Functional assessment of CTCF sites at cytokine-sensing mammary enhancers using CRISPR/Cas9 gene editing in mice.
The zinc finger protein CTCF has been invoked in establishing boundaries between genes, thereby controlling spatial and temporal enhancer activities. However, there is limited genetic evidence to support the concept that these boundaries restrict the search space of enhancers. We have addressed this question in the casein locus containing five mammary and two non-mammary genes under the control of at least seven putative enhancers. We have identified two CTCF binding sites flanking the locus and two associated with a super-enhancer. Individual deletion of these sites from the mouse genome did not alter expression of any of the genes. However, deletion of the border CTCF site separating the Csn1s1 mammary enhancer from neighboring genes resulted in the activation of Sult1d1 at a distance of more than 95 kb but not the more proximal and silent Sult1e1 gene. Loss of this CTCF site led to de novo interactions between the Sult1d1 promoter and several enhancers in the casein locus. Our study demonstrates that only one out of the four CTCF sites in the casein locus had a measurable in vivo activity. Studies on additional loci are needed to determine the biological role of CTCF sites associated with enhancers.
Laboratory of Genetics and Physiology, National Institute of Diabetes, Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, MD 20892, USA.|Department of Cell and Developmental Biology & Dental Research Institute, Seoul National University, Seoul 110-749, Korea.
Hye Kyung
HK
Lee
Laboratory of Genetics and Physiology, National Institute of Diabetes, Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, MD 20892, USA.|Division of Bioinformatics, Biocenter, Medical University of Innsbruck, 6020 Innsbruck, Austria.
Michaela
M
Willi
Laboratory of Genetics and Physiology, National Institute of Diabetes, Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, MD 20892, USA.
Chaochen
C
Wang
Laboratory of Genetics and Physiology, National Institute of Diabetes, Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, MD 20892, USA.
Chul Min
CM
Yang
National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, USA.
Harold E
HE
Smith
Transgenic Core,National Heart Lung and Blood Institute, US National Institutes of Health, Bethesda, MD 20892, USA.
Chengyu
C
Liu
Laboratory of Genetics and Physiology, National Institute of Diabetes, Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, MD 20892, USA.
Lothar
L
Hennighausen
D000818:Animals; D001665:Binding Sites; D000076246:CCCTC-Binding Factor; D064113:CRISPR-Cas Systems; D002364:Caseins / Q000235:genetics / Q000378:metabolism; D016207:Cytokines / Q000235:genetics* / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D005260:Female; D000072669:Gene Editing; D005786:Gene Expression Regulation; D056426:Genetic Loci; D016678:Genome; D008321:Mammary Glands, Animal / Q000378:metabolism; D051379:Mice; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism
D000076246:CCCTC-Binding Factor; D002364:Caseins; C000619698:Ctcf protein, mouse; D016207:Cytokines; D012097:Repressor Proteins; C431463:SULT1D1 sulfotransferase; D015238:Sulfotransferases; C020122:estrone sulfotransferase