High Resolution Epigenomic Atlas of Early Human Craniofacial Development

Abstract

12 13 Defects in embryonic patterning resulting in craniofacial abnormalities are common birth 14 defects affecting up to 1 in 500 live births worldwide, and are mostly non-syndromic. 15 The regulatory programs that build and shape the craniofacial complex are thought to 16 be controlled by information encoded in the genome between genes and within intronic 17 sequences. Early stages of human craniofacial development have not been interrogated 18 with modern functional genomics techniques, preventing systematic analysis of genetic 19 associations with craniofacial-specific regulatory sequences. Here we describe a 20 comprehensive resource of craniofacial epigenomic annotations and systematic, 21 integrative analysis with a variety of human tissues and cell types. We identified 22 thousands of novel craniofacial enhancers and provide easily accessible genome 23 annotations for craniofacial researchers and clinicians. We demonstrate the utility of our 24 data to find likely causal variants for craniofacial abnormalities and identify a large 25 enhancer cluster that interacts with HOXA genes during craniofacial development.

7q11.23 Syndromes Reveal BAZ1B as a Master Regulator of the Modern Human Face and Validate the Self-Domestication Hypothesis

“…The 7dupASD NCSC KD lines took longer to fill the wound when compared to the respective 20 control lines (scr), as indicated by images taken at 8 and 16 hours after a gap was created on the plate surface ( Fig. 1C, S1F).…”

“…A core insight from our previous work had been the discovery that already at the pluripotent state 15 7q11.23 CNVs cause transcriptional dysregulation in disease-relevant pathways, which are then selectively amplified along development in a tissue-specific manner (13). Given the critical role of BAZ1B as transcriptional regulator in different cell and animal models (17-19), as well as its functional involvement in human NC from the above described findings, we thus hypothesized that its dosage could significantly contribute to the transcriptional dysregulation in 7q11.23 CNV- 20 NCSCs. To test this hypothesis and gain mechanistic insights into the specific BAZ1B dosagedependent downstream circuits, we subjected 32 interfered NCSC lines to high coverage RNA sequencing (RNA-seq) analysis.…”

“…As shown in Fig. S2A, a manually curated signature from an 8 extensive literature review (20)(21)(22)(23)(24)(25) In order to dissect it, we thus resorted to a combination of classical pairwise comparative analysis, contrasting shBAZ1B-interfered NCSC lines (sh1+sh2) with their respective controls (scr), with a 10 complementary regression analysis using BAZ1B expression levels as independent variables, subtracting the contribution of individual genetic backgrounds. This design increases robustness and sensitivity in the identification of genes that, across multiple genetic backgrounds and target gene dosages, might have a different baseline (scr) across individuals while still being robustly dysregulated upon BAZ1B interference.…”

“…S1A) and cognitive/behavioral traits (4,5) are uniquely and symmetrically aligned to the domesticationrelated traits of AMHs. Structural variants in WBS genes have been shown to underlie stereotypical hypersociability in domestic dogs (6) and within the WBSCR the following lines of evidence point to the chromatin regulator BAZ1B as a key gene underlying domestication-relevant 15 craniofacial features: i) its crucial role in NC maintenance and migration in Xenopus Laevis and the craniofacial defects observed in knock-out mice (7,8); ii) the observation that its expression is impacted by domestication-related events in canines (9); iii) the first formulation of the neurocristopathic hypothesis of domestication, which included BAZ1B among the genes influencing NC development (3); iv) the most comprehensive studies focusing on regions of the 20 modern human genome associated with selective sweep signals compared to Neanderthals/Denisovans (hereafter 'archaics') (10,11), one of which specifically included BAZ1B within the detected portions of the WBSCR; and v) the thus far most detailed study 5 systematically exploring high frequency (> 90%) changes in modern humans for which archaic humans carry the ancestral state, which found BAZ1B enriched for mutations in modern humans (most of which fall in the regulatory regions of the gene) (12). Through the thus far largest cohort of 7q11.23 patient-derived induced pluripotent stem cell (iPSC) lines, our previous work had revealed major disease-relevant dysregulation that was already apparent at the pluripotent state 5 and was further exacerbated upon differentiation (13).…”

“…15 lines In order to dissect the role of BAZ1B in the paradigmatic craniofacial dysmorphisms that characterize WBS and 7dupASD, we started from our previous characterization of WBS and 7dupASD patient-specific iPSC lines and differentiated derivatives (13) and selected a cohort of 11 NCSC lines (4 from WBS patients, 3 from 7dupASD patients and 4 from control individuals). 20 These represent the largest cohort of patient-specific NCSCs described so far and we used transcriptomic profiling to validate their cranial identity as a privileged entry point into diseasepathogenesis, given the centrality of the cranial NC in the development of the face. We then 6 knocked-down (KD) BAZ1B via RNA interference in all lines across the three genetic conditions, including also NCSCs derived from a particularly informative atypical WBS patient (hereafter atWBS) bearing a partial deletion of the region that spares BAZ1B and six additional genes (14) ( Fig.…”