3, once with lysis buffer 3 with 500 mM NaCl, one time with LiCl wash buffer and one time with TE plus 50 mM NaCl. DNA was eluted in elution buffer. The eluted complexes were de-crosslinked overnight and RNA and protein were digested using RNase A and Proteinase K. Immunoprecipitated DNA fragments were purified by phenol chloroform extraction and ethanol precipitation and used for qPCR analysis.

For TIP60V5 ChIP assays were performed as described (Adli and Bernstein, 2011; Nelson et al., 2006) with minor modifications. For dual cross-linking Tip60-/-; Tip60V5 MEFs were fixed with 1.5 mM ethylene glycol bis(succinimidylsuccinate) (EGS) (Pierce) for 30 min followed by 1% formaldehyde for another 15 min. After quenching the reaction by adding glycine to a final concentration of 125 mM, cells were lysed in SDS lysis buffer on ice for 10 min, with brief vortexing. Samples were diluted 10-fold in ChIP dilution buffer and sonicated (Bioruptor; 30 s ON/30 s OFF) to obtain chromatin fragments of 150–600 bp length. V5 (R96025) antibody was added to the chromatin solution and incubated overnight at 4°C on a rotating wheel. Immunoprecipitated chromatin complexes (Dynabead Protein G Beads) were washed twice with ice cold low-salt wash buffer, twice with ice cold lithium chloride wash buffer, and twice with ice cold TE buffer. 100 µl 10% Chelex 100 Slurry (Bio Rad) was added directly to the washed beads, briefly vortexed, and boiled for 10 min. Input DNA samples were ethanol precipitated and washed with 70% ethanol. DNA pellets were dissolved in 100 µl 10% Chelex 100 suspension and boiled for 10 min. Samples were Proteinase K treated and boiled for 10 min. Supernatant was transferred to a new tube and used for qPCR analysis.

Primer pair that targets an unbound region was used as internal control (Duong et al., 2011) and fold enrichment was calculated using the 2^(-ΔΔCT) method. Experiments were performed with biological independent triplicates and data normalized to the mean value obtained from the locus with the strongest enrichment. Primer sequences for ChIP-qPCR are listed in Supplementary file 1.

Bioluminescence recording

After synchronization of cells with dexamethasone medium was changed to recording medium (DMEM with high glucose, without glutamine and phenol red; 2 mM GlutaMAX; 10 mM HEPES; 5% FBS; 100 U/ml penicillin and 100 µg/ml streptomycin [all Thermo Fisher Scientific] and 0.1 mM D-luciferin [Synchem]). Petri dishes were sealed with transparent plastic film and bioluminescence was recorded in a LumiCycle luminometer (Actimetrics).

For JQ1 and flavopiridol dose dependence experiments, NIH-3T3 Bmal1-LUC fibroblasts were plated on 35 mm dishes and cultured for 2 days to reach confluency. Cells were synchronized and medium was replaced with the recording medium containing various concentrations of JQ1, flavopiridol or vehicle (final 0.08% DMSO). For calculation of period, luminescence intensity, and damping rate parameters the curve fitting program LumiCycle (Actimetrics) was used and data obtained from the first day were excluded from analysis because of transient luminescence changes upon the medium change.

In vitro acetylation assay

Recombinant TIP60 (SignalChem) and BMAL1 (Abnova) proteins were mixed in acetyltransferase assay buffer (50 mM Tris-HCl, pH 8.0; 0.1 mM EDTA; 0.4 mM DTT; 25 ng/ µl BSA; 5% glycerol) in the presence or absence of 10 µM acetyl-CoA and incubated for 1 hr at 30°C. The reaction was stopped by denaturation in SDS sample buffer and proteins were analyzed by western blotting.

Flow cytometry

Cells were stained using the SYTOX AADvanced Dead Cell Stain Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions and samples were analyzed using the Accuri C6 Flow Cytometer (BD). To provide a positive control cells were boiled for 3 min.

Acknowledgements

We thank Dr. JW Lough and Dr. B Amati for antibodies, Dr. MP Antoch for Clock-/- and Bmal1-/- MEFs and Dr. SA Kay, Dr. P Sassone-Corsi and MJ Rossner for sharing vectors.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Gregor Eichele, Email: gregor.eichele@mpibpc.mpg.de.

Asifa Akhtar, Max Planck Institute for Immunobiology and Epigenetics, Germany.

K VijayRaghavan, National Centre for Biological Sciences, Tata Institute of Fundamental Research, India.

Funding Information

This paper was supported by the following grants:

  • Volkswagen Foundation Lichtenberg Fellowship to Henrik Oster.

  • Max-Planck-Gesellschaft Open-access funding to Gregor Eichele.

Additional information

Competing interests

No competing interests declared.

Author contributions

Conceptualization, Data curation, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing, Designed and supervised research, Prepared the manuscript with input from all authors.

Generated mouse model, Performed behavior analysis.

Methodology, Generated mouse model.

Data curation, Validation, Investigation, Methodology, Writing—original draft, Writing—review and editing.

Conceptualization, Resources, Supervision, Funding acquisition, Validation, Writing—original draft, Project administration, Writing—review and editing, Design and supervise research, Prepared the manuscript with input from all authors.

Ethics

Animal experimentation: Mouse handling was carried out in accordance with the German Law on Animal Welfare and was ethically approved and licensed by the Office of Consumer Protection and Food Safety of the State of Lower Saxony (license numbers 33.11.42502-04/072/07 and 33.9-42502-04-12/0719).

Additional files

Supplementary file 1. Two-way ANOVA statistical analysis.
elife-43235-supp1.docx (13.7KB, docx)
DOI: 10.7554/eLife.43235.017
Supplementary file 2. Primer sequences.
elife-43235-supp2.docx (14.6KB, docx)
DOI: 10.7554/eLife.43235.018
Supplementary file 3. Key resources table.
elife-43235-supp3.xlsx (25.5KB, xlsx)
DOI: 10.7554/eLife.43235.019
Transparent reporting form
elife-43235-transrepform.docx (244.5KB, docx)
DOI: 10.7554/eLife.43235.020

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

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Decision letter

Editor: Asifa Akhtar1

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for sending your article entitled "Acetylation of BMAL1 by TIP60 controls BRD4-P-TEFb recruitment to circadian promoters" for peer review at eLife. Your article is being evaluated by two peer reviewers, and the evaluation is being overseen by a Reviewing Editor and K VijayRaghavan as the Senior Editor.

The points that need to be addressed are given in the reviews below. A very important one to address is the conflict with what was previously known for BMAL1 acetylation of K538 by CLOCK leading to repression (Sassone-Corsi and co-workers) in comparison to the rather unconvincing effect of Tip60 KO on BMAL1 acetylation and interaction with Brd4 (IP-WB). Further, the way the data- analysis is presented, it is not possible to know how the ChIPs were calculated/normalized, unless this information has been missed.To reiterate, the authors would really need to address the weakness of their biochemical data. One supplementary data set arguing that CLOCK does not acetylate BMAL1 is not compelling to revise earlier reports.

Reviewer #1:

This interesting study presents data supporting a role of Tip60-dependant acetylation of BMAL1 to allow expression of circadian regulated genes. The acetylation of a previously identified single lysine on BMAL1 is argued to be at the center of a mechanism controlling Brd4/P-TEFb recruitment, allowing RNAPII pause release and productive transcription elongation. A large amount of data is presented to support the conclusions and endogenous acetylation site mutant of BMAL1 (K538R) is characterized after CRISPR-mediated genome editing.

While the model presented appears well supported by the data, it is very strange that it is not put in the context of known important literature directly linked to what is studied here. For example, BMAL1 acetylation on K537 (mouse, K538 in human) was published in Nature 2007 (Sassone-Corsi lab), shown to be done by CLOCK lysine acetyltransferase activity (BMAL1 heterodimer partner) and oscillates during the circadian cycle, peaking with the repression phase. It was argued that BMAL1 acetylation on K537 leads to repression of circadian regulated genes, through recruitment of CRY1. Ectopic expression of BMAL1 K537R mutant was shown to disrupt circadian rhythm. This is very different to what is proposed here.

Related to that, the authors state that they tested Tip60 as the KAT for BMAL1 because it was previously shown to co-IP with CLOCK/BMAL1 (Sassone-Corsi lab, Cell 2006). Well, that report also showed co-IP with 2 other major KATs, CBP and PCAF, all acetyltransferases previously shown to be present on E-box promoters in vivo… In fact Tip60 and PCAF histone acetyltransferase complexes are well-known co-factors (through their common TRRAP subunit) of Myc transcription factor that binds the same E-box sequences as CLOCK/BMAL1. Interestingly, elevated Myc expression was shown to disrupt the circadian clock (Cell Metabolism 2015).

Tip60/KAT5 is known to prefer the "GGK" sequence as substrate and this is what is found for K538 (BMAL1 sequence "GGKKI"). So modification in vitro with recombinant proteins (or through joint over-expression in transient transfection) makes sense but does not prove per se in vivo physiological relevance (see below).

Specific points about the data:

– Figure 2A:

The effect of K538R on co-IP of CDK9/Brd4 is difficult to judge since there seems to be less BMAL1 signal in the input and the BMAL1 WB signal is saturated. I suspect the effect is, in fact, minor if any.

– Figure 3—figure supplement 1:

This is a key experiment in the context of the literature that showed CLOCK-dependent acetylation of BMAL1. The data presented here raise some questions as there seems to be higher and more constant acBMAL1 signal in the CLOCK KO cells. From the band patterns on the CLOCK KO membrane it seems that the same membrane was used for both total BMAL1 and acBMAL1. Is it possible that the acBMAL1 signal is, in fact, residual total BMAL1 signal after stripping the membrane? Or non-specific acBMAL1 signal because of the high amount of IPed BMAL1 on the membrane?

– Figure 4C:

The effect of Tip60 induced KO on BMAL1 acetylation in vivo is not convincing since the level of total BMAL1 IPed is lower in the KO sample. The graph below based on n=3 shows a decrease after normalization but since the shown WB is not convincing I do not know what to think. Quantification of single point WB signals is known to be misleading as ECL is not linear. The decrease of acBMAL1 seems similar to the one shown for total BMAL1.

– Figure 5A:

The exact same thing can be said here as for Figures 2A and 4C. The effect of Tip60 KO on BMAL1 acetylation/interaction with Brd4 is not convincing as there is less BMAL1 in the input and the IPed signal is saturated but still points to less IPed BMAL1 in Tip60 KO). An identical problem can be noted in 5C as much less Brd4 is IPed, making the conclusion on BMAL1 interaction being dependent on Tip60 not valid.

In any case, it is clear that BMAL1 still gets acetylated in the absence of Tip60? As the TIP60 coactivator complex is present on the enhancers and promoters of a large number of transcribed genes, the effect of its induced KO can lead to indirect effects on gene transcription.

– Figure 5D:

To distinguish effects on txn initiation vs elongation the authors perform ChIP-qPCR with RNAPII-Ser2ph and TFIIE Abs. To really argue about initiation vs elongation, or pause release, the real readout should be RNAPII-Ser5ph vs Ser2ph?

Overall, all ChIP-qPCR experiments are difficult to efficiently judge as it is not clear from the Materials and methods/Figure legends how these values were calculated. 6B acBMAL1 oscillation not convincing with load. The graphs indicate "relative enrichment" and the Materials and methods state "to unbound regions". But since the values are 1 and below this would mean no enrichment vs the control unbound regions? The ChIP-qPCR methods also state n=3, is this biological independent triplicate experiments? Or technical replicates (same chromatin, 3 IPs)? Or PCR replicates?

Reviewer #2:

This is an interesting paper that explores mechanisms that contribute to circadian regulation of clock gene transcription, using experiments performed in cultured cells and mutant mice

In the first section of the paper, the authors show that periodic expression of a BMAL1-driven luciferase reporter and of several endogenous clock-regulated genes is inhibited by the Cdk9 inhibitor flavopiridol and by JQ1, a BET inhibitor that preferentially targets BRD4, which contributes to P-TEFb recruitment to genes. Consistent with these observations, JQ1 treatment also leads to loss of BRD4 and Cdk9 at the clock gene Dbp and to a decreased in phosphorylation of the Pol II CTD on Ser2. Based on these observations, the authors propose that BRD4-dependent P-TEFb recruitment is a rate limiting step in Dbp regulation. In further experiments, they present evidence that (i) BRD4 (and Cdk9) bind BMAL1 acetylated at lysine 538; (ii) Tip60 acetylates BMAL1 at K538; (iii) occupancy of BRD4, Cdk9, and Ser2P-phosphorylated Pol II at clock genes is reduced in cells expressing BMAL K538R, as is expression of Dbp mRNA and a BMAL1 luciferase reporter; (iv) Tip60 deficiency gives rise to a circadian phenotype in mice, dampening or disruption of cyclic clock gene expression in SCN and in fibroblasts, (v) Tip60 deficiency also leads to decreased BMAL1 acetylation, BRD4, Cdk9, and Ser2P-phosphorylated Pol II at clock genes.

Overall, the data are consistent with the authors' model that the histone acetyl transferase TIP60 contributes to circadian gene regulation by acetylating BMAL1, which in turn leads to recruitment of BRD4 and Cdk9/P-TEFb and release of promoter proximally paused Pol II into productive elongation of several circadian transcripts. I do, however, have a number of minor comments.

Comments:

1) Relevant to Figure 1 and supplemental figures: It would perhaps be more surprising if blocking P-TEFb/Cdk9 or BRD4 activity didn't interfere with clock gene regulation than that it does since they have very widespread roles in gene regulation.

2) The authors used the ratio of Ser2P Pol II/total Pol II as a measure of release from promoter-proximal pausing into productive elongation, but they don't show effects of drug treatments or BMAL1 / Tip60 mutations on total Pol II distribution. I think it would be better to show not only the ratio of these two ChIP signals but also each of them individually so the reader can assess the degree to which the mutations alter Pol II distribution vs Pol II CTD phosphorylation.

3) The authors conclude that BMAL or Tip60 mutation doesn't affect initiation based on evidence that the mutations don't lead to changes in TFIIE occupancy measured by ChIP. Strictly speaking, this is consistent with idea that PIC assembly is unlikely to be affected but doesn't rule out changes in initiation rate.

eLife. 2019 Jul 11;8:e43235. doi: 10.7554/eLife.43235.023

Author response

Reviewer #1:

[…] While the model presented appears well supported by the data, it is very strange that it is not put in the context of known important literature directly linked to what is studied here. For example, BMAL1 acetylation on K537 (mouse, K538 in human) was published in Nature 2007 (Sassone-Corsi lab), shown to be done by CLOCK lysine acetyltransferase activity (BMAL1 heterodimer partner) and oscillates during the circadian cycle, peaking with the repression phase. It was argued that BMAL1 acetylation on K537 leads to repression of circadian regulated genes, through recruitment of CRY1. Ectopic expression of BMAL1 K537R mutant was shown to disrupt circadian rhythm. This is very different to what is proposed here.

We are aware of the proposed model of Sassone-Corsi and co-workers that rests on their finding that acetylation of Lys538 by CLOCK peaks within the repression phase. This enables recruitment of CRY expressed in the repression phase. Specifically, Hirayama et al., 2007, show in their Figure 1A that BMAL1 acetylation peaks at Zeitgeber time 15 (ZT15), which is in the repression phase. However, please note that in a subsequent publications from the Sassone-Corsi laboratory (Nakahata et al., 2008), BMAL1 acetylation is most pronounced during the activation phase (left panel of Figure 1B).

These data are for the liver, the same is also true for fibroblasts. Nakahata et al., 2008, show that BMAL1 acetylation peaks ~18 h post serum shock (Figure 2A) which is the time when the BMAL1 direct target gene Dbp (for a discussion about direct targets see the Schibler lab paper by Stratmann et al., 2008) shows peak expression (Figure 2B). Accordingly, BMAL1 acetylation peaks during the activation phase at which time CRYs are of low abundance.

Nakahata et al., 2009, show that maximal BMAL1 acetylation levels again coincide with peak expression of Dbp (Figure 3).

To “put [our approach] in the context of known important literature” is rather difficult because the data from the Sassone-Corsi lab is not internally consistent. Note, however, that their 2008 and 2009 publications are consistent with our finding that maximal BMAL1 acetylation falls into the activation phase of the clock cycle.

In the Introduction we address the Sassone-Corsi results (Hiarayama et al., 2007) in the context of reviewing the ChIP-seq data of Takahashi’s group (paragraph three). We also comment on the apparent contradictions of the different papers published by the Sassone-Corsi lab (Hiarayama et al., 2007 vs. Nakahata et al., 2008/9). We come back to the point of when BMAL1 is acetylated in the Discussion.

Related to that, the authors state that they tested Tip60 as the KAT for BMAL1 because it was previously shown to co-IP with CLOCK/BMAL1 (Sassone-Corsi lab, Cell 2006). Well, that report also showed co-IP with 2 other major KATs, CBP and PCAF, all acetyltransferases previously shown to be present on E-box promoters in vivo.

We focus on TIP60 because knocking it out in the SCN leads to a severe circadian phenotype resembling that seen in BMAL1 deficiency. Of course, it is possible that CBP/p300 or PCAF acetylate BMAL1 at Lys538. Note, however, that CBP/p300 acetylates Lys500 of BMAL1 (Weinert et al., 2018). At the present time there is no knock out model that suggests that CBP/p300 or PCAF play a key role in circadian clock function.

We refer to the Weinert paper in the Discussion.

In fact Tip60 and PCAF histone acetyltransferase complexes are well-known co-factors (through their common TRRAP subunit) of Myc transcription factor that binds the same E-box sequences as CLOCK/BMAL1. Interestingly, elevated Myc expression was shown to disrupt the circadian clock (Cell Metabolism 2015).

If we understand the reviewer correctly, the reviewer suggests that TIP60-deficency would result in dysregulation of Myc expression and this would disrupt the circadian clock. Overexpression of MYC leads to enhanced expression of clock controlled genes (Altman et al., Cell Metabolism 2015; 22(6): 1009-19), a phenotype that we do not observe in TIP60-deficient cells (see Figure 3D and 3F).

Tip60/KAT5 is known to prefer the "GGK" sequence as substrate and this is what is found for K538 (BMAL1 sequence "GGKKI"). So modification in vitro with recombinant proteins (or through joint over-expression in transient transfection) makes sense but does not prove per se in vivo physiological relevance (see below).

Correct, in-vitro assays and overexpression in transient transfection experiments are supportive “only”, but we also showed that TIP60 deficiency in MEFs (derived from floxed mouse embryos) exhibited markedly reduced BMAL1 acetylation and diminished recruitment of BRD4 and p-TEFb to BMAL1 with the effect of preventing pause release.

Specific points about the data:

– Figure 2A:

The effect of K538R on co-IP of CDK9/Brd4 is difficult to judge since there seems to be less BMAL1 signal in the input and the BMAL1 WB signal is saturated. I suspect the effect is, in fact, minor if any.

Detection is based on chemiluminescence and measured with a GE ImageQuant LAS 4010 instrument that yields a quantitative output. Unlike X-ray films the instrument gives a linear correlation between protein quantity and signal intensity over 4 orders of magnitude. We measured the BMAL1 input signals and observed only a minor change (1.00 vs. 0.87). In a revised manuscript, we will provide numerical values next to the WB bands (see e.g. Zhang et al., eLife 2017; 6: e24466).

We provide numerical values for signal intensity next to the WB bands.

– Figure 3—figure supplement 1:

This is a key experiment in the context of the literature that showed CLOCK-dependent acetylation of BMAL1. The data presented here raise some questions as there seems to be higher and more constant acBMAL1 signal in the CLOCK KO cells. From the band patterns on the CLOCK KO membrane it seems that the same membrane was used for both total BMAL1 and acBMAL1. Is it possible that the acBMAL1 signal is, in fact, residual total BMAL1 signal after stripping the membrane? Or non-specific acBMAL1 signal because of the high amount of IPed BMAL1 on the membrane?

Yes, the same membrane was used for total BMAL1 and acetylated BMAL1. Unlike the referee suspected, we first detected acetylated BMAL1 followed by a stripping step and reaction with the pan BMAL1 antibody. We will add this information to “Materials and methods”.

The CLOCK-deficient cells were a gift from M.P. Antoch but were re-genotyped using primers as described in Debruyne et al., 2006. These cells shown in Figure 3—figure supplement 1 are uncontestable CLOCK-deficient.

One could argue that the Millipore antibody we used is not specific for acetylated BMAL1. However, the strong specificity of this reagent is demonstrated in Figure 2—figure supplement 1D where we detect K538 acetylated BMAL1 only in cells expressing wild type BMAL1.

We propose to include a second line of evidence, a co-transfection experiment, that further tests the hypothesis that BMAL1 is a CLOCK substrate. The experiment goes as follows. BMAL1 is transfected into HEK293 cells and after 2 days of culture the extent of acetylation of BMAL1 is determined by quantitative chemiluminescence. This defines base-line Lys538 acetylation levels of BMAL1. In parallel, BMAL1 and CLOCK or CLOCK-mutA are co-transfected (CLOCK-mutA is a catalytically inactive CLOCK that was obtained from the Sassone-Corsi lab and was re-sequenced by us for verification). If CLOCK acetylates BMAL1 the level of acetylated BMAL1 should increase, while CLOCK-mutA should not have this effect. Lastly, we will co-transfect TIP60 with BMAL1 and CLOCK or CLOCK-mutA. Here we expect that TIP60 will markedly increases BMAL1 acetylation.

We note in Materials and methods that we first detected acetylated BMAL1, subsequently carried out a stripping step followed by an incubation with the pan BMAL1 antibody.

BMAL1 is transfected into HEK293 cells and after 2 days of culture the extent of acetylation of BMAL1 was determined by quantitative chemiluminescence (new Figure 3B). This defines base-line Lys538 acetylation levels of BMAL1. In parallel, BMAL1 and CLOCK or CLOCK-mutA were co-transfected. We found that both clock variants showed a 2-fold increase of BMAL1 acetylation. By contrast with TIP60 this increase was 8-fold.

– Figure 4C:

The effect of Tip60 induced KO on BMAL1 acetylation in vivo is not convincing since the level of total BMAL1 IPed is lower in the KO sample. The graph below based on n=3 shows a decrease after normalization but since the shown WB is not convincing I do not know what to think. Quantification of single point WB signals is known to be misleading as ECL is not linear. The decrease of acBMAL1 seems similar to the one shown for total BMAL1.

A dilution series would address this critique. In fact, Figure 4C where we show the dependency of BRD4 recruitment on the acetylation status of BMAL1, provides such a dilution series and thus confirms the marked reduction of BMAL1 acetylation upon AdCre-mediated TIP60 deletion. We propose that in a revision, we will generate a dilution series with 3 additional biological replicates and revise Figure 4C accordingly. At the same time we will also measure TIP60 levels so as to address critique 9 below.

The new data (Figure 5C) are based on a dilution series and a small experimental modification in that compared to our previous experiment we used 25% more AdCre s and an incubation period of 6 instead of 4 days.

– Figure 5A:

The exact same thing can be said here as for Figures 2A and 4C. The effect of Tip60 KO on BMAL1 acetylation/interaction with Brd4 is not convincing as there is less BMAL1 in the input and the IPed signal is saturated but still points to less IPed BMAL1 in Tip60 KO). An identical problem can be noted in 5C as much less Brd4 is IPed, making the conclusion on BMAL1 interaction being dependent on Tip60 not valid.

Please see our reply to critique point 5. We will provide numerical values demonstrating that protein loads were comparable between samples. We will repeat the experiment of Figure 5B with more similar sample concentrations.

The experiment was repeated so that the experimental and control lanes now show a similar signal strength that was quantified.

In any case, it is clear that BMAL1 still gets acetylated in the absence of Tip60?

Yes, in the absence of TIP60, BMAL1 acetylation is reduced to about 35%. It should be recalled how this experiment is carried out. First, fibroblasts are grown to confluence at which time they stop proliferation. AdCre or AdGFP is added which will then lead to the excision of the floxed TIP60 with the first vector. Eventually, TIP60 protein will also disappear. At day 3 cells are synchronized by dexamethasone treatment and lysed 24 h later. Then the acetylation status of BMAL1 is checked. This is a standard protocol, but it does not “remove” all TIP60 protein. In fact, Figure 5A shows that with this protocol there is residual TIP60 (18%). We will address the issue of residual TIP60 in the context of Critique 7 above where we quantify TIP60.

See our reply above. The reduction of BMAL1 acetylation was > than 85% in three independent biological replicate experiments.

As the TIP60 coactivator complex is present on the enhancers and promoters of a large number of transcribed genes, the effect of its induced KO can lead to indirect effects on gene transcription.

TIP60 is an enzyme and as such TIP60 has multiple substrates that play a role in diverse cellular functions. This is reflected in the early embryonic lethality of Tip60-deficient flies or mice. The challenge we were facing was to set up experiments in which we could study a specific TIP60 function. We did this by studying postmitotic cells (neurons of the SCN [using the CRE-LoxP system] and growth-arrested fibroblasts) and we focused on a single substrate – BMAL1 – that is part of the circadian pacemaker. Our “bias” for BMAL1 and the clock was due to the fact, that our genetic experiments revealed a strong impairment of the circadian clock (and nothing else). Hence it makes sense to look for substrates of TIP60 among the clock proteins. Three major additional pieces of information prompted us to follow this path. First, Sassone-Corsi and associates showed that BMAL1 was acetylated and that such acetylation was functionally important. Second, we had some doubts about the possibility that CLOCK acetylates BMAL1 at lysine 538, since in CLOCK-deficient fibroblasts that particular lysine was still acetylated. Nonetheless, even in this situation there could be additional TIP60 substrates that contribute to the circadian phenotype such as hypoacetylation of certain histones. We comment on this particular point in our Discussion (paragraph five).Third, in growth-arrested Tip60-deficient MEFs we found no change in transcription of Gapdh, 18S, Actb, and 36b4 genes Figure 3 (now Figure 4F and Figure 4 —figure supplement 1D). This would argue against a pleiotropic effect of TIP60.

– Figure 5D:

To distinguish effects on txn initiation vs elongation the authors perform ChIP-qPCR with RNAPII-Ser2ph and TFIIE Abs. To really argue about initiation vs elongation, or pause release, the real readout should be RNAPII-Ser5ph vs Ser2ph?

Characterization of initiation and elongation require complex multi-omics approaches that are not just done by compering Ser2 and Ser5 phosphorylation of CTD (Gressel et al., eLife 2017; 6: e29736). Nevertheless, in this manuscript we clearly show that TIP60-mediated acetylation of BMAL1 regulates Pol II pause release and that preinitiation complex (PIC) assembly is not affected by TIP60 deficiency. Our paper pioneers the idea that TIP60-mediated control of BRD4-P-TEFb recruitment is a novel temporal checkpoint in the circadian cycle. Obviously, our finding will require further mechanistic analyses that investigate the complex interplay of transcription initiation and elongation.

Overall, all ChIP-qPCR experiments are difficult to efficiently judge as it is not clear from the Materials and methods/Figure legends how these values were calculated.

6B acBMAL1 oscillation not convincing with load… The graphs indicate "relative enrichment" and the Materials and methods state "to unbound regions". But since the values are 1 and below this would mean no enrichment vs the control unbound regions? The ChIP-qPCR methods also state n=3, is this biological independent triplicate experiments? Or technical replicates (same chromatin, 3 IPs)? Or PCR replicates?

We apologize that the way the data analysis was presented, did not include a description of how the ChIP experiments were calculated and normalized. Biological independent triplicate experiments were performed and data were first normalized to an internal control and maximum values were set to 1. We missed to give this information in “Materials and methods” section and the requested information is now found in the Materials and method section.

Reviewer #2:

[…] Overall, the data are consistent with the authors' model that the histone acetyl transferase TIP60 contributes to circadian gene regulation by acetylating BMAL1, which in turn leads to recruitment of BRD4 and Cdk9/P-TEFb and release of promoter proximally paused Pol II into productive elongation of several circadian transcripts. I do, however, have a number of minor comments.

Comments:

1) Relevant to Figure 1 and supplemental figures: It would perhaps be more surprising if blocking P-TEFb/Cdk9 or BRD4 activity didn't interfere with clock gene regulation than that it does since they have very widespread roles in gene regulation.

JQ1 and Flavopiridol affect the expression of many genes, but rather than inhibiting transcription, they prevent Pol II pause release. Thus, the effect of these pharmacological agents on the clock cycle and amplitude suggests that Pol II pause and pause release are likely to be regulatory steps in the circadian clock.

A brief assessment of the usefulness and meaning of the inhibitor experiment is provided in the first paragraph of the Results section.

2) The authors used the ratio of Ser2P Pol II/total Pol II as a measure of release from promoter-proximal pausing into productive elongation, but they don't show effects of drug treatments or BMAL1 / Tip60 mutations on total Pol II distribution. I think it would be better to show not only the ratio of these two ChIP signals but also each of them individually so the reader can assess the degree to which the mutations alter Pol II distribution vs Pol II CTD phosphorylation.

We have these data and we will include them in the revision in Figures 2 and 5 and in the associated supplemental figures.

These data are include in the revision in the supplement figures of Figures 1, 2, 6, and 7.

3) The authors conclude that BMAL or Tip60 mutation doesn't affect initiation based on evidence that the mutations don't lead to changes in TFIIE occupancy measured by ChIP. Strictly speaking, this is consistent with idea that PIC assembly is unlikely to be affected but doesn't rule out changes in initiation rate.

Yes, recent studies from the Cramer and Zeitlinger laboratories show that promoter-proximal pausing of Pol II sets a limit to the frequency of transcription initiation in both human and Drosophila cells (Gressel et al., eLife 2017; 6: e29736; Shao and Zeitlinger, Nat. Genet. 2017; 49: 1045–1051). Pol II pausing inhibits new transcription initiation. Therefore, recruitment of P-TEFb to the promoters, as described in our study, plays a central role in controlling pause duration and thereby also the productive initiation frequency (Gressel et al., eLife 2017; 6: e29736).

Associated Data

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    Supplementary Materials

    Supplementary file 1. Two-way ANOVA statistical analysis.
    elife-43235-supp1.docx (13.7KB, docx)
    DOI: 10.7554/eLife.43235.017
    Supplementary file 2. Primer sequences.
    elife-43235-supp2.docx (14.6KB, docx)
    DOI: 10.7554/eLife.43235.018
    Supplementary file 3. Key resources table.
    elife-43235-supp3.xlsx (25.5KB, xlsx)
    DOI: 10.7554/eLife.43235.019
    Transparent reporting form
    elife-43235-transrepform.docx (244.5KB, docx)
    DOI: 10.7554/eLife.43235.020

    Data Availability Statement

    All data generated or analysed during this study are included in the manuscript and supporting files.

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