Characterization and target genes of nine human PRD-like homeobox domain genes expressed exclusively in early embryos

Abstract

PAIRED (PRD)-like homeobox genes belong to a class of predicted transcription factor genes. Several of these PRD-like homeobox genes have been predicted in silico from genomic sequence but until recently had no evidence of transcript expression. We found recently that nine PRD-like homeobox genes, ARGFX, CPHX1, CPHX2, DPRX, DUXA, DUXB, NOBOX, TPRX1 and TPRX2, were expressed in human preimplantation embryos. In the current study we characterized these PRD-like homeobox genes in depth and studied their functions as transcription factors. We cloned multiple transcript variants from human embryos and showed that the expression of these genes is specific to embryos and pluripotent stem cells. Overexpression of the genes in human embryonic stem cells confirmed their roles as transcription factors as either activators (CPHX1, CPHX2, ARGFX) or repressors (DPRX, DUXA, TPRX2) with distinct targets that could be explained by the amino acid sequence in homeodomain. Some PRD-like homeodomain transcription factors had high concordance of target genes and showed enrichment for both developmentally important gene sets and a 36 bp DNA recognition motif implicated in Embryo Genome Activation (EGA). Our data implicate a role for these previously uncharacterized PRD-like homeodomain proteins in the regulation of human embryo genome activation and preimplantation embryo development.

Figure 1. Exon-intron structure of PRD-like homeobox genes.

Exons are drawn to scale and represented by horizontal boxes. 5′ and 3′ UTRs are shown as thinner boxes, protein coding region as thicker boxes with homeobox sequence colored grey. Introns are represented by solid lines. Chromosomal locations and Gene IDs, if available, are given on the right. *Indicates the isoform used in the overexpression experiment. NA, not available.

Figure 2. Expression of PRD-like homeobox genes in 8-cell blastomeres and hESCs.

STRT RNAseq expression values are shown for two 8-cell stage blastomeres (14 single cells in total) and for two different hESCs (HS980 and HS983a, 15 single cells each) at single cell resolution. Each dot indicates a single cell: 8-cell (filled circle), hES cell line HS983a (not filled circle) or hES cell HS980 (star). Y-axis shows log10 transformed spike-in normalized expression values. Vertical line indicates mean values. ND, not detected, indicates the number of cells in which the gene of interest was not detectable over the total number of cells analyzed with the threshold of one sequencing read per cell.

Figure 3. Target genes of the PRD-like homeobox genes in hESCs.

The homeobox genes were overexpressed in hESCs followed by transcriptional profiling by RNAseq. (a) The number of up- and down-regulated target genes following transfection for 9–11 hours. (b) The most commonly occurring up- and down-regulated target genes are shown as a gene network. (c) Similarity of the down-regulated target gene lists as observed by performing chi-squared test to the pairwise overlap of all the gene sets. (d) Similarity of the up-regulated target genes lists as observed by performing chi-squared test to the pairwise overlap of all the gene sets. The color indicates logarithmic value of the chi-squared test statistics, and the significance of higher than expected overlap is shown by asterisk according to the multiple-testing corrected Fisher’s exact test value (p < 10⁻²*, p < 5 × 10⁻⁵**, p < 5 × 10⁻⁸***). (e) Alignment of the amino acid sequences for all homeodomains in the CPHX1, CPHX2, ARGFX, DUXB, NOBOX, DUXA, TPRX2 and DPRX genes. Conserved residues are marked in blue, homeodomain helices have a green background. A conserved splice-site is marked by a red line. *Mark the position of variable amino acids in the DNA-binding domain that are indicated in (c,d). Dotted line around the amino acid stretch shows grouping of similar genes (c,d). The red line marks the splice site characteristic for PRD-like homeobox genes.

Figure 4. Overlap of target genes with developmentally important gene sets.

Chi-squared test was performed for identifying significant number of intersecting genes. Target genes from our overexpression experiment were compared to the developmentally important datasets of genes up-regulated in embryonic genome activation (EGA) (two independent datasets for EGA.Yan and EGA.Töhönen3), epiblast (Epi), Trophectoderm (TE) or inner cell mass (ICM). The number of observed divided by expected number of intersecting genes is indicated by color scale, and written on the plot followed by multiple-testing corrected p-value in brackets.

Figure 5. Promoter properties of the homeodomain genes.

(a) Enrichment of a 36 bp DNA motif in the promotor region (−2,000 ~ + 500 bp distance around the center of the TFE) of the PRD-like homeodomain transcription factor target genes. The motif was enriched upstream of the promoters of genes during human pre-implantation development (red line). The motif is over-represented upstream of the up- and down-regulated genes by homeobox genes and under-represented from about 0 to 500 bp downstream from the TFE position. The motif is not enriched in any specific regions among random start-sites from the FANTOM database (blue dotted line). (b) Luciferase expression Fold Change between ZSCAN4 promoter-containing vector and corresponding empty vector (pGL4.25) with transfection by activators CPHX1, CPHX2 and ARGFX in HEK293 cell lines. The average values from three biological replicas are shown, error bars represent standard deviation. The values are normalized to corresponding vector without transcription factor overexpression.