Rhythmic SAF-A binding underlies circadian transcription of the Bmal1 gene

Abstract

Although Bmal1 is a key component of the mammalian clock system, little is understood about the actual mechanism of circadian Bmal1 gene transcription, particularly at the chromatin level. Here we discovered a unique chromatin structure within the Bmal1 promoter. The RORE region, which is a critical cis element for the circadian regulation of the Bmal1 gene, is comprised of GC-rich open chromatin. The 3'-flanking region of the promoter inhibited rhythmic transcription in the reporter gene assay in vitro even in the presence of RORalpha and REV-ERBalpha. We also found that the nuclear matrix protein SAF-A binds to the 3'-flanking region with circadian timing, which was correlated with Bmal1 expression by footprinting in vivo. These results suggest that the unique chromatin structure containing SAF-A is required for the circadian transcriptional regulation of the Bmal1 gene in cells.

FIG. 1.

Effect of 3′-flanking region on Bmal1 transcription. (A) Oscillatory transcription is disturbed by the 3′-flanking region of the Bmal1 promoter. NIH 3T3 cells were transfected with the indicated Bmal1 promoter constructs and stimulated with dexamethasone, and then bioluminescence was measured. The analyzed regions were as follows: A, nucleotides −497 to +473 (blue); B, −497 to +74 (black); C, −275 to +27 (magenta); D, −275 to −74 (green); E, −197 to +27 (red); F, −197 to −74 (cyan); and G, −101 to +27 (yellow). Relative luciferase units (RLU) and rhythmicity results are summarized. Raw traces of all clones (bottom left) and detrended results for clones A, B, and E (bottom right) are representative of three independent experiments that generated similar results. (B) The effects of ROREs are diminished by the 3′-flanking region. Transcriptional assays were performed by using constructs containing regions A (blue), B (black), C (magenta), D (green), E (red), F (cyan), and G (yellow). RORα (ROR) and REV-ERBα (REV) expression plasmids were also introduced into NIH 3T3 cells. Normalized expression levels were calculated relative to the luciferase activities of mock transfectants. Values are means ± standard error of the mean (SEM) from triplicate assays. (C) Transcription of Bmal1 suppressed by the 3′-flanking region. The reporter plasmid pGL3- promoter, containing the 3′-flanking region (+27 to +473) and oriented as indicated by arrows, was transfected into NIH 3T3 cells. Normalized expression levels were calculated relative to the luciferase activity of pGL3-promoter. Values are means ± SEM from triplicate assays.

FIG. 2.

Analysis of transcription initiation sites. After stimulation with 100 nM dexamethasone for 2 h, NIH 3T3 cells were incubated for the indicated times, and mRNA was prepared for primer extension analysis (left). Arrows show the positions of the extended products. Primers for extension and ROREs are underlined and in bold, respectively (top right). The transcription initiation site of the longest product is designated nucleotide +1. The band intensity was measured, and the relative amounts of products are shown (bottom right).

FIG. 3.

The 3′-flanking region of the Bmal1 promoter prevents nucleosome formation in vitro. Nucleosomes were reconstituted in vitro using 1,260-bp DNA fragments; digested with MNase at a final concentration of 15 units/ml at 25°C for 2, 5, and 10 min; and analyzed by Southern blotting using probe A (nucleotides −497 to −236). Fragments on the same membrane were rehybridized with probe B (290-bp luciferase [Luc] gene fragment). Deduced nucleosome positions in reconstituted chromatin are shown as ovals. The dashed ovals show the region that has little nucleosome structure. tri, di, and mono, tri-, di-, and mononucleosome respectively.

FIG. 4.

Unique chromatin structure of the Bmal1 promoter in vivo. (A) Nucleosome positions in the Bmal1 promoter. NIH 3T3 cells were cultured for the indicated periods after stimulation with dexamethasone (Dex), and then isolated nuclei and naked genomes were incubated with MNase. DNA was purified, digested with SacI, and blotted onto the membrane that also hybridized with probes A (nucleotides −691 to −497), B (−497 to −236), and G3PDH. The image of the agarose gel which was used for the Southern blotting is shown in the left panel. Deduced nucleosome positions in vivo are shown as ovals. (B) Nucleosomes are not featured in the 5′-flanking region around ROREs. ChIP assays using NIH 3T3 cells were performed without antibody (−Ab) and with antihistone antibody (α-Histone). (C) The intranuclear 3′-flanking region of the Bmal1 promoter is protected from DNase I digestion. “Halo” nuclei were prepared from NIH 3T3 cells and digested with DNase I, and purified residual DNA was used as a template for PCR. The control was G3PDH.

FIG. 5.

Tandem ROREs are accessible for transcriptional regulators in vivo. (A) Tandem ROREs in the hypomethylated CpG island. The examination of the Bmal1 promoter sequence revealed a prominent CpG island situated at nucleotides −351 to +494 (shaded area) using the algorithm at MethPrimer (www.urogene.org/methprimer). The genomic sequence of NIH 3T3 cells was analyzed after modification with bisulfite. Vertical lines indicate CpG sites in the Bmal1 promoter region. Filled and open circles indicate methylated and unmethylated CpG sites, respectively. (B) Binding of transcriptional regulators to tandem ROREs in NIH 3T3 cells. Flag-RORα or Myc-REV-ERBα was expressed in NIH 3T3 cells and analyzed by ChIP assays 24 h later, using anti-Flag and anti-Myc antibodies. −Ab, without antibody; α-Flag, with anti-Flag antibody; α-Myc, with anti-Myc antibody.

FIG. 6.

SAF-A binds to the region protected from DNase I digestion. (A) Nuclear complex containing SAF-A. Nuclear complexes prepared from NIH 3T3 cells with (+) or without (−) His₆-tagged SAF-A expression plasmids were resolved by 7% SDS-PAGE and Western blotting against anti-SAF-A antibody. Arrowhead, SAF-A. (B) SAF-A complexes bound to the 3′-1 region. The EMSA was performed using SAF-A-containing nuclear complex. The positions of probes 5′ (nucleotides −197 to +39), 3′-1 (−27 to +266), and 3′-2 (+262 to +473) are shown as arrows. Cold, 200-fold molar excess of unlabeled probe; NE, nuclear complex with (+SAF-A) or without (−SAF-A) SAF-A. (C) SAF-A bound directly to the 3′-1 region. The EMSA was performed using the 3′-1 probe and purified SAF-A protein. IgG, control immunoglobulin G; α-SAF-A, anti-SAF-A antibody. (D) SAF-A binding sites in the 3′-1 region were determined by EMSA using purified SAF-A protein and a 200-fold molar excess of competitors (Comp). Arrows show the positions of the probe and competitors (1 to 7). Competitors were as follows: cold, nucleotides −27 to +266; 1, −27 to +23; 2, +14 to +63; 3, +54 to +103; 4, +94 to +143; 5, +134 to +183; 6, +174 to +223; and 7, +214 to +263. Arrowhead, shifted band.

FIG. 7.

SAF-A is involved in oscillatory Bmal1 transcription. (A) Analysis of the chromatin structure of integrated genes. Indirect end labeling was performed using stable clones 6 and 7. Arrow, probe for Southern blotting; arrowheads, MNase-sensitive sites; ovals, nucleosomes; dashed ovals, nucleosomes in small amounts. Luc, luciferase. (B) SAF-A does not bind to the 3′-end-flanking region of the integrated gene. Stable clones 6 and 7 were analyzed using ChIP assays with anti-SAF-A antibody, and integrated gene-specific or luciferase (A) regions were analyzed by PCR. −Ab, without antibody; α-SAF-A, with anti-SAF-A antibody. (C) The functions of ROREs are inhibited in integrated genes. RORα (filled bars) and REV-ERBα (hatched bars) expression plasmids were introduced into stable clones 6 and 7 and analyzed by luciferase gene assays. Normalized expression levels were calculated relative to the luciferase activities of mock transfectants (open bars). Values are means ± SEM from triplicate assays. (D) Transcription from the promoter in integrated genes without circadian oscillation. Stable clones 6 (blue) and 7 (red) were analyzed by real-time reporter gene assays. Actual bioluminescence is shown.

FIG. 8.

The binding of SAF-A correlates with Bmal1 transcription. (A) Oscillatory transcription of the Bmal1 gene. NIH 3T3 cells were stimulated with 100 nM dexamethasone, and then transcripts were analyzed by a real-time quantitative RT-PCR. The levels of RNA were normalized to the level of G3PDH expression, and the peak value was set at 1. Values are means ± SEM from triplicate assays. (B) SAF-A protein in the NIH 3T3 cell nuclei. NIH 3T3 cells were stimulated with 100 nM dexamethasone (Dex), and then nuclei were isolated and analyzed by Western blotting. The levels of SAF-A protein were normalized to lamin protein, and the peak value was set to 1. Values are means ± SEM from triplicate assays. SAF-A, with anti-SAF-A antibody; Lamin, with anti-lamin A/C antibody. (C) Oscillatory binding of SAF-A to the 3′-1 region. After stimulation with dexamethasone, NIH 3T3 cells were analyzed by ChIP assays of the 3′-1 region, followed by a real-time quantitative PCR (left). PCR products were also analyzed on a 2% agarose gel (right). Binding values were normalized to input DNAs, and the peak value was set at 1. Values are means ± SEM from triplicate assays. −Ab, without antibody; α-SAF-A, with anti-SAF-A antibody.

FIG. 9.

Rhythmic alterations of chromatin structure around the Bmal1 promoter. Footprinting in vivo in the region corresponding to competitors 1 (A) and 4 (B). Brackets indicate regions showing rhythmic footprinting alterations. (C) Footprinting in vivo around the ROREs. The arrowhead indicates bands with rhythmic changes of intensity. Numbers and “N” above the gels indicate time after stimulation and naked DNA, respectively. Results are representative of three independent experiments that generated similar results. Dex, dexamethasone.

FIG. 10.

Model of transcriptional regulation of the Bmal1 gene. The Bmal1 promoter region has a unique chromatin structure, with open chromatin around ROREs and a nuclear matrix-like structure at the 3′-flanking region. First, SAF-A protein binds to the 3′-flanking region with circadian rhythm and alters the local chromatin structure. Thereafter, RORα (ROR) binds to ROREs and activates transcription. In contrast, SAF-A protein deviates from the 3′-flanking region under a repressive state and gains changes in the chromatin structure. Thereafter, the negative transcription factor REV-ERBα (REV) binds to ROREs with the reverse rhythmic phase of SAF-A to repress transcription. Finally, the Bmal1 gene is transcribed with accurate circadian rhythm. Gray ovals, nucleosomes; yellow region, the nuclear matrix-like structure.