Global gene expression profiling of JMJD6- and JMJD4-depleted mouse NIH3T3 fibroblasts

Emerging evidence suggests Jumonji domain-containing proteins are epigenetic regulators in diverse biological processes including cellular differentiation and proliferation. RNA interference-based analyses combined with gene expression profiling can effectively characterize the cellular functions of these enzymes. We found that the depletion of Jumonji domain-containing protein 6 (JMJD6) and its paralog protein Jumonji domain-containing protein 4 (JMJD4) individually by small hairpin RNAs (shRNAs) slowed cell proliferation of mouse NIH3T3 fibroblasts. We subsequently performed gene expression profiling on both JMJD6- and JMJD4-depleted mouse NIH3T3 fibroblasts using the Affymetrix GeneChip Mouse Exon 1.0 ST Array. Here we report the gene profiling datasets along with the experimental procedures. The information can be used to further investigate how JMJD6 and JMJD4 affect gene expression and cellular physiology.


Background & Summary
Jumonji domain-containing proteins are iron-and 2-oxoglutarate-dependent oxygenases that act on diverse substrates including proteins, nucleic acids and small molecules 1,2 . These enzymes either hydroxylate or demethylate their substrates in an oxygen-dependent manner. Many JmjC domain proteins play a key role in the epigenetic regulation of mammalian development and of diseases such as cancer 2,3 .
Among the members of this functionally diverse subgroup, JMJD6 was recently characterized as a crucial regulator for gene expression at the level of histone modification 10,13 , transcriptional elongation 14 and RNA splicing 11,15,16 . Jmjd6-deficient mice show various developmental defects and perinatal lethality 17,18 . Moreover, JMJD6 is required for angiogenesis 15 , adipocyte differentiation 19 and T cell proliferation 20 . Elevation of JMJD6 expression has been observed in breast 21,22 , lung 23 , colon 24 , and oral cancer 25 . Depletion of JMJD6 by RNA interference reduced the proliferation of human cancer cell lines 21,24 . Therefore, JMJD6 is essential for cellular proliferation and differentiation. In contrast, the paralog protein JMJD4 that shares 34% sequence identity with JMJD6 was shown to be involved in translational termination by hydroxylating the translational termination factor eRF1 (ref. 7). Other biological functions of JMJD4 have not been demonstrated.
We conducted a loss-of-function study using an RNA interference approach to reduce the levels of endogenous JMJD6 and JMJD4 in proliferating mouse NIH3T3 fibroblasts. We found that depletion of either JMJD6 or JMJD4 alone significantly reduced cell proliferation. In order to determine whether and how these JmjdC domain proteins affect gene expression on a global scale, we performed gene expression profiling on the JMJD6 and JMJD4 knockdown NIH3T3 cells using the Affymetrix Exon Arrays. In this report, we provide a detailed description of the gene expression profiling datasets.

Methods
The scheme of the experimental procedures is presented in Fig. 1.

Cell culture
The mouse NIH3T3 fibroblast cell line was maintained in DMEM high glucose medium (Invitrogen) containing 10% calf serum (Sigma) and 100 U ml −1 penicillin/streptomycin (Invitrogen). Human 293T cells were maintained in DMEM high glucose medium containing 10% fetal calf serum (Sigma) and 100 U ml −1 penicillin/streptomycin (Invitrogen). To evaluate cell proliferation, 1 × 10 5 cells were seeded in 6-well plates, and the number of viable cells was counted every other day with a hemocytometer.

Plasmid construction and virus transduction
The preparation of small hairpin RNA (shRNA) lentiviral constructs was performed as previously described (Campeau, 2009). The shJMJD6 target sequence is 5′-GCTGACACCCAGAGAACAA-3′. The shJMJD4 target sequence is GAAGAATTCTGGAGAGCAT. The control shRNA sequence is TGGCGGCGAGTGAAGTACGTGATAA. The shRNA lentiviral constructs were co-transfected with pLP1, pLP2 and pVSVG packaging vectors into 293T cells with Lipofectamine 2000 reagent (Invitrogen). The viral supernatant was harvested after 48 h incubation and filtered through a 0.45 μm syringe filter (Millipore). To infect NIH3T3 cells, one million cells were incubated with 2 ml of the filtered viral supernatant supplemented with 4 μg ml −1 of polybrene (Sigma) for 48 h. The infected cells were subsequently selected in media containing 2.5 μg ml −1 puromycin (Invitrogen) for 7 days.

RNA isolation and real-time qPCR analysis
One million cells were plated overnight in 10 cm culture dishes. The cells were washed with 5 ml cold PBS and then lysed directly in the culture dish by adding 1 ml of TRIzol reagent (Invitrogen). Total RNA was isolated from TRIzol lysates using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. An on-column DNAse digestion step is included in this protocol. cDNA was prepared from 1 μg of total RNA by Superscript III reverse transcriptase kit (Invitrogen). Quantitative PCR was performed on a StepOne Plus real-time PCR machine with Fast SYBR Green Master mix (Applied Biosystems). The relative gene expression levels were calculated as 2^(Ct Eef1a1 − Ct gene ) and were normalized to the scrambled shRNA control as indicated. The primers are listed below: Eef1a1 (

Data Records
Gene expression profiling on RNA samples collected from the control shRNA-treated (shCtrl_R1 and shCtrl_R2), JMJD6 knockdown (shJMJD6_R1 and shJMJD6_R2), JMJD4 knockdown (shJMJD4_R1 and shJMJD4_R2) cells was performed using GeneChip Mouse Exon 1.0 ST Arrays. Two biological replicates were performed. Both gene level and exon level expression were analyzed. All samples and datasets are described in Table 1. The primary data are available at the NCBI Gene Expression Omnibus (GEO) under the accession numbers GSE76758 (Data Citation 1).

Technical Validation
Confirmation of the shRNA-mediated knockdown The shRNA-mediated knockdown reduced cell proliferation is shown in Fig. 2a. The signal intensity values of expression from the Jmjd6 and Jmjd4 genes in the microarray datasets are shown in Fig. 2b. The expression levels from the endogenous Jmjd6 and Jmjd4 genes were confirmed by real-time qPCR and are shown in Fig. 2c.

Quality control of microarray data
The probe cell intensity values in each individual array were generated from the Affymetrix Expression Console software and are presented in the box plot (Fig. 3a). The data indicate that the probe cell intensities from the individual arrays were similar. The reproducibility of the microarray results is shown by the correlation analyses on the biological replicates. The correlation between the samples at the gene level (Fig. 3b) and at the exon level (Fig. 3c) are presented in the scatter plots with the squared Pearson correlation coefficient (R 2 ). All pairs of biological replicates have very high correlation (R 2 ≥ 0.98 for gene level analysis; R 2 ≥ 0.96 for exon level analysis). Knockdown of JMJD4 has more profound impact on gene expression than knockdown of JMJD6 (R 2 = 0.97 versus R 2 = 0.99 for gene level analysis; R 2 = 0.96 versus R 2 = 0.98 for exon level analysis).

Usage Notes
The full quality control report can be accessed using the Affymetrix Expression Console software. The differentially expressed genes and exons can be identified using the Affymetrix Transcriptome Analysis Console software.