Disrupting the three-dimensional regulatory topology of the Pitx1 locus results in overtly normal development

Abstract

Developmental gene expression patterns are orchestrated by thousands of distant-acting transcriptional enhancers. However, identifying enhancers essential for the expression of their target genes has proven challenging. Maps of long-range regulatory interactions may provide the means to identify enhancers crucial for developmental gene expression. To investigate this hypothesis, we used circular chromosome conformation capture coupled with interaction maps in the mouse limb to characterize the regulatory topology of , which is essential for hindlimb development. We identified a robust hindlimb-specific interaction between and a putative hindlimb-specific enhancer. To interrogate the role of this interaction in regulation, we used genome editing to delete this enhancer in mouse. Although deletion of the enhancer completely disrupts the interaction, expression in the hindlimb is only mildly affected, without any detectable compensatory interactions between the promoter and potentially redundant enhancers. enhancer null mice did not exhibit any of the characteristic morphological defects of the mutant. Our results suggest that robust, tissue-specific physical interactions at essential developmental genes have limited predictive value for identifying enhancer mutations with strong loss-of-function phenotypes.

A novel enhancer near the Pitx1 gene influences development and evolution of pelvic appendages in vertebrates

“…Black bars show BAC locations. Gray bars denote orthologous positions of other known or suspected cis regulatory regions, including: sequences removed by the avian deletion associated with feathered feet in pigeons ( Domyan et al, 2016 ), the PDE element ( Sarro et al, 2018 ), the Pit enhancer ( Kragesteen et al, 2018 ), human deletions associated with Liebenberg syndrome ( Spielmann et al, 2012 ), and the hs1473 limb enhancer ( Pennacchio et al, 2006 ; Spielmann et al, 2012 ), now also referred to as the pan-limb enhancer , or Pen ( Kragesteen et al, 2018 ). ( B-P ) Whole-mount lacZ staining of transgenic embryos carrying RP23-55J18 ( B-D ), RP24-296G14 ( E-G ), RP24-353M13 ( H-J , only left side of embryo showed limb staining), RP23-231P13 ( K-M ), or RP23-233P6 ( N-P ), with close ups of the forelimb (‘FL’) and hind limb (‘HL’) below.…”

“…These two BACs overlap in a 29 kb region, suggesting this shared region may contain a pituitary enhancer. Interestingly, this shared region also contains a sequence called the Pitx1 Distal Enhancer ( PDE , Figure 1 ), a region that shows significant chromatin interactions in limb tissues with the promoter of Pitx1 ( Sarro et al, 2018 ). This region has been knocked out in mice, leading to modest reductions in Pitx1 expression in limbs and mandibles, but no detectable limb or jaw skeletal phenotypes ( Sarro et al, 2018 ).…”

“…( B ) H3K27ac ChIP-seq signal from stage 7 Anolis carolinensis forelimbs (blue) and hind limbs (red); AnoCar2 genome coordinates: ChrUn_GL343223:324,575–981,559. H3K27ac data are from genome-wide analysis in Infante et al (2015) , and black bars are added to show the locations of PelB sequences (this study) and the previously described PDE region ( Sarro et al, 2018 ). Note that in the PelB region, but not the PDE region, hind limb-enriched H3K27ac signals are conserved between mice and lizards.…”

“…Pel enhancer orthologs can be identified in other fish, but not in most other vertebrates, including mammals ( Chan et al, 2010 ). Conversely, genetic studies in birds, mice, and humans suggest multiple upstream regions may be involved in Pitx1 limb expression, but it has been difficult to identify any individual Pitx1 enhancers that drive hind limb-specific expression ( Pennacchio et al, 2006 ; Spielmann et al, 2012 ; Domyan et al, 2016 ; Kragesteen et al, 2018 ; Sarro et al, 2018 ). To identify possible mammalian hind limb enhancers in Pitx1 , we have now surveyed a large region surrounding the mouse Pitx1 locus for sequences that drive limb expression in transgenic mice.…”

Genetics of scapula and pelvis development: An evolutionary perspective

“…This research has identified distinct enhancer sequences that act as key musculoskeletal regulators, including specific long bone and joint regulatory sequences for genes such as Gdf5 (Capellini et al, 2017), Gdf6 (Mortlock, Guenther, & Kingsley, 2003), Bmp5 (Guenther, Pantalena-Filho, & Kingsley, 2008), Fgf8 (Marinić, Aktas, Ruf, & Spitz, 2013), and Myf5 and Myf6 (Summerbell et al, 2000; Vinagre et al, 2010). However, aside from the developmental work on the biochemical and genetic interaction of Pbx1 and Emx2 on an Alx1 regulatory element during scapula patterning (Capellini et al, 2010) and studies on the cisregulatory structure of the Pitx1 locus (Chan et al, 2010; Sarro et al, 2018; Spielmann et al, 2012), few tissue-specific regulatory elements have been identified so far during scapula and pelvic development.…”