Promoter Frame Position Affects Strength and Nature of Circadian Oscillations in hPER2 Luciferase Reporters

We obtained two truncated sequences of the hPER2 promoter [38,39], and generated lentiviral luciferase promoter reporters using each, resulting in hPER2.1:luc and hPER2.2:luc, respectively (Figure 1A). To compare the frames of the two promoter fragments, we performed sequence alignment of the two promoter sequences against the full-length hPER2 promoter, using the T-Coffee multiple sequence alignment (MSA) program [40]. We identified the full-length hPer2 promoter by comparing the genomic sequence and the mRNA transcript sequence for the hPER2 gene. We found the +1_h site by analyzing the functional and regulatory elements in the hPER2 genomic sequence (Figure S1), which aligned with previous literature [21,41]. The hPER2.1 promoter sequence aligned with the upstream region (−1114 to −333, relative to the hPER2 transcription start site, +1_h), while the hPER2.2 fragment aligned with the downstream region (−752 to +101, relative to +1_h) of the full-length hPER2 promoter (Figure 1B). There was a 419-base pair (bp) region of overlap between hPER2.1 and hPER2.2 sequences, which contained E’-box (E2 enhancer; 5′-CACGTT-3′) and hypoxia response element (HRE) sequences and aligned with the consensus sequence. Further alignment with the truncated mPer2 promoter from a previously established mPer2:luc promoter reporter [10,21] revealed a 34 bp conserved region with it, the E’-box, HRE, and the mPer2 transcription start site (TSS), +1_m (Figure 1C). We identified the conserved +1_m in both hPER2.1 and hPER2.2 sequences. In addition to the +1_m, the hPER2.2 sequence also contained the +1_h site [41]. We also found a non-canonical E-box (5′-CAGGTG-3′; five bases upstream of +1_h) in hPER2.2, which closely resembled the “minimal proximal promoter” organization described previously for mPer2 [21]. Additionally, there were significantly longer frames of unconserved regions in both of the hPER2 promoter sequences, which could contain regulatory elements unique to hPER2. Both hPER2.1:luc and hPER2.2:luc promoter reporter constructs were validated, by Sanger sequencing and whole plasmid sequencing, respectively.

Following validations, we stably transfected the hPER2.1:luc and hPER2.2:luc reporter constructs into U2OS (human bone osteosarcoma) cells using lentiviral transductions. After selection of the positively transfected cells, we performed a luciferase assay to validate the generation of a functional luciferase (Figure 2). We compared the bioluminescence intensities of U2OS-hPER2.1:luc, U2OS-hPER2.2:luc, and a U2OS-mPer2:luc cell line previously generated in our lab [26]. We observed that the normalized bioluminescence intensities of U2OS-mPer2:luc, U2OS-hPER2.1:luc, and U2OS-hPER2.2:luc were approximately 35-, 42-, and 153-fold, respectively, higher than the non-transfected U2OS control. The promoter activities of mPer2:luc and hPER2.1:luc have been established previously [26,38]. To discern the promoter activity of the newly established hPER2.2:luc promoter-reporter from baseline, we compared the luminescence signal from hPER2.2:luc against the same lentiviral vector backbone containing a non-specific (scrambled) insert upstream of the luciferase gene. A luciferase assay in U2OS cells transiently transfected with the two vectors revealed a significantly higher bioluminescence intensity in the U2OS-hPER2.2 reporter cells, indicative of efficient hPER2.2 promoter activity (Figure S2).

We performed luminometry assays to monitor the circadian behavior of our hPER2 promoter-reporters and compare the nature(s) of their oscillations against the mPer2 promoter reporter. We concurrently synchronized U2OS-mPer2:luc, U2OS-hPER2.1:luc, and U2OS-hPER2.2:luc cells for 2 h using a dexamethasone pulse, a synchronization method widely used for U2OS cells [25,26,31,42]. We recorded the bioluminescence signals for each cell line for seven days. The raw data was pre-processed by removing the first 24 h to eliminate the signal from transient peak expression. The data were detrended by subtracting a 24 h window moving average (Figure 3 and Figures S3–S5). The detrended data was then fit to a damped cosine curve.

The average bioluminescence intensity and oscillations of mPer2 (Figure 3A,B) were consistent with our previously reported results [25,26,31]. Both hPER2.1 and hPER2.2 also exhibited robust oscillations (Figure 3C–F). We noted minor differences in the signals observed across different experiments (see Figures S3–S5); however, they remained largely consistent within each experiment. Our results showed that the amplitudes of the oscillations were stronger in both hPER2.1 and hPER2.2 when compared with the mPer2 promoter reporter. This could be attributed to the use of longer lengths of the hPER2 promoters (781 bp for hPER2.1 and 853 bp for hPER2.2) compared to ~200 bp of the mPer2. While previous reports suggest that the use of the minimal mPer2 promoter with the E’-box is sufficient to elicit rhythmic oscillations [21], there may be additional elements in the promoter that might be responsible for regulating the strengths and amplitudes of oscillations. Another reason for the higher amplitude of the hPER2.1 promoter could be the presence of a miniCMV promoter downstream of the hPER2.1 promoter. Furthermore, we used lentiviral transduction to generate stable reporter cell lines; however, we cannot control the number of promoter-reporter copies transfected per cell, which could be a contributing factor resulting in higher amplitudes. Off-target insertion of the reporter sequence into the genome is another pitfall of lentiviral transduction and could result in disruptions to normal cellular processes. We note that the cells did not have any detrimental changes to their physical (i.e., changes in cell morphology or cell division, vacuole indicative of cell stress, and cell death), or circadian attributes.

We then evaluated circadian parameters, including period and phase offsets (Figure 4 and Figure S6). Both were estimated by fitting a damped cosine curve to the data (see Section 3). The phase offset, measured in radians, can be interpreted as the portion of a cycle between the time of synchronization (end of dexamethasone pulse) and the time of the first peak. Time-series were labeled as outliers if the period or phase offset values were more than two standard deviations away from the mean calculated across all of the replicates for a given reporter. Excluding outliers (one replicate from mPer2:luc and two replicates from the hPER2.2:luc time-series), we found the period of hPER2.1 to be slightly longer than that of mPer2, while the period of hPER2.2 was similar to mPer2. The average period of mPer2 was determined to be 23.68 ± 0.72 h, which aligned with mPer2 period values previously calculated in U2OS cells [25,26,31,43]. The average periods of hPER2.1 and hPER2.2 were determined to be 24.22 ± 0.51 h and 23.66 ± 0.13 h, respectively (Figure 4 and Figure S6A,B). We also determined periods by estimating the average differences in timing of the first four peaks starting 24 h after the end of the dexamethasone treatment. In these instances, the periods were found to be 23.83 ± 0.89 h, 24.65 ± 0.57 h, and 23.75 ± 0.15 h for mPer2, hPER2.1, and hPER2.2, respectively (Figure S6C).

The phase offset values for mPer2 and hPER2.2 were found to be similar (0.16π ± 0.07π rad and 0.17π ± 0.09π rad, respectively), while we observed a phase delay for hPER2.1 (Figure 4; phase offset = 0.41π ± 0.15π rad). These results align with observations previously reported using mPer2 promoter sequences [44]. We note that in one experiment presented here, mPer2 periods were shorter but their phase offsets had little effect on the mean of standard deviation of the distribution. Excluding this “short-period” experiment led to an mPer2 period of 24.2 ± 0.15 h and a phase offset of 0.12π ± 0.04π rad. We also determined the phases by calculating the difference between the first peak and its ideal timing, given a peak of bioluminescence at t = 0 h and the period estimated with damped cosine-fitting (Figure S6D). Including all experiments, those phase estimates were 2.00 ± 0.85 h for mPer2, 4.35 ± 1.92 h for hPER2.1, and 1.47 ± 1.07 h for hPER2.2. Excluding the short mPer2-period experiment, the alternate phase estimate for mPer2 was 1.62 ± 0.77 h. Our phase offset estimates capture the phase at the end of the synchronization treatment, so period length alone does not explain the differences. In fact, the short-period mPer2 time-series were not phase-advanced in comparison to the remaining mPer2 time-series. hPER2.1 time-series exhibited both delays and longer periods.

While the period and phase offsets showed variations across experiments, the values within each experiment were tightly grouped. The similarities in the period and phases of mPer2 and hPER2.2 can be attributed to the conserved “rhythm-generating element” located upstream of +1_m [21]. We hypothesize that the presence of regulatory elements in the upstream region of hPER2.1, in addition to the rhythm-generating element, could be responsible for the longer periods and phase delays.

The mPer2:luc reporter construct (pMA3160 lentiviral backbone) was previously generated by our group [10]. The hPER2.1:luc lentiviral reporter construct containing a 781 bp truncated human PER2 promoter fragment (−1114 to −333; relative to +1_h) was obtained from Dr. Benedetto Grimaldi (Istituto Italiano di Tecnologia; Genova, Italy) [38]. For generating the hPER2.2:luc promoter reporter plasmid, a 941 bp truncated human PER2 promoter fragment (−752 to +101, relative to +1_h) was PCR-amplified from a pGL4[Luc2P/Neo] backbone obtained from Dr. Helmut Zarbl (Rutgers University; New Brunswick, NJ, USA) [39]. The primers used for PCR were: forward primer (containing EcoRI restriction site, underlined) = 5′-ccg gaa ttc AGG TGG AGG TCT CCC TCG TCC GGC T-3′; reverse primer (containing NotI restriction site, underlined) = 5′-AAA TAT gcg gcc gcG GAG GGT TCC CAA AAG AGA A-3′. The EcoRI/NotI containing hPER2.2 promoter fragment was subcloned into a pMA3160 lentiviral vector (Addgene plasmid #35043, deposited by Dr. Mikhail Alexeyev) [45], thereby generating the hPER2.2:luc promoter reporter construct. The ligated plasmid DNA was transformed into the electrocompetent Stbl3 strain of E. coli (Thermo Fisher, Waltham, MA, USA), plated on LB agar plates containing ampicillin, and incubated for approximately 16–20 h at 37 °C. The recombinant clones were further expanded, and plasmid DNA was isolated using a GeneJET plasmid midiprep kit (Thermo Fisher Scientific, #K0481). The reporter plasmid was validated using Sanger sequencing and whole plasmid sequencing (Azenta Life Sciences, South Plainfield, NJ, USA).

HEK293T (ATCC) cells were maintained in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F12 (DMEM/F12; Gibco, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Corning, Corning, NY, USA) and 100 U/mL penicillin-streptomycin (Gibco). U2OS (ATCC) cells and derived reporter cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% FBS, 100 U/mL penicillin-streptomycin (Gibco), 2mM glutamine (Gibco), 1mM sodium pyruvate (Gibco), and 1% non-essential amino acids (Cytiva, Marlborough, MA, USA). The cells were grown in an incubator maintained at 37 °C under 5% CO₂ atmosphere.

The generation of the U2OS-mPer2:luc cell line has been previously described [26]. For U2OS-hPER2.1:luc and -hPER2.2:luc cells, the procedure was as follows. HEK293T cells were used as the viral packaging cell line and seeded into 60 mm dishes at a density of 2.5 × 10⁶ cells per dish. At 70–90% confluence (approximately 24 h), the cell culture media was replaced with fresh HEK293T culture media. The cells were further treated with a transfection mixture containing 5 μg of the target DNA (hPER2.1:luc, or hPER2.2:luc), 3 μg of lentiviral packaging plasmid (psPAX2; Addgene plasmid #12260, deposited by Dr. Didier Trono), and 2 μg of lentiviral envelope plasmid (pMD2.G; Addgene plasmid #12259, deposited by Dr. Didier Trono), along with Lipofectamine 3000 (Invitrogen, Waltham, MA, USA), following the manufacturer’s protocol. Meanwhile, the target cell line, U2OS, was seeded into T25 flasks at a density of 6 × 10⁵ cells/flask and grown to 60–70% confluence.

After 48 h incubation, the viral supernatant from HEK293T cells was collected and sterile-filtered using a 0.45 μm syringe filter before combining it with U2OS culture media containing 4 μg/mL polybrene (Sigma-Aldrich, St. Louis, MO, USA) in a 1:1 ratio. Culture media from the U2OS cells was replaced with 6 mL of viral media, and the infections were repeated every 12 h over two days. The viral media was replaced with fresh media 24 h following the final infection. U2OS-hPER2.1:luc cells were passaged and screened for copGFP using flow cytometry (BD FACSAria Fusion at the Flow Cytometry Core Facility, Institute for Applied Life Sciences, UMass Amherst) to select for stably transfected cells, which were further cultured for future use. U2OS-hPER2.2:luc cells were treated with U2OS media containing 4 μg/mL of puromycin (Gibco) for 3–5 weeks, before expanding and freezing for future use.

For luminometry experiments, the U2OS-mPer2:luc, -hPER2.1:luc, and -hPER2.2:luc cells were seeded in 35 mm dishes at a density of 4 × 10⁵ cells per dish. After approximately 24 h, the cells were rinsed with PBS and replaced with synchronization media containing 100 nM dexamethasone (Sigma-Aldrich) in U2OS cell culture media for two hours. After synchronization, the cells were rinsed with phosphate-buffered saline (PBS) and treated with recording media. Recording media was prepared by dissolving powdered DMEM (Sigma-Aldrich) in water (11.25 mg/mL). The resulting solution was sterile-filtered using a 0.22 μm syringe filter and further supplemented with 5% FBS, 10 mM HEPES (Cytiva), 4 mM sodium bicarbonate (Fisher Bioreagents, Waltham, MA, USA), 500 U/mL penicillin-streptomycin, and 500 μM D-luciferin (Thermo Scientific) dissolved in water. After the addition of recording media, the dishes were sealed with 40 mm cover glasses using autoclaved silicone vacuum grease, and the bioluminescence recordings were monitored using a Lumicycle 32 system (Actimetrics, Wilmette, IL, USA) for 7 days at 37 °C.

The bioluminescence recordings were preprocessed to exclude the first 24 h of transient expression and discard the oscillations after 6 days. The data was then detrended by removing a 24 h sliding window and fit into a damped cosine curve with a linear baseline Ae−λtcos2πτ−θ+co+c1t, where τ is the period in hours, θ is the phase in radians, and t is the time in hours. The bioluminescence time series was considered an outlier if the goodness-of-fit value was less than 0.8, or if the period and phase offset values were greater than two standard deviations away from the mean for all the replicates, for a given reporter.

The original data presented in this study are openly available via the Data Repository at ScholarWorks. The URL is https://hdl.handle.net/20.500.14394/57756↗ (accessed on 24 September 2025).