Functional characterisation ofcis-regulatory elements governing dynamicEomesexpression in the early mouse embryo.
The T-box transcription factor (TF) Eomes is a key regulator of cell fate decisions during early mouse development. Thecis-acting regulatory elements that direct expression in the anterior visceral endoderm (AVE), primitive streak (PS) and definitive endoderm (DE) have yet to be defined. Here, we identified three gene-proximal enhancer-like sequences (PSE_a, PSE_b and VPE) that faithfully activate tissue-specific expression in transgenic embryos. However, targeted deletion experiments demonstrate that PSE_a and PSE_b are dispensable, and only VPE is required for optimalEomesexpressionin vivoEmbryos lacking this enhancer display variably penetrant defects in anterior-posterior axis orientation and DE formation. Chromosome conformation capture experiments reveal VPE-promoter interactions in embryonic stem cells (ESCs), prior to gene activation. The locus resides in a large (500 kb) pre-formed compartment in ESCs and activation during DE differentiation occurs in the absence of 3D structural changes. ATAC-seq analysis reveals that VPE, PSE_a and four additional putative enhancers display increased chromatin accessibility in DE that is associated with Smad2/3 binding coincident with transcriptional activation. By contrast, activation of theEomestarget genesFoxa2andLhx1is associated with higher order chromatin reorganisation. Thus, diverse regulatory mechanisms govern activation of lineage specifying TFs during early development.
The Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK.
Claire S
CS
Simon
MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK.
Damien J
DJ
Downes
MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK.
Matthew E
ME
Gosden
MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK.
Jelena
J
Telenius
MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK.
Douglas R
DR
Higgs
MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK.
Jim R
JR
Hughes
The Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK.
Ita
I
Costello
The Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK.
Elizabeth K
EK
Bikoff
The Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK elizabeth.robertson@path.ox.ac.uk.
Elizabeth J
0000-0001-6562-0225
EJ
Robertson
D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D002843:Chromatin / Q000378:metabolism; D004622:Embryo, Mammalian / Q000378:metabolism*; D004707:Endoderm / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D005260:Female; D051858:Forkhead Transcription Factors / Q000378:metabolism; D054262:Gastrulation / Q000235:genetics; D017353:Gene Deletion; D018507:Gene Expression Regulation, Developmental; D018390:Gene Targeting; D017930:Genes, Reporter; D005838:Genotype; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D063146:Polycomb-Group Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics; D051899:Smad2 Protein / Q000378:metabolism; D020825:T-Box Domain Proteins / Q000235:genetics* / Q000378:metabolism
D002843:Chromatin; C491198:Eomes protein, mouse; D051858:Forkhead Transcription Factors; C491117:Foxh1 protein, mouse; D063146:Polycomb-Group Proteins; D051899:Smad2 Protein; D020825:T-Box Domain Proteins
Capture-C; Definitive endoderm; Enhancer; Eomesodermin; Nodal signalling