Human-Specific Gain of Function in a Developmental Enhancer Shyam Prabhakar,1* Axel Visel,1 Jennifer A. Akiyama,1 Malak Shoukry,1 Keith D. Lewis,1 Amy Holt,1 Ingrid Plajzer-Frick,1 Harris Morrison,2 David R. FitzPatrick,2 Veena Afzal,1 Len A. Pennacchio,1,3 Edward M. Rubin,1,3 James P. Noonan1 1 Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA 2 MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK. 3 United States Department of Energy Joint Genome Institute, Walnut Creek, CA, USA Evolution … of News • Yale Researchers Find “Junk DNA” May Have Triggered Key Evolutionary Changes in Human Thumb and Foot ▫ Office of Public Affairs (Yale University), Sept 4 • “Junk DNA” key to human evolution? ▫ World Science, Sept 4 • Opposable Thumbs and Upright Walking Caused By "Junk DNA" ▫ Slashdot, Sept 7 • Junk DNA may have handed us a gripping future ▫ New Scientist, Sept 4 Or Revolution? • It's the junk that makes us human ▫ Nature (2006) • Accelerated Evolution of Conserved Noncoding Sequences in Humans ▫ Shyam Prabhakar, James P. Noonan, Svante Pääbo, Edward M. Rubin HACNS1 • Human-accelerated conserved non-coding sequence 1 • 546 bp region • Well-conserved among terrestrial mammals • 16 human- specific mutations (since chimpanzee) Experiment • Transgenic mouse assay • β-galactosidase (lacZ) reporter gene • Minimal Hsp68 promoter • 1.2 kb fragment encompassing HACNS1 ▫ Human, Chimp, Rhesus In Vivo Results (E11.5) Reproducibility In Vivo (E13.5) Money Shot “Humanized” Element • Synthesize chimeric element with 16 human- specific bases on chimpanzee background Testing the 13 substitution region Evolution Math • Evidence of Adaptive Evolution ▫ Rate of change is approx 4 times background rate (conserved, non-coding region) • Not explained by biased gene conversion ▫ Increase rate of AT→GC substitution Substitutions concetrated in HACNS1 • AT to GC fraction in 9kb sliding window Guessing/Conclusion • HACNS1 is exciting because of unique human digit and limb patterning • HACNS1 is in the intron of CENTG2 ▫ Role of CENTG2 is unknown in limb development • Next closest gene is GBX2 ▫ Expressed in developing limb but null mice appear normal • HACNS1 in mouse may not be equivalent to human.