A massively parallel reporter assay reveals context-dependent activity of homeodomain binding sites in vivo

Genome Research
Andrew E O HughesJoseph C Corbo

Abstract

Cone-rod homeobox (CRX) is a paired-like homeodomain transcription factor (TF) and a master regulator of photoreceptor development in vertebrates. The in vitro DNA binding preferences of CRX have been described in detail, but the degree to which in vitro binding affinity is correlated with in vivo enhancer activity is not known. In addition, paired-class homeodomain TFs can bind DNA cooperatively as both homodimers and heterodimers at inverted TAAT half-sites separated by 2 or 3 nucleotides. This dimeric configuration is thought to mediate target specificity, but whether monomeric and dimeric sites encode distinct levels of activity is not known. Here, we used a massively parallel reporter assay to determine how local sequence context shapes the regulatory activity of CRX binding sites in mouse photoreceptors. We assayed inactivating mutations in more than 1700 TF binding sites and found that dimeric CRX binding sites act as stronger enhancers than monomeric CRX binding sites. Furthermore, the activity of dimeric half-sites is cooperative, dependent on a strict 3-bp spacing, and tuned by the identity of the spacer nucleotides. Saturating single-nucleotide mutagenesis of 195 CRX binding sites showed that, on average, changes in ...Continue Reading

References

Nov 1, 1993·Genes & Development·D WilsonC Desplan
Apr 12, 2006·Journal of Molecular Medicine : Official Organ of the Gesellschaft Deutscher Naturforscher Und Ärzte·Gilles ChatelainThomas Lamonerie
Jul 27, 2007·PloS One·Timothy H-C HsiauJoseph C Corbo
Jan 31, 2009·Proceedings of the National Academy of Sciences of the United States of America·Karen Alvarez-DelfinJames M Fadool
May 16, 2009·Science·Gwenael BadisMartha L Bulyk
Aug 10, 2010·Genome Research·Joseph C CorboThomas Langmann
Feb 19, 2011·Bioinformatics·Charles E GrantWilliam Stafford Noble
Jul 7, 2011·Journal of Visualized Experiments : JoVE·Cynthia L MontanaJoseph C Corbo
Aug 26, 2011·The Journal of Biological Chemistry·Cynthia L MontanaJoseph C Corbo
Mar 1, 2012·Nature Biotechnology·Rupali P PatwardhanJay Shendure
Sep 8, 2012·Nature·UNKNOWN ENCODE Project Consortium
Nov 7, 2012·Proceedings of the National Academy of Sciences of the United States of America·Jamie C KwasnieskiBarak A Cohen
Jan 19, 2013·Science·Cosmas D ArnoldAlexander Stark
Jan 22, 2013·Cell·Arttu JolmaJussi Taipale
Jun 19, 2013·Nucleic Acids Research·Christopher Fletez-BrantMichael A Beer
Jul 3, 2013·Proceedings of the National Academy of Sciences of the United States of America·Michael A WhiteBarak A Cohen
Jul 18, 2014·PLoS Computational Biology·Mahmoud GhandiMichael A Beer
May 29, 2015·PLoS Computational Biology·Manu Setty, Christina S Leslie
Jun 16, 2015·Nature Genetics·Dongwon LeeMichael A Beer
Aug 25, 2015·Nature Methods·Jian Zhou, Olga G Troyanskaya
Nov 19, 2015·Genome Research·Susan Q ShenJoseph C Corbo
Mar 8, 2016·ELife·Alisa MoJeremy Nathans
May 7, 2016·Bioinformatics·Dongwon Lee
May 7, 2016·Bioinformatics·Mahmoud GhandiMichael A Beer
Nov 24, 2016·Cell Reports·Jung-Woong KimAnand Swaroop
Feb 1, 2017·Proceedings of the National Academy of Sciences of the United States of America·Sharon R GrossmanEric S Lander

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