CLOCK and TIMELESS regulate rhythmic occupancy of the BRAHMA chromatin-remodeling protein at clock gene promoters

Abstract

Circadian clock and chromatin-remodeling complexes are tightly intertwined systems that regulate rhythmic gene expression. The circadian clock promotes rhythmic expression, timely recruitment, and/or activation of chromatin remodelers, while chromatin remodelers regulate accessibility of clock transcription factors to the DNA to influence expression of clock genes. We previously reported that the BRAHMA (BRM) chromatin-remodeling complex promotes the repression of circadian gene expression in Drosophila. In this study, we investigated the mechanisms by which the circadian clock feeds back to modulate daily BRM activity. Using chromatin immunoprecipitation, we observed rhythmic BRM binding to clock gene promoters despite constitutive BRM protein expression, suggesting that factors other than protein abundance are responsible for rhythmic BRM occupancy at clock-controlled loci. Since we previously reported that BRM interacts with two key clock proteins, CLOCK (CLK) and TIMELESS (TIM), we examined their effect on BRM occupancy to the period (per) promoter. We observed reduced BRM binding to the DNA in clk null flies, suggesting that CLK is involved in enhancing BRM occupancy to initiate transcriptional repression at the conclusion of the activation phase. Additionally, we observed reduced BRM binding to the per promoter in flies overexpressing TIM, suggesting that TIM promotes BRM removal from DNA. These conclusions are further supported by elevated BRM binding to the per promoter in flies subjected to constant light and experiments in Drosophila tissue culture in which the levels of CLK and TIM are manipulated. In summary, this study provides new insights into the reciprocal regulation between the circadian clock and the BRM chromatin-remodeling complex.

Fig 1. BRM binding to clock gene promoters in fly heads is rhythmic despite constitutive BRM protein expression.

(A) Western blot validation of the BRM antibody detecting proteins extracted from Drosophila S2 cells and heads of flies collected at ZT16 on LD3 (light-dark cycle day 3) subsequent to 2-day entrainment at 12h:12h LD. S2 cells were either untransfected or transfected with pAc-brm-3XFLAG-His. The two fly lines used for validation are flies expressing either endogenous levels of BRM (w; tim(UAS)-Gal4 parental driver line referred to as TUG) or flies expressing FLAG-His-tagged BRM (referred to as brm^OE) (top panel). FLAG epitope was simultaneously detected to confirm expression of FLAG-tagged BRM (middle panel). HSP70 was used as a loading control (bottom panel). (B) Quantification of BRM signal shown in Fig 1A. Each data point represents a biological replicate. Error bars represent ±SEM (S2 cells: n = 3; Fly heads: n = 4). Asterisks denote significant p-values: *p<0.05. (C-H) BRM occupancy at the promoters of (C) period (per), (D) timeless (tim), (E) vrille (vri), (F) clockwork orange (cwo), (G) heat shock protein 27 (hsp27), and (H) glycine transporter (glyT) was detected in heads of w¹¹¹⁸ (WT) flies collected at the indicated time points on LD3 subsequent to 2-day entrainment at 12h:12h LD. The grey background denotes the dark phase of the LD cycle. Each data point represents a biological replicate (n = 4), and each biological replicate is an average of 2 technical replicates of qPCR. RAIN: (C) p = 0.0053; peak: ZT16, (D) p = 0.0487; peak: ZT16, (E) p = 0.0124; peak: ZT16, (F) p = 0.0005; peak: ZT16, (G) p = 0.9543, and (H) p = 0.3140 (I) Western blot showing BRM expression in heads of w¹¹¹⁸ flies (top panel) collected at the indicated time points on LD3. HSP70 was used as a loading control (bottom panel). (J) Quantification of BRM signal normalized to HSP70 as shown in Fig 1I (n = 3, RAIN p = 0.5811).

Fig 2. CLK promotes BRM occupancy at the per promoter.

(A) BRM and (B) Histone H3 occupancy at the perCRS in head tissues of w¹¹¹⁸ (black) and clk^out (red) flies (A: n = 4; w¹¹¹⁸ RAIN p = 0.0056, clk^out RAIN p = 0.7915; B: n = 3; w¹¹¹⁸ RAIN p = 0.0488, clk^out RAIN p = 0.2080). Each data point represents a biological replicate, and each biological is an average of at least 2 technical replicates of qPCR. Asterisks denote significant p-values: *p<0.05, ***p<0.001. Error bars represent ±SEM. The grey background denotes the dark phase of the LD cycle. (C) BRM binding at the perCRS in S2 cell nuclear extracts expressing either brm alone (white) or brm co-expressed with clk (grey). Relative fold change of ChIP signal is calculated with amount of BRM binding in the brm alone condition equal to 1(n = 6). (D) BRM (black) and CLK (blue) occupancy at the perCRS in heads of w¹¹¹⁸ flies collected at the indicated time points on LD3 (n = 3; BRM ChIP: RAIN p = 0.0016, phase = ZT14; CLK ChIP: RAIN p = 6.17e-6, phase = ZT12; DODR p = 0.0033). Trendlines connect the mean relative ChIP signal of each time point.

Fig 3. CLK stabilizes BRM protein.

(A-B) BRM protein and (C) brm mRNA levels in the heads of w¹¹¹⁸ (black) and clk^out (red) flies collected at the indicated time points on LD3. (B) BRM signal (A: top panel) was quantified and normalized to HSP70 (A: bottom panel) (n = 3). The grey background denotes the dark phase of the LD cycle. Each data point represents a biological replicate. Error bars represent ±SEM. Asterisks denote significant p-values: *p<0.05. (C) Steady state brm mRNA was normalized to cbp20 mRNA expression. Each biological replicate (n = 4) is an average of 2 technical replicates of qPCR. (D) Western blot detecting FLAG-tagged BRM (top panel) every 2 hours (hrs) post-cycloheximide (CHX) addition to S2 cells expressing brm alone or brm co-expressed with clk. HSP70 was used as a loading control (middle panel). V5 was detected to confirm CLK-V5 expression (bottom panel). (E) BRM expression was normalized to HSP70 (n = 3). Asterisks denote significant p-values: ****p<0.0001.

Fig 4. TIM promotes the reduction of BRM occupancy at the per promoter.

(A) BRM and (B) Histone H3 occupancy at the perCRS in head nuclear extracts of w¹¹¹⁸ (black) and w¹¹¹⁸;ptim(WT) (referred to as tim^OE) (red) flies collected at the indicated time points on LD3. Each data point represents a biological replicate (n = 3), and each biological replicate is an average of at least 2 technical replicates of qPCR. Error bars represent ±SEM. The grey background denotes the dark phase of the LD cycle. Asterisks denote significant p-values: *p<0.05. (C) BRM occupancy at the perCRS in head extracts of TUG flies entrained for 3 days in 12:12LD and collected on LD4 (white) or LL1 (yellow) (n = 3). (D) BRM binding at the perCRS in S2 cell nuclear extracts expressing either brm alone (white) or brm co-expressed with tim (grey) (n = 4). ChIP signal is relative to the amount of BRM binding in the brm alone condition. (E-F) Steady state mRNA expression of per in the heads of TUG (black) and TUG>brm^GOF (blue) flies entrained in LD for 3 days and collected on (E) LD4 and (F) DD1. Steady state cbp20 mRNA levels were used for normalization. Each biological replicate (n = 3) is an average of at least 2 technical replicates of qPCR. The light grey background denotes subjective day, and the dark grey background denotes subjective night in complete darkness (DD) conditions. CircaCompare: (E) MESOR: 9.997e-8, amplitude: 0.0026 and (F) MESOR: 0.688, amplitude: 0.597. (G) Histone H3 occupancy at the perCRS in TUG and TUG>brm^GOF flies (n = 3). Asterisks denote significant p-values: *p<0.05 and ****p<0.0001.

Fig 5. Model depicting the impact of CLK and TIM on BRM occupancy at the per promoter.

CLK-CYC heterodimers bind to the E-box of per to activate transcription. At the peak of transcription, CLK promotes BRM binding to the chromatin. While bound, BRM condenses the chromatin and recruits repressors to reduce gene transcription levels. When PER-TIM complexes are in the nucleus to repress CLK-CYC activated transcription, TIM promotes the removal of BRM from the DNA to reset the chromatin for the next cycle of transcription. This figure was created with BioRender.com (license to lab of JCC).