Nkx2.5, a vertebrate homologue of the Drosophila tinman gene, is expressed throughout the developing heart during embryogenesis and is a direct regulator of many genes determining the differentiated heart muscle phenotype (Jay and Izumo,2002). Despite its broad expression, loss-of-function experiments in mice reveal particularly important roles for Nkx2.5 in second heart field (SHF) regulation and in conduction system development. For example, hearts of Nkx2.5 null embryos display a hypoplastic outflow tract (OFT) and a single amorphous chamber of left ventricular character (Lints et al.,1993; Tanaka et al.,1999), indicating an almost complete loss of SHF specification or expansion (Prall et al.,2007). This severe SHF phenotype is partially rescued in mice expressing low levels of Nkx2.5 (approximately 25% of normal): the hearts of these mice exhibit more limited arterial pole defects of OFT rotation, alignment and septation, i.e., double oultlet right ventricle (DORV) and ventricular septal defects (VSD; Prall et al.,2007). Finally, hearts of mice heterozygous for Nkx2.5 gene deletion, which express 50% of normal Nkx2.5 mRNA levels, develop normally with the exception of a variably penetrant central conduction system hypoplasia and atrial septal defects (ASD; Biben et al.,2000; Jay et al.,2004; Prall et al.,2007).
Point mutations in the human NKX2-5 gene have been implicated in the occurance of familial, sporadic, syndromic, and nonsyndromic congenital heart disease (CHD) that includes isolated conotruncal OFT anomalies such as tetralogy of Fallot, a cardiac alignment defect leading to pulmonary artery stenosis with secondary right ventricle (RV) hypertrophy, VSD and overriding aortic valve, as well as other anomalies such as secundum ASD with or without atrioventricular delay (Schott et al.,1998; Goldmuntz et al.,2001; McElhinney et al.,2003). Several of the better characterized mutations directly or indirectly affect the ability of NKX2-5 to bind DNA and, hence, may affect the ability of NKX2-5 to regulate target genes (Kasahara et al.,2000,2001; McElhinney et al.,2003).
Despite the high prevalence of OFT anomalies in CHD and the strong linkage of Nkx2.5 to these defects, our knowledge of direct transcriptional targets of Nkx2.5 in the SHF remains scant. Herein, we define a set of genes regulated by Nkx2.5 in pharyngeal arch (PA) tissue encompassing the SHF by an analysis that combines new and existing transcriptomic data. Of these Nkx2.5-regulated genes, we establish the transcriptional regulator Jarid2 as a direct target gene of Nkx2.5 in the SHF.
Identification of Nkx2.5-Regulated Genes Using Microarray-Based Gene Expression Analysis
To identify Nkx2.5 target genes relevant specifically to the SHF, we developed a data analysis schema that would identify genes satisfying three criteria: (1) genes must exhibit differential expression in RNA samples from the cardiothoracic region of embryonic day (E) 9.5 wild-type mice in comparison to mice heterozygous or homozygous for Nkx2.5-gene deletion, (2) gene transcripts must be elevated in the PA-containing region from wild-type E10.5 mice in comparison to hearts from wild-type E12.5 mice, and (3) the genes must show a response to graded changes in Nkx2.5 expression.
Using this schema, we performed a microarray analysis of PA tissue from wild-type E10.5 mouse embryos vs. hearts from E12.5 mouse embryos, identifying 684 genes expressed in the PA but absent in the heart (data not shown). We then examined the comparative expression levels of the resulting 684 genes in the cardiothoracic tissues of E9.5 embryos wild-type, and heterozygous or homozygous for targeted deletion of the Nkx2.5 gene as reported in a publicly available database (http://www.cardiogenomics.org; Genomics of Cardiovascular Development, Adaptation, and Remodeling. NHLBI Program for Genomic Applications, Harvard Medical School). As shown in Figure 1A, these first two analyses together identified 28 PA-expressed genes that displayed a significant increase (7 of 28) or decrease (21 of 28) in expression in Nkx2.5 null vs. heterozygote and wild-type embryos.
To determine if these 28 genes additionally exhibited dynamic responses to graded changes in Nkx2.5 expression in a heterologous system, we conducted a microarray study of the cardiogenic embryonal carcinoma cell line P19CL6 (Habara-Ohkubo,1996) during differentiation in vitro. Notably, Nkx2.5 is initially expressed at low levels in proliferating P19CL6 cells but is substantially induced during the late stages of a 12-day differentiation period when beating cardiac myocytes appear (Fig. 1D). Examination of the temporal expression profiles of the 28 genes during differentiation revealed that 11 exhibited patterns consistent with activation/de-repression (seven genes) or down-regulation/repression (four genes) in response to increasing Nkx2.5 mRNA expression (Fig. 1B–D). Among the seven candidates activated/de-repressed in differentiating P19CL6 cells, three exhibited an early, transient activation (i.e., Elovl2, Lrrn1, Safb) while the other four (i.e., Slc39a6, Khdrbs1, HoxB4, Fez1) showed late activation. Of the four genes whose expression decreased concomitant with the induction of Nkx2.5 expression, two showed relatively rapid down-regulation (i.e., Nrcam, Enpp3) while the other two showed gradual down-regulation (i.e., Ccdc117, Jarid2) during the P19CL6 differentiation period.
Examination of expression profiles for the 11 putative Nkx2.5 target genes in the heterologous system revealed that their responses to changes in Nkx2.5 expression were concordant with the responses observed in cardiothoracic tissue from E9.5 embryos (Fig. 1D). Specifically, all genes activated/de-repressed in vitro in cells expressing high levels of Nkx2.5 were down-regulated by loss of Nkx2.5 expression in Nkx2.5 null E9.5 cardiothoracic tissue, while all genes repressed in cells with high Nkx2.5 expression levels were up-regulated by loss of Nkx2.5 expression in cardiothoracic tissue of E9.5 Nkx2.5 nulls (Fig. 1D).
Promoter Analyses Highlight Potential Direct Gene Regulation by Nkx2.5
Unbiased in silico promoter analyses using the Transfac Public database of transcription factor (TF) binding sequences detected 27 statistically overrepresented TF binding sequences in the 5′ flanking regions of the 11 genes. Of these 27 sites, a binding sequence for Nkx2.5 was found at the highest frequency, constituting 10 of the 27 sites. Furthermore, Nkx2.5 binding sites were the most prevalent in terms of number of promoters containing a given TF site (7 of 11 promoters) and scored at the highest overall significance (P = 0.0061) (Fig. 1E). Because the Transfac Public database uses only two of the several sites reported for Nkx2.5, we manually screened the gene flanking sequences for other known Nkx2.5 binding sequences (Chen and Schwartz,1995). This analysis revealed additional Nkx2.5 binding sites in the four remaining genes (i.e., Lrrn1, Safb, Khdrbs1, and Jarid2) (see below, and data not shown). Together, these findings support the possibility that these 11 genes represent direct downstream targets of Nkx2.5.
Jarid2 mRNA Expression in SHF Mesoderm, Pharyngeal Endoderm, OFT Endocardium, and Cranial Mesenchyme Is Augmented in Nkx2.5 Nulls
Of the 11 putative Nkx2.5 targets identified, the nuclear protein Jarid2 has been shown to be critical to OFT morphogenesis (Lee et al.,2000; Takeuchi et al.,2006). We, therefore, performed a more in-depth evaluation of Jarid2 as a candidate Nkx2.5 target gene. In situ hybridization analysis (ISH) was performed to assess the expression of Jarid2 in cranial and caudal PA regions at a stage when the SHF is actively contributing to morphogenesis of the OFT, and to assess the effect of Nkx2.5 loss of function on Jarid2 expression in this population. ISH of embryos at the E9.5 (19-somite) stage showed modest Jarid2 mRNA expression in pharyngeal endoderm and mesoderm and extending into OFT myocardium and endocardium of both the OFT and right ventricle (Fig. 2A,C). In later stage embryos (29–30 somite/E10.5), Jarid2 ISH signal was more widespread throughout the heart in wild-type mouse embryos, consistent with previously published data regarding Jarid2 expression (Lee et al.,2000) (data not shown). No ISH signal was observed with control sense Jarid2 probes at any stage (data not shown). The PA-specific Jarid2 mRNA expression observed at E9.5 stages is consistent with lacZ expression patterns reported in mice bearing a gene trap insertional mutation of the Jarid2 locus (Takeuchi et al.,1995,1999). Collectively, these data confirm that Jarid2 is expressed in the SHF in regions critical for the morphogenic contribution of OFT progenitors (Waldo et al.,2001; Goddeeris et al.,2007; Park et al.,2008). The apparent overlap of Jarid2 expression with previously published studies of Nkx2.5 expression in this region (Komuro and Izumo,1993; Lints et al.,1993; Tanaka et al.,1999; Prall et al.,2007) is also consistent with a functional relationship between the two genes in the context of SHF regulation and OFT morphogenesis.
The expression pattern of Jarid2 in Nkx2.5 null embryos at the E9.5 (19-somite) stage was qualitatively similar to the pattern seen in wild-type embryos, occurring in pharyngeal endoderm and mesoderm and OFT myocardium and endocardium (Fig. 2B,D). However, Jarid2 expression was elevated in the null embryos in comparison to the wild-type embryos. The observed expression domain is highly similar, if not identical to the pattern of lacZ expression observed in E9.5 Nkx2.5 homozygous null embryos bearing one Nkx2.5-lacZ knock-in allele (Prall et al.,2007). It was not possible to analyze Jarid2 mRNA expression in Nkx2.5−/− null embryos at later stages (i.e., after E10.5) due to the lethality resulting from Nkx2.5 deficiency.
Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) expression analysis confirmed that Jarid2 expression was increased >2-fold in PA and heart-containing tissue from E9.5 Nkx2.5 nulls as compared to wild-type embryos (Fig. 2E). These quantitative increases in levels of Jarid2 mRNA observed in SHF regions of Nkx2.5 null hearts support a model of repression of Jarid2 by Nkx2.5 in SHF-containing PA regions during OFT morphogenesis.
ChIP Analysis Shows Nkx2.5 Interaction With Predicted Binding Elements in the Jarid2 Promoter and in Intron 2 in PA Cells
We next investigated whether the apparent repression of Jarid2 by Nkx2.5 was through a direct interaction of Nkx2.5 with Jarid2 control regions. A multi-species sequence alignment of the Jarid2 promoter region and nearby genomic flanking regions revealed two conserved Nkx2.5 NK homeodomain binding elements (NKEs) of the consensus TNNAGTG (Chen and Schwartz,1995) present in the region 5′ to exon 1 (−203) and within the second intron (+1872) (Fig. 3A and Supp. Fig. S1, which is available online). We therefore used chromatin immunoprecipitation (ChIP) analysis to determine whether Nkx2.5 binds to these NKE sites in vivo in cells from SHF. As shown in Figure 3B, PCR performed on chromatin fragments derived from E9.5 PA tissue and precipitated using Nkx2.5 antibodies generated amplicons containing the Jarid2 −203 NKE as well as the +1872 NKE. As a control, PCR performed with material immunoprecipitated using nonimmune rabbit IgG (Control IgG) showed no evidence of these amplicons. Additionally, no amplicons were generated in control reactions with these chromatin samples using primers specific for exon 1 sequences that lacked potential Nkx2.5 binding sites (data not shown). Reactions performed with E9.5 embryonic heart-derived chromatin fragments precipitated using Nkx2.5 antibodies were also negative, indicating that Nkx2.5 associates with the Jarid2 5′ element (−203) and the intron 2 element (+1872) in PA cells, but not in heart cells at this stage of development. Findings from quantitative PCR analysis using these primers show a 4.5-fold enrichment of the −203 NKE in PA samples, with a more modest (∼2-fold) enrichment of the +1872 NKE (Fig. 3C). Again, no apparent enrichment for these elements was detected in chromatin from hearts at this stage. These results suggest that Jarid2 up-regulation in the setting of Nkx2.5 deficiency is due to loss of direct repression by Nkx2.5 acting on Jarid2 control regions specifically in SHF cells of the PA.
Overexpression of Nkx2.5 Suppresses Jarid2 Promoter Activity and Represses Jarid2 mRNA Expression
To examine the relationship between Nkx2.5 and Jarid2 mRNA expression more directly, we performed transfection assays using an Nkx2.5 overexpression construct and a Jarid2 reporter gene construct. The Jarid2 promoter encompassed nt −997 to +964 relative to the Jarid2 transcriptional initiation site, which notably includes the −203 NKE but not the +1872 NKE. As shown in Figure 3D, when transfected into P19CL6 cells, Nkx2.5 overexpression repressed the Jarid2 reporter activity by 50%. We next examined the consequence of Nkx2.5 overexpression on endogenous Jarid2 expression. Undifferentiated (growth phase) P19CL6 cells normally express high amounts of Jarid2 mRNA. However, overexpression of Nkx2.5 in these cells resulted in approximately a 50% decrease in endogenous Jarid2 mRNA expression (Fig. 3E). These findings in combination with our ChIP data showing direct association of Nkx2.5 to Jarid2 promoter regions further support the hypothesis that Jarid2 is directly and negatively regulated by Nkx2.5.
Identification of Nkx2.5 Target Genes in the SHF
Here, we define a set of genes expressed in SHF-containing pharyngeal arch tissue at E10.5 whose regulation is dependent on Nkx2.5 expression. These genes represent candidate mediators of the critical effects that Nkx2.5 has on OFT morphogenesis. The genes identified by our analysis fall into several broad categories reflecting diverse functions in metabolism and ion transport (Slc39a6, Elovl2, Enpp3; Taylor and Nicholson,2003; Jakobsson et al.,2006; Rucker et al.,2007), cell structure, growth, or adhesion (Lrrn1, Safb, Fez1, Ccdc117, Nrcam; Grumet et al.,1991; Ishii et al.,1999; Haines et al.,2005; Ivanova et al.,2005), and transcription or RNA metabolism (Khdrbs1, Hoxb4, Jarid2; Lee et al.,2000; Brend et al.,2003; Richard et al.,2005). Several have been defined as having cardiac-specific function (Enpp3) or expression in early heart or pharyngeal arch precursors (Lrnn1, Safb, Khdrbs1, Hoxb4, Jarid2; Brend et al.,2003; Haines et al.,2005; Ivanova et al.,2005; Richard et al.,2005). However, detailed expression analysis and gain- or loss-of-function studies have not been done for most of these genes, particularly with respect to the SHF.
Nkx2.5 Negatively Regulates Jarid2 in SHF Mesoderm
One of our identified candidate genes, Jarid2, stood out as having a known function in cardiac development. Jarid2 is a member of the AT-rich interactive domain (ARID)2 family of transcriptional regulators, and is known to be critical to OFT morphogenesis as evidenced by the fact that Jarid2 nulls display DORV and associated VSD. Our findings collectively indicate that Jarid2 is directly and negatively regulated by Nkx2.5: (1) Jarid2 mRNA expression was quantitatively increased in SHF regions of Nkx2.5 null hearts; (2) Nkx2.5 was found to bind NKEs in the Jarid2 promoter in chromatin from E9.5 pharyngeal arch cells; (3) Nkx2.5 overexpression repressed the transcriptional activity of Jarid2 promoter sequences containing an Nkx2.5 consensus binding site; and, (4) Nkx2.5 overexpression repressed endogenous Jarid2 expression in a heterologous cell culture system.
The potential relevance of Nkx2.5 regulation of Jarid2 to OFT development is underscored by their coincident expression in the SHF region at E9.5 within the pharyngeal mesoderm and pharyngeal endoderm proximal to the aortic sac. Intercommunication between these tissues and adjacent pharyngeal ectoderm is critical for the SHF and its morphogenic contribution of OFT progenitors (Waldo et al.,2001; Goddeeris et al.,2007; Park et al.,2008). Published Nkx2.5 in situ hybridization studies (Komuro and Izumo,1993; Lints et al.,1993) confirm that Nkx2.5 is also expressed in pharyngeal mesoderm and endoderm at this stage. Furthermore, the expression pattern of lacZ in embryos (E9.5) homozygous for a Nkx2.5-lacZ knock-in (Prall et al.,2007) is highly similar if not identical to the Jarid2 expression pattern we observed in the Nkx2.5 nulls (E9.5). Thus, the overlap of Jarid2 mRNA expression with Nkx2.5 expression domains in wild-type and Nkx2.5 null embryos, respectively, are consistent with a close functional relationship between Nkx2.5 and Jarid2. These observations, in addition to our data demonstrating both the physical association of Nkx2.5 with Jarid2 promoter regions in SHF-containing PA cells and the ability of overexpressed Nkx2.5 to repress Jarid2 promoter activity and mRNA expression in cell assays reinforce our conclusion that Nkx2.5 is a direct negative regulator of Jarid2 in the SHF.
Jarid2 Expression in the SHF: Implications for OFT Morphogenesis
The augmentation of Jarid2 expression observed in Nkx2.5 nulls highlights an apparent need to normally suppress Jarid2 in the cardiogenic tissues (i.e., SHF). Jarid2 nulls display DORV and associated VSD; however, the significance of Jarid2 overexpression in the SHF is not yet known. Jarid2, like its other jmjC domain-containing family members, exhibits histone modifying capabilities, and suppresses cardiomyocyte proliferation through its ability to repress cyclinD by means of a histone methyltransferase activity (Shirato et al.,2009). Secondary to this repression, Jarid2 nulls display increased cardiac myocyte proliferation in some genetic backgrounds (Takeuchi et al.,1999; Toyoda et al.,2003; Ohno et al.,2004). Given that reduced proliferation in the SHF is observed in both Nkx2.5 null mutants and Nkx2.5 hypomorphs (Prall et al.,2007), it is plausible that overexpression of Jarid2 in the SHF contributes to the decreased SHF precursor proliferation observed Nkx2.5 nulls. Previous studies of Jarid2 nulls did not assess proliferation rates of OFT cardiomyocytes or SHF progenitors at stages during OFT morphogenesis (i.e., E8.5–E10.5), so it remains to be established whether or not Jarid2 overexpression leads to reduced proliferation in the SHF and the subsequent and RV/OFT hypoplasia and DORV.
As well as potentially regulating proliferation of SHF progenitor cells, modulation of Jarid2 expression levels may play a yet broader role in the SHF. Several recent studies have highlighted a major role for Jarid2 in mediating the recruitment of histone methylase-containing PRC2 repressor complexes to multiple loci in embryonic stem (ES) cells. Undifferentiated ES cells, like the P19CL6 EC cells used in our study, maintain high levels of Jarid2 expression that decrease upon the initiation of in vitro differentiation. In the absence of Jarid2, this systematic gene regulation is disrupted with delayed and defective development of the resultant embryoid bodies (Peng et al.,2009; Shen et al.,2009; Li et al.,2010; Pasini et al.,2010). It is likely that Jarid2-mediated function in SHF cells will involve a distinct set of target genes and activator/repressor complexes as compared to ES cells. Nevertheless, these studies raise the interesting possibility that Jarid2 expression systematically regulates SHF progenitor specification or their rate of differentiation, potentially through recruitment to target loci by means of cardiac specifying TFs like Nkx2.5.
Embryo and Cell Harvest, RNA Extraction, and Probe Preparation
SHF and hearts were collected by dissection from wild-type mouse embryos at E10.5 and E12.5. P19CL6 cells were harvested after 4, 6, 8, 10, or 12 days in differentiation medium (Habara-Ohkubo,1996; Monzen et al.,1999,2008). RNA was prepared from individual tissue isolates and from P19CL6 cells by Trizol extraction and purification with the RNeasy Mini kit (Qiagen, Valencia, CA). Following RNA sample assessment, for mouse tissue samples, total RNA (10 ng) of each sample (n = 5) was converted into biotin-labeled cDNA suitable for hybridization to Affymetrix GeneChips (Affymetrix, Santa Clara, CA) using the WT-Ovation Pico amplification kit and the FL-Ovation cDNA Biotin V2 kit (NuGen, San Carlos, CA). For P19CL6 cells, 2 μg of each sample (n = 3 for each time point) were converted into biotin-labeled cRNA using the SuperScript II cDNA synthesis kit (Invitrogen, Carlsbad, CA) and the Enzo HighYield in vitro transcription amplification kit (Enzo Life Sciences, Farmingdale, NY). Tissue samples were hybridized to Affymetrix Mouse Genome 430A GeneChips; P19CL6 samples were hybridized to Affymetrix Mouse 430 2.0 GeneChips. Hybridization, post hybridization washing, staining, and fluorescence scanning were performed using Affymetrix instrumentation in accordance with manufacturer recommendations.
Microarray Data Processing
DNA microarray hybridization data (processed by MAS4 algorithm) for Nkx2.5 wild-type, heterozygous, and homozygous cardiothoracic regions from E9.5 embryos was obtained directly from the CardioGenomics website (http://www.cardiogenomics.org; Genomics of Cardiovascular Development, Adaptation, and Remodeling. NHLBI Program for Genomic Applications, Harvard Medical School). DNA microarray hybridization data for P19CL6 cells was normalized using the ArrayQuest Web tool (Argraves et al.,2005) to implement the Bioconductor (Gentleman et al.,2004) build of GCRMA (Wu et al.,2004). Affymetrix detection (presence and absence) scores and normalized hybridization values for E10.5 SHF and E12.5 heart samples were obtained using the Bioconductor MAS5 algorithm. Raw and processed expression data were deposited at NCBI Gene Expression Omnibus (accession GSE17936). Normalized hybridization data and detection call data were analyzed with dChip software (Li and Wong,2001). Criteria for potential Nkx2.5 targets were as follows: (1) differential expression between wild-type and Nkx2.5 null E9.5 SHF, defined by fold change >2, P < 0.05 (Student's t-test, unpaired); (2) majority “present” detection scores for E10.5 SHF and majority “absent” detection scores for wild-type E12.5 heart.
In Silico Promoter Analysis
Candidate Nkx2.5 targets identified by microarray analysis were analyzed to find those having significant Nkx2.5 binding sites in their promoters. Promoter sequences were obtained by automated retrieval from the Ensembl database using the Toucan Web tool (Aerts et al.,2003,2005) configured to find genomic sequence encompassing the transcription start site (−2000 to +500). These sequences were reviewed manually to ensure that accurate segments of the gene were obtained. Canonical TF binding sites within the promoter sequences were identified with the PAINT v3.5 Web tool (Vadigepalli et al.,2003) using the TransFac Public database of TF binding sites and the following settings for variable parameters: (1) minimize false positives; (2) core similarity threshold = 1.0; and (3) find binding sites on complementary strands. Overrepresentation of TF binding sites was evaluated using PAINT, which calculates a significance score (P value) by hypergeometric distribution that reflects the likelihood that a TF site occurs by random chance, using as reference a list of TF binding site occurrences (approximately 30,000 total) found in the extant mouse promoter database based on comparable promoter length and identical settings for variable parameters; significance was assessed at P < 0.05.
cDNA was prepared from total RNA from pools of four to six hearts and PA regions from E9.5 embryo wild-type, heterozygous, and null for Nkx2.5 using the iScript cDNA synthesis kit and quantitative RT-PCR (qRT-PCR) for Jarid2 performed using the iQ SYBR green/iCycler amplification system (Bio-Rad, Hercules, CA) and Jarid2 primers: 5′-AGG AGA CTG GAA GAG GCA CA-3′ (nucleotide positions 1497–1516) and 5′-GCT TGT TTG CCC AGC ATA TT-3′ (nucleotide positions 1701–1720; based on accession no. NM 021878). Control reactions were performed using β-actin primers 5′-CGG GAC CTG ACA GAC TAC CTC-3′ (nucleotide positions 2126–2152) and 5′-AAC CGC TCG TTG CCA ATA-3′ (nucleotide positions 2352–2343; based on accession no. NC000071.5; Integrated DNA Technologies, Coralville, IA). Relative quantification and standard deviations were calculated using the ΔΔC(t) method as previously described (Allen et al.,2009). qPCR assays were performed in triplicate and results presented are representative of three independent assays.
Candidate Gene Validation by In Situ Hybridization
ISH of wild-type and Nkx2.5 null E9.5 mouse embryos was performed using antisense and sense (control) digoxigenin (DIG) -labeled riboprobes according to established protocols (Hogan,1994). Probes were reverse transcribed from a linearized form of a full-length Jarid2 cDNA clone in pCMV-SPORT6 (Thermo Scientific/Open Biosystems, Huntsville, AL) using the DIG RNA Labeling Kit Sp6/T7 (Roche, Indianapolis, IN). Hybridization was performed at 63°C. For section analysis, whole-mount stained embryos were Paraplast embedded, sectioned, and mounted in Permount (Sigma, St. Louis, MO) before digital photography.
Tissue Harvest and Chromatin Immunoprecipitation(ChIP)
PA-containing regions and hearts were dissected from four wild-type E9.5 embryos (FVB strain). ChIP was performed using reagents from the Upstate (Millipore) ChIP Assay Kit (Millipore, Billerica MA) according to manufacturer's protocols. Samples were sheared with a Misonix 3000 sonicator using 10 × 10-sec pulses on power setting 6 with 10-sec cooling intervals. Chromatin shearing was assessed by agarose gel electrophoresis of de-crosslinked DNA input samples and chromatin yield was quantified by protein assay determination using the BCA protein assay kit (Pierce/Thermo Scientific, Rockford, IL). ChIP was performed using the H-114 anti-Nkx2.5 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or control normal rabbit IgG (Dako, Denmark) in ChIP dilution buffer at a protein concentration of 1 μg/μl and an antibody concentration of 4 μg/ml overnight at 4°C. Purified genomic fragments were subject to 37–39 cycles of PCR amplification with Taq polymerase (Qiagen) using the following primers and annealing temperatures: Jarid2 promoter: 5′-AAA AGG GAG TTG AGT GAC AGG A-3′ and 5′-CCC TTG ATC TTC TGG AAG TTG T-3′ amplifying nt (−)212–(−)124 relative to transcription start site (based on accession no. BC052444); 59°C; Jarid2 intron 2: 5′-TGG TTT CTA GTT TGA GGG GAA A-3′and 5′-ACC TAA CCA TCA CAA CCC AAT C-3′ amplifying nt (+)1602–(+)1729 relative to the transcription start site; 59°C; Jarid2 exon 2: 5′-CCC GTG GTC AGA AGA GAG AG-3′ and 5′-GGC ACA GAA AGA CTC CAT CC-3′ amplifying nt (+)92,495–(+)92,652 relative to the transcription start site; 61°C. qPCR of ChIP-recovered amplicons was accomplished using the above primers and the iQ SYBR green/iCycler amplification system. Fold enrichment was calculated by a ΔC(t) method by comparison of cycle number of linear amplification for anti-Nkx2.5 immunoprecipitated genomic DNA vs. genomic DNA immunoprecipitated by control Ig. Triplicate assays were performed and results expressed as mean fold difference calculated as 2−ΔC(t) and maximum and minimum fold differences calculated as 2−(ΔC(t)+SD) and 2−(ΔC(t)−SD), respectively; SD = (S + S)1/2 where S1 is the standard deviation of the C(t) values obtained for amplification of genomic fragments from ChIP material recovered with anti-Nkx2.5 antibody and S2 is the standard deviation of the C(t) values obtained for amplification of genomic fragments from ChIP material recovered with control IgG. Results shown are representative of two independent ChIP experiments.
Jarid2 Promoter Assay
A 1,955-bp 5′ flanking region of the Jarid2 locus representing nt −997 to +964 relative to the transcriptional start site (based on accession no. BC052444) was cloned by PCR from BAC clone RP24-125d22 (CHORI, Oakland, CA) using LongAmp Taq polymerase (NEB, Beverly, MA) and oligonucleotides 5′-CAT CTC GAG TCT CGG TCG CGG ACA-3′ (forward) and 5′-ATG CCA TGG TGA GAT CCA AAT GCT GAT TG-3′ (reverse). The resulting promoter fragment was digested with XhoI and NcoI and ligated into the pGL3-Control luciferase vector (Promega, Madison, WI) digested with XhoI and NcoI, therefore replacing the SV40 promoter region with the Jarid2 promoter fragment. Promoter assays were performed in P19CL6 cells as described (Lee et al.,2004) using 25 ng of Jarid2 promoter reporter, 5 ng of control TK-Renilla luciferase reporter (Promega), and 25 ng of either control pCS2 expression vector or pCS2-Nkx2.5 (a kind gift from S. Izumo) using 1 μl FuGene6 (Roche) per 24-well plate well. Following 24 hr of incubation, samples were lysed and assayed for normalized luciferase activity using a dual luciferase assay system (Promega). Results show mean and standard deviation of duplicate well assays normalized to Renilla luciferase activity and are representative of three independent assays. P values were calculated using Student's unpaired t-test.
In Vivo Nkx2.5 Overexpression Assays
P19CL6 cells were cultured in nondifferentiating conditions as previously described (Habara-Ohkubo,1996) in αMEM with high glutamine (GibcoBRL-Invitrogen) with 10% ES cell-grade fetal bovine serum (GibcoBRL-Invitrogen) and 1× Pen-strep. Following seeding overnight in 60-mm dishes at a density of approximately 1 × 105 cells/ml, cells were transfected with 1 μg pCS2 or pCS2-Nkx2.5 and 1 μg pGreenLantern enhanced green fluorescent protein (eGFP) expression plasmid (Gibco-BRL) using FuGene (Roche) according to manufacturer's protocol. Cells were harvested by trypsinization following 24-hr incubation and subject to fluorescence-activated cell sorting (FACS) sorting on a BD FACS-Aria2 flow cytometry cell sorter (BD Biosciences, San Jose, CA) optimized for eGFP fluorescence wavelengths. RNA was collected from control and Nkx2.5 co-expressing eGFP (+) cell samples using Trizol extraction (Invitrogen), followed by isopropanol precipitation, resuspension, then DNAse digestion and re-purification using RNeasy mini columns (Qiagen). cDNA was synthesized from purified RNA samples and subject to qPCR for Jarid2 and control β-actin as described above. Results shown are representative of three independent experiments; standard deviation and significance score calculation are as above; P values were calculated using Student's unpaired t-test.
We thank Dr. Marion A. Cooley for advice with in situ hybridization methodology, Dr. Donald R. Menick for advice and reagents related to anti-Nkx2.5 ChIP methodology, Saurin Jani for bioinformatics assistance, Dr. Richard Peppler for FACS sorting assistance, and Marie M. Lockhart for technical assistance. K.L.H., J.L.B., and W.S.A. were funded by the NIH. This work was supported by NCRR grant P20 RR015434.