Chromatin Architecture of the Pitx2 Locus Requires CTCF- and Pitx2-Dependent Asymmetry that Mirrors Embryonic Gut Laterality

Abstract

Summary Expression of Pitx2 on the left side of the embryo patterns left-right (LR) organs including the dorsal mesentery (DM), whose asymmetric cell behavior directs gut looping. Despite the importance of organ laterality, chromatin-level regulation of Pitx2 remains undefined. Here we show that genes immediately neighboring Pitx2 in chicken and mouse, including a long noncoding RNA, Playrr (Pitx2 locus asymmetric regulated RNA), are expressed on the right side and repressed by Pitx2. CRISPR/Cas9 genome editing of Playrr, 3D fluorescent in situ hybridization (FISH) and variations of chromatin conformation capture (3C), demonstrate that mutual antagonism between Pitx2 and Playrr is coordinated by asymmetric chromatin interactions dependent on Pitx2 and CTCF. We demonstrate that transcriptional and morphological asymmetries driving gut looping are mirrored by chromatin architectural asymmetries at the Pitx2 locus. We propose a model where Pitx2 auto-regulation directs chromatin topology to coordinate LR transcription of this locus essential for LR organogenesis.

Mutations in zebrafish pitx2 model congenital malformations in Axenfeld-Rieger syndrome but do not disrupt left-right placement of visceral organs

“…The close proximity of pitx2 and elovl6 suggests left-sided expression of these genes is controlled by a Nodal-responsive enhancer on chromosome 14. Intriguingly, Elovl6 is asymmetrically expressed opposite of Pitx2 in the right dorsal mesentery in the chicken embryo during gut looping (Welsh et al, 2015). It is not clear whether asymmetric Elovl6 expression in chicken is Nodal-dependent, but studies in chicken and mouse indicate chromatin conformation at the Pitx2 locus plays an important role in directing left-sided expression of Pitx2 and right-sided expression of downstream genes Enpep and Elovl6 (Welsh et al, 2015).…”

“…Intriguingly, Elovl6 is asymmetrically expressed opposite of Pitx2 in the right dorsal mesentery in the chicken embryo during gut looping (Welsh et al, 2015). It is not clear whether asymmetric Elovl6 expression in chicken is Nodal-dependent, but studies in chicken and mouse indicate chromatin conformation at the Pitx2 locus plays an important role in directing left-sided expression of Pitx2 and right-sided expression of downstream genes Enpep and Elovl6 (Welsh et al, 2015). In contrast to chicken and mouse, the zebrafish pitx2 locus lacks an enpep gene and the pitx2 and elovl6 genes are very close together.…”

Hemispheric asymmetry in the human brain and in Parkinson’s disease is linked to divergent epigenetic patterns in neurons

“…During early development, hemispheric asymmetry in DNA methylation is linked to the lateralization of nervous system organization, which affects hemisphere dominance for handedness, cognitive processes, and language [25][26][27]. Furthermore, DNA methylation status governs the activity of gene regulatory elements such as enhancers and promoters, which affect the establishment of left-right asymmetries in various tissues [28][29][30]. Hence, epigenetic variation may influence asymmetrical gene expression patterns in the brain, which if pathogenic could contribute to PD.…”

Epigenetic contributions to hemisphere asymmetry in healthy brain, aging, and Parkinson’s disease

“…During early development, hemispheric asymmetry in DNA methylation contributes to the lateralization of nervous system organization, which affects hemisphere dominance for handedness, cognitive processes, and language [25][26][27] . Furthermore, DNA methylation status governs the activity of gene regulatory elements such as enhancers and promoters, which affect the establishment of left-right asymmetries in various tissues [28][29][30] . Hence, epigenetic variation may influence asymmetrical gene expression patterns in the brain, which if pathogenic could contribute to PD.…”