Dynamic interaction between HDAC and JARID1a correlates with normal histone acetylation at Per promoter. (A) H3K9Ac detected by ChIP-QPCR in Jarid1a^−/− or WT cells (average +SEM, n = 3; **P < 0.05). (B) H3K9 acetylation at the endogenous Per2 promoter after overexpression of JARID1a or HDAC1 (**P < 0.001, ***P < 0.0001, n = 3 per group, one-way ANOVA). (C) ChIP-QPCR quantification of HDAC1 at E-box of Per2 promoter in mouse liver. For (A) to (C), chromatin modifications are presented as percent of total chromatin used for immunoprecipitation (% input). (D) Effect of various concentrations of TSA on Per2:Luc in Jarid1a^−/− cells. Representative real-time traces are shown. (E) Acetylated histone H3 (lane 1) was incubated with 100 ng HDAC1 (lane 2), along with purified JARID1a (lane 3) or JMJD6 (lane 4), and analyzed by immunoblot.

Circadian rhythm disruption in lid¹⁰⁴²⁴ flies. (A) RT-QPCR of lid, per, cry, and tim mRNA in the heads of y;ry (black) and lid¹⁰⁴²⁴/CyO (red) flies collected every 4 hours after 72 hours in constant darkness (±SEM, n = 3). (B) Representative circadian double-plotted actograms of wild-type lid^+/+ (y;ry), lid¹⁰⁴²⁴/CyO, lid¹⁰⁴²⁴/lid¹⁰⁴²⁴;gLid, and lid¹⁰⁴²⁴/lid¹⁰⁴²⁴;gJmjC* flies under LD or DD conditions. Arrowheads indicate transition from LD to DD. (C) (Left) Normalized average activity of y;ry (black) and lid¹⁰⁴²⁴/CyO flies (red) (n = 33) under LD and DD conditions. (Right) Summary of the circadianactivity rhythms in various Drosophila strains tested. Histogram shows the percentage of flies with normal (R, rhythmic), weak (W) or nodetectable(A, arrhythmic) circadian activity rhythms (+SEM). (D) Normalized average activity (+SEM; n = 16 to 75) in 30-min bins during the light:dark period show two major phases of activities in y;ry flies at morning and evening.

JARID1a is required for normal circadian function. (A) Real-time bioluminescence from Jarid1a^+/+ (black) and Jarid1a^−/− (red) mouse fibroblasts stably expressing a Per2:Luc construct. (Middle and right) Average period lengths (+SEM, n = 10) and amplitude estimates (***P < 0.001, two-tailed Student’s t test). (B) Per2, Cry1, Dbp, and SDF1 mRNA abundance in Jarid1a^+/+cells (black) and Jarid1a^−/− cells (red). Forty-eight hours after circadian synchronization, cells were collected at 3-hour intervals over the course of 24 hours, and the mRNA abundance was analyzed by reverse transcription (RT)-QPCR and normalized to that of Gapdh. Average normalized values (±SEM, n = 3) are plotted at various times after cell synchronization. (C) Representative real-time luminescence from U2OS cells stably expressing Bmal1:Luc and transfected with scrambled (S, black) or Jarid1a siRNA (red). Effects of different Jarid1a siRNA concentrations (10, 20, and 40 nM) on the endogenous Jarid1a mRNA abundance (average + SEM, n = 3) and period length (average + SEM, n = 5) relative to those of cells transfected with scrambled siRNA (40 nM). Representative results from at least three independent repetitions are shown.

JARID1a coactivates CLOCK-BMAL1-mediated transcription of Per genes. (A) H3K4 trimethylation and H3K9 acetylation oscillations at the Per2 promoter E-box in mouse liver shown alongside Per2 mRNA expression (secondary y axis). Normalized average (+SEM, n = 3) of chromatin immunoprecipitations (ChIPs) performed with antibodies against H3K4me3 or H3K9Ac followed by QPCR analysis. Chromatin modifications shown as a percentage of total chromatin used (input) normalized to the corresponding lowest value (H3K4me3 = 0.27%, H3K9Ac = 0.25%) for ease of comparison. (B) Abundance of BMAL1 and JARID1a in mouse liver at the Per2 promoter E-box and flanking regions. (C) Absence of JARID1a from the Per2 promoter E-box in Bmal1^−/− cells. (D) Association of endogenous JARID1a and CLOCK-BMAL1. Immunoprecipitates (IPs) obtained with the indicated antibodies from asynchronous U2OS (a human osteosarcoma cell line) nuclear extracts were immunoblotted (IB) with antibodies against JARID1a, CLOCK, BMAL1, or βACTIN. (E) Effect of JARID1a on CLOCK-BMAL1-dependent transcription from a Per2:Luc reporter. Luciferase activity (light counts) from HEK293T cells transiently expressing full-length JARID1a cDNA and Per2:Luc reporter construct are shown. (F) Wild-type (WT) or demethylase-mutant JARID1a H483A (MUT) rescues CLOCK-BMAL1 activation of a Per2 reporter construct in Jarid1a^−/− cells.