01742nas a2200301   4500000000100000008004100001260001500042100001700057700001600074700002000090700001600110700002300126700001900149700002300168700001900191700001700210700002300227700002700250700001900277700002500296700001700321700002500338245009400363856005600457300001300513490000800526520090600534       2024                            d  c2024-05-241 aChengyu Deng1 aSean Whalen1 aMarilyn Steyert1 aRyan Ziffra1 aPawel F. Przytycki1 aFumitaka Inoue1 aDaniela A. Pereira1 aDavide Capauto1 aScott Norton1 aFlora M. Vaccarino1 aPsychENCODE Consortium1 aAlex A. Pollen1 aTomasz J. Nowakowski1 aNadav Ahituv1 aKatherine S. Pollard00aMassively parallel characterization of regulatory elements in the developing human cortex  uhttps://www.science.org/doi/10.1126/science.adh0559  aeadh05590 v3843 aNucleotide changes in gene regulatory elements are important determinants of neuronal development and diseases. Using massively parallel reporter assays in primary human cells from mid-gestation cortex and cerebral organoids, we interrogated the cis-regulatory activity of 102,767 open chromatin regions, including thousands of sequences with cell type–specific accessibility and variants associated with brain gene regulation. In primary cells, we identified 46,802 active enhancer sequences and 164 variants that alter enhancer activity. Activity was comparable in organoids and primary cells, suggesting that organoids provide an adequate model for the developing cortex. Using deep learning we decoded the sequence basis and upstream regulators of enhancer activity. This work establishes a comprehensive catalog of functional gene regulatory elements and variants in human neuronal development.