Description

This 313pb sequence, located in an intronic region of Ephrin3 (KH.C7.568), drives reporter gene expression in notochord, mesenchyme and head epidermis. It contains 2 Myb #2, 1 E-box, 1 Sp1/Kif, 2 Brachyury and 3 Fox putative binding sites and one AC repeat (six repeats). Second Brachyury and seconf Fox putative binding sites were mutated : TCACAC replaced by TCAaga and TGTTTAT replaced by TGggccg respectively

Hierarchy of Regulatory Regions

Modification of Cirobu.REG.KH2012.C7:3853910-3854222|Ephrin3 by substitution

Regulated gene(s)

KH2012:KH.C7.568 (EPHA4; EPHA7; EPHB2)

Construct(s) made to test this region

CONS00001302: p.Cirobu.REG.KH2012.C7:3853910-3854222_Bra2Fox2mut>LacZ

Overview of functional TF binding sites in this region

3,853,9103,854,222

	Motif Name	Binding Factor(s)	Sequence	Position in Region	Comment
1	Helix Turn Helix / Tryptophane clusters	n/a	attgaa	[3,853,923 / 3,853,929]	This is one of the two Myb#2 binding sites located in the notochord-active enhancer of KH.C7.568. Deletion of the region containing these two binding sites does not affect the enhancer activity in the notochord.
2	Helix Turn Helix / Tryptophane clusters	n/a	attgaa	[3,853,942 / 3,853,948]	This is one of the two Myb#2 binding sites located in the notochord-active enhancer of KH.C7.568. Deletion of the region containing these two binding sites does not affect the enhancer activity in the notochord.
3	Fox	n/a	tatttat	[3,853,959 / 3,853,966]	This Fox binding site is one of the 3 putative binding sites located in the 313pb enhancer of Ephrin3 (KH.C7.568).
4	ZnF_(C2H2)	n/a	ccacc	[3,853,992 / 3,853,997]	This Sp1/Klf binding site is located in the intronic enhancer of Ephrin3 (KH.C7.568).
5	T-Box	n/a	tcacac	[3,854,037 / 3,854,043]	This Brachyury binding site is located in the intronic enhancer of Ephrin3 (KH.C7.568).
6	Fox	n/a	tatttgt	[3,854,049 / 3,854,056]	This Fox binding site is located in the intronic enhancer of Ephrin3 (KH.C7.568).
7	bHLH	n/a	cagatg	[3,854,195 / 3,854,201]	This E-box binding site is located in the intronic enhancer of Ephrin3 (KH.C7.568).

Sequence

Aniseed Coordinates: [3,853,910 / 3,854,222] on scaffold KhC7

Sequence of the primers used : Forward : CAATCTAGGTCAagaACACACACAC Reverse 1: GTGTGTGTGTtctTGACCTAGATTG Reverse 2 : GAccatggCAATACTACAATGgggcgATTACATCTG Lowercase letters correspond to XhoI/XbaI restriction site in Forward and Reverse 1, and they correspond to subsitutions in Reverse 2 (mutation of Bra and Fox putative binding sites). This sequence was retrieved using ANISEED Blast tool, thanks to the information provided in FigS3.a and TableS5 of Jose-Edwards et al. 2015.

Reference(s)

José-Edwards et al. (2015). Brachyury, Foxa2 and the cis-Regulatory Origins of the Notochord. PLoS Genet. 11(12):e1005730