A distant, cis-acting enhancer drives induction of Arf by Tgfβ in the developing eye

Abstract

The Arf tumor suppressor represents one of several genes encoded at the Cdkn2a and Cdkn2b loci in the mouse. Beyond its role blunting the growth of incipient cancer cells, the Arf gene also plays an essential role in development: Its gene product, p19Arf, is induced by Tgfβ2 in the developing eye to dampen proliferative signals from Pdgfrβ, which effect ultimately fosters the vascular remodeling required for normal vision in the mouse. Mechanisms underlying Arf induction by Tgfβ2 are not fully understood. Using the chr4Δ70kb/Δ70kb mouse, we now show that deletion of the coronary artery disease (CAD) risk interval lying upstream of the Cdkn2a/b locus represses developmentally-timed induction of Arf resulting in eye disease mimicking the persistent hyperplastic primary vitreous (PHPV) found in Arf-null mice and in children. Using mouse embryo fibroblasts, we demonstrate that Arf induction by Tgfβ is blocked in cis to the 70 kb deletion, but Arf induction by activated RAS and cell culture “shock” is not. Finally, we show that Arf induction by Tgfβ is derailed by preventing RNA polymerase II recruitment following Smad 2/3 binding to the promoter. These findings provide the first evidence that the CAD risk interval, located at a distance from Arf, acts as a cis enhancer of Tgfβ2-driven induction of Arf during development.

Identification of De Novo Enhancers Activated by TGFβ to Drive Expression of CDKN2A and B in HeLa Cells

“…7). Focusing first on TGFb, which guides mouse Arf expression in a process that is required for proper mouse eye development and vision (12)(13)(14), we demonstrate that the signaling pathway, generally conserved between mouse and human cells (43), includes SMAD2/3 and p38MAPK activation as well as SP1 in both species. However, the kinetics of mouse and human ARF mRNA induction and RNAPII binding differ in early-passage MEFs and HeLa cells: Arf is essentially silenced in mouse embryos and early-passage MEFs (13,53) and its induction requires recruitment of RNAPII to the promoter (16), whereas we show here that ongoing ARF transcription in HeLa cells positions them to respond to TGFb without that step.…”

“…Total RNA extraction and cDNA preparation were carried out as described previously (14). Quantitative mRNA analysis was performed using KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems) and the CFX96 Touch System (Bio-Rad) for real-time PCR detection.…”

“…For example, a number of genetically engineered mouse models show that Arf is required for normal eye development. Germline deletion of Cdkn2a exon 1b, unique to p19 Arf (12), or exon 2, which encodes both p19 Arf and p16 Ink4a , or genetic changes silencing Arf expression (13,14), results in hyperplastic expansion of cells in the developing primary vitreous, rendering the animals blind (12,15). Pursuing the molecular basis for this developmental eye disease revealed a previously unrecognized signaling pathway that selectively controls Arf transcription in which extracellular cues from Tgfb2 are essential for Arf expression and proper mouse eye development (13).…”

9p21.3 Coronary Artery Disease Risk Variants Disrupt TEAD Transcription Factor–Dependent Transforming Growth Factor β Regulation of p16 Expression in Human Aortic Smooth Muscle Cells

“…48,49 However, there is no evidence that supports an a priori hypothesis to tie the 9p21.3 locus to these phenotypes. Therefore, our hypothesis was evidence-based to explain the link between the disruption of TEAD binding with the known effect of the 9p21.3 risk locus on p15 and p16 levels and their associated phenotypes of proliferation and senescence [10][11][12]17 Deletion of the 9p21.3 homologous sequence in the mouse is associated with an impaired Smad-dependent response to TGF-β 10,27,39 and with failure of cells to enter senescence. 12 A recent study reported that mice with a deletion of the 9p21.3 homologous region (chr4 Δ70kb/Δ70kb mice) have reduced p15 transcripts in the aorta, with undetectable levels of p16.…”

“…27,37,38 Deletion of the 9p21.3 homologous sequence in the mouse is associated with an impaired Smad-dependent response to TGF-β 10,27,39 indicating that this region contains DNA sequences targeted by SMAD proteins. However, by gel shift, the 2 putative SMAD target sequences disrupted by rs10757275 (data not shown) and rs1333045 ( Figure II in the online-only Data Supplement) did not show differential binding by gel shift assay in response to TGF-β stimulation.…”