Jarid2 is among a set of genes differentially regulated by Nkx2.5 during outflow tract morphogenesis

Abstract

Nkx2.5, a transcription factor implicated in human congenital heart disease, is required for regulation of second heart field (SHF) progenitors contributing to outflow tract (OFT). Here, we define a set of genes (Lrrn1, Elovl2, Safb, Slc39a6, Khdrbs1, Hoxb4, Fez1, Ccdc117, Jarid2, Nrcam, and Enpp3) expressed in SHF containing pharyngeal arch tissue whose regulation is dependent on Nkx2.5. Further investigation shows that Jarid2, which has been implicated in OFT morphogenesis, is a direct target of Nkx2.5 regulation. Jarid2 expression was up-regulated in SHF mesoderm of Nkx2.5-deficient embryos. Chromatin immunoprecipitation analysis showed Nkx2.5 interaction with consensus binding sites in the Jarid2 promoter in pharyngeal arch cells. Finally, Jarid2 promoter activity and mRNA expression levels were down-regulated by Nkx2.5 overexpression. Given the role of Jarid2 as a regulator of early cardiac proliferation, these findings highlight Jarid2 as one of several potential mediators of the critical role played by Nkx2.5 during OFT morphogenesis.

Figure 1. Identification of Nkx2.5 regulated genes in the SHF

A, Shown is a heatmap of the expression intensities of 28 gene transcripts satisfying two criteria: 1) differential expression in E9.5 cardiothoracic region of wild-type, Nkx2.5-heterozygous and homozygous null mice; and, 2) presence in the PA-containing region from wild-type E10.5 embryos, but absent in hearts from E12.5 embryos. B and C, Schematic depiction of temporal expression profiles of Nkx2.5 targets responding rapidly (“Early”) or slowly (“Late”) to Nkx2.5 induction. D, Eleven of 28 genes shown in A exhibit corroborative expression patterns in differentiating P19CL6 (P19) cells. Genes are color-coded to match the potential patterns shown in B and C. Nkx2.5 expression in differentiating P19 cells is shown in the bottom of the panel; Nkx2.5 data is not available for the 9.5 cardiothoracic regions because of the microarray format used in that study (Affymetrix Mu11k Sub A and Sub B). E, Analysis of promoter sequences (−2000 to +500 relative to transcriptional start) for genes depicted in D detected 27 TF binding sequences (columns correspond to individual motifs). An Nkx2.5 binding element (Nkx2.5_01) was present one or more times in 7 of the 11 sequences analyzed and scored highly significant (p=0.0061).

Figure 2. Jarid2 mRNA expression in wild-type and Nkx2.5 null embryos

Wholemount and sectioned wholemount ISH for Jarid2 mRNA are shown for wild-type Nkx2.5 (A, C) and null Nkx2.5 (B, D) E9.5 embryos. Jarid2 mRNA is detected in SHF-related splanchnic mesoderm (open arrowheads) and pharyngeal endoderm (black arrowheads) as well as in the endocardium (black arrows) and myocardium of the OFT (bracket) in wild-type and Nkx2.5^−/− embryos. Staining is more easily detectable in SHF-related splanchnic mesoderm and pharyngeal endoderm as well as OFT endocardium of null embryos. E, results of qRT-PCR for Jarid2 mRNA in combined heart and PA regions of E9.5 embryos wild-type (WT), heterozygous (Het) or mutant null (Null) for Nkx2.5. Results are expressed as fold change relative to WT mRNA levels, and were normalized with respect to qRT-PCR for β-actin mRNA in these samples. Error bars represent standard deviation as calculated according to the ΔΔC(t) method (Allen et al., 2009). *, significant by t-test (P <0.05). Abbreviations: OFT: outflow tract; LV: left ventricle; RV: right ventricle; V: common ventricle; NT: neural tube.

Figure 3. Nkx2.5 binds Jarid2 promoter region in vivo in E9.5 PA cells and represses Jarid2 promoter activity and mRNA expression

A, Mouse Jarid2 has one Nkx2.5 binding element 5′ and proximal to the transcription start site and a second in intron 2. Shown is a schematic diagram of the Jarid2 mouse gene (top of panel) with the region encompassing exons 1 and 2 expanded (2.3 kb). Arrows indicate position and directionality of the Nkx2.5 binding elements (TNNAGTG) (NKE). Numeric positions of the two elements (−203 and +1872) are relative to the transcriptional start site annotated for Jarid2 mRNA (accession no. BC052444.1). B, Data are shown for ChIP assay for binding of Nkx2.5 to Jarid2 promoter proximal and intron 2 regions bearing conserved binding consensus sites for Nkx2.5 in SHF-containing PA regions (PA) and heart (Hrt) of wild-type E9.5 mouse embryos. PCR assay generates amplicons for both 5′ and intron 2 candidate regulatory regions containing NKEs shown in A using antibody against Nkx2.5 (anti-Nkx2.5) versus control total rabbit immunoglobulin (Control IgG) using PA-derived chromatin (PA) but not heart chromatin (Hrt). Controls shown include DNA from non-selected input chromatin (Input, PA and Hrt) and a no-template (No DNA) control for background amplification. C, qPCR quantitation of above ChIP experiments are shown in graphical fashion. Results are expressed as fold enrichment for genomic fragments containing Nkx2.5 binding consensus sites using anti-Nkx2.5 antibody over control IgG. Error bars represent minimum and maximum fold enrichment as calculated by the ΔC(t) method and based upon standard deviation of C(t) values at linear amplification (see Methods). D, Results of promoter-reporter assays comparing activity of control nt −997 to +964 Jarid2 promoter-luciferase (white bar above) to activity of nt −997 to +964 Jarid2 promoter-luciferase in the presence of Nkx2.5 overexpression (black bar below). Results are expressed as normalized fold change relative to WT luciferase levels. E, Results of qRT-PCR for Jarid mRNA in control eGFP-transfected growth-phase P19CL6 cells (white bar above) and eGFP and Nkx2.5 co-transfected growth-phase P19CL6 cells (black bar below) following FACS sorting for eGFP fluorescence. Results are expressed as normalized fold change relative to WT mRNA levels. *, significant by t-test (P <0.05).