Global gene expression profiling of jmjd6- and jmjd4-depleted mouse nih3t3 fibroblasts

ABSTRACT 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. Design Type(s) parallel group design • RNA Interference Measurement Type(s) transcription profiling assay Technology Type(s) DNA microarray Factor Type(s) biological replicate

role • shRNA Sample Characteristic(s) Mus musculus • NIH-3T3 cell Machine-accessible metadata file describing the reported data (ISA-Tab format) SIMILAR CONTENT BEING VIEWED BY OTHERS

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BACKGROUND & SUMMARY Jumonji domain-containing proteins are iron- and 2-oxoglutarate-dependent oxygenases that act on diverse substrates including proteins, nucleic acids and small

molecules1,2. 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 cancer2,3. Phylogenetic analysis classifies the Jumonji domain containing proteins into several subgroups3. One subgroup comprises the

asparaginyl hydroxylase FIH4,5, the ribosomal hydroxylases MINA53 and NO66 (ref. 6) , the lysyl hydroxylase JMJD4 (ref. 7), the lysine demethylase/hydroxylase JMJD5 (refs 8,9) , the lysyl

hydroxylase/arginine demethylase JMJD6 (refs 10,11) , the tRNA hydroxylase TYW5 (ref. 12), and the enzymatically uncharacterized proteins JMJD8 and HSPBAP1. Although these enzymes share the

conserved JmjC domain with the histone lysine demethylases, they generally lack chromatin-binding domain, indicating potential functions other than modifying histones. Among the members of

this functionally diverse subgroup, JMJD6 was recently characterized as a crucial regulator for gene expression at the level of histone modification10,13, transcriptional elongation14 and

RNA splicing11,15,16. _Jmjd6_-deficient mice show various developmental defects and perinatal lethality17,18. Moreover, JMJD6 is required for angiogenesis15, adipocyte differentiation19 and

T cell proliferation20. Elevation of JMJD6 expression has been observed in breast21,22, lung23, colon24, and oral cancer25. Depletion of JMJD6 by RNA interference reduced the proliferation

of human cancer cell lines21,24. 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, 1×105 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_ (forward): 5′-AGCTTCTCTGACTACCCTCCACTT-3′ _Eef1a1_ (reverse): 5′-GACCGTTCTTCCACCACTGATT-3′

_Jmjd6_ (forward): 5′-GTTCCAGCTCGTCAGACTCG-3′ _Jmjd6_ (reverse): 5′-TGCCCCTAAGACATGACCAC-3′ _Jmjd4_ (forward): 5′-TCCTGCTGGAATGTCGCACCTGT-3′ _Jmjd4_ (reverse): 5′-ACCCCAAATAGGGACCGGAGGC-3′

MICROARRAY ANALYSIS The RNA samples were processed at the Genomics Core Facility of UMass Medical School. RNA quantity and quality were assessed using an Agilent Bioanalyzer 2100. cDNA was

synthesized from 500 ng RNA using the Ambion WT kit (Life Technologies) and the single stranded cDNA was prepared using the Affymetrix GeneChip WT Terminal Labeling Kit (Affymetrix)

according to the manufacturer’s instructions. The labeled mix was hybridized to GeneChip Mouse Exon 1.0 ST Array (Affymetrix) in the Gene Chip Hybridization Oven 640 overnight. Probe

intensities were measured using the Affymetrix GeneChip Scanner 3000 7G. The probe cell intensity data (CEL) from GeneChip Mouse Exon 1.0 ST Arrays was analyzed in the Affymetrix Expression

Console software to generate CHP files using the Robust Multichip Analysis (RMA)-sketch algorithm workflow. The transcript structure confidence levels for both gene and exon level analyses

were set as Core, which limits analysis to exon-level probe sets that map to BLAT alignments of mRNA with annotated full-length coding sequence (CDS) regions. Differentially expressed genes

and exons were identified by Transcriptome Analysis Console (TAC) 3.0 software (Affymetrix). 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(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 (R2). All pairs of biological replicates have very high correlation

(R2≥0.98 for gene level analysis; R2≥0.96 for exon level analysis). Knockdown of JMJD4 has more profound impact on gene expression than knockdown of JMJD6 (R2=0.97 versus R2=0.99 for gene

level analysis; R2=0.96 versus R2=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. ADDITIONAL INFORMATION HOW TO CITE THIS ARTICLE: Hu, Y.-J. &

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(2015). Article  Google Scholar  DATA CITATIONS * Hu, Y. J., & Imbalzano, A. N. _Gene Expression Omnibus_ GSE76758 (2016) Download references ACKNOWLEDGEMENTS We thank P. Spatrick and R.

Barutcu for technical assistance. This work was supported by funding from NIH grant GM56244 to A.I. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Cell and Developmental

Biology, University of Massachusetts Medical School, Worcester, 01655, Massachusetts, USA Yu-Jie Hu & Anthony N. Imbalzano Authors * Yu-Jie Hu View author publications You can also

search for this author inPubMed Google Scholar * Anthony N. Imbalzano View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Y.H. and A.I.

designed the experiments and wrote the manuscript. Y.H. generated RNA samples and performed data analyses. CORRESPONDING AUTHOR Correspondence to Anthony N. Imbalzano. ETHICS DECLARATIONS

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