Transfer learning reveals sequence determinants of the quantitative response to transcription factor dosage.

Deep learning approaches have made significant advances in predicting cell type-specific chromatin patterns from the identity and arrangement of transcription factor (TF) binding motifs. However, most models have been applied in unperturbed contexts, precluding a predictive understanding of how chromatin state responds to TF perturbation. Here, we used transfer learning to train and interpret deep learning models that use DNA sequence to predict, with accuracy approaching experimental reproducibility, how the concentration of two dosage-sensitive TFs (TWIST1, SOX9) affects regulatory element (RE) chromatin accessibility in facial progenitor cells. High-affinity motifs that allow for heterotypic TF co-binding and are concentrated at the center of REs buffer against quantitative changes in TF dosage and strongly predict unperturbed accessibility. In contrast, motifs with low-affinity or homotypic binding distributed throughout REs lead to sensitive responses with minimal contributions to unperturbed accessibility. Both buffering and sensitizing features show signatures of purifying selection. We validated these predictive sequence features using reporter assays and showed that a biophysical model of TF-nucleosome competition can explain the sensitizing effect of low-affinity motifs. Our approach of combining transfer learning and quantitative measurements of the chromatin response to TF dosage therefore represents a powerful method to reveal additional layers of the cis-regulatory code.

DNA-guided transcription factor cooperativity shapes face and limb mesenchyme.

Transcription factors (TFs) can define distinct cellular identities despite nearly identical DNA-binding specificities. One mechanism for achieving regulatory specificity is DNA-guided TF cooperativity. Although in vitro studies suggest that it may be common, examples of such cooperativity remain scarce in cellular contexts. Here, we demonstrate how "Coordinator," a long DNA motif composed of common motifs bound by many basic helix-loop-helix (bHLH) and homeodomain (HD) TFs, uniquely defines the regulatory regions of embryonic face and limb mesenchyme. Coordinator guides cooperative and selective binding between the bHLH family mesenchymal regulator TWIST1 and a collective of HD factors associated with regional identities in the face and limb. TWIST1 is required for HD binding and open chromatin at Coordinator sites, whereas HD factors stabilize TWIST1 occupancy at Coordinator and titrate it away from HD-independent sites. This cooperativity results in the shared regulation of genes involved in cell-type and positional identities and ultimately shapes facial morphology and evolution.

Joint multi-ancestry and admixed GWAS reveals the complex genetics behind human cranial vault shape.

The cranial vault in humans is highly variable, clinically relevant, and heritable, yet its genetic architecture remains poorly understood. Here, we conduct a joint multi-ancestry and admixed multivariate genome-wide association study on 3D cranial vault shape extracted from magnetic resonance images of 6772 children from the ABCD study cohort yielding 30 genome-wide significant loci. Follow-up analyses indicate that these loci overlap with genomic risk loci for sagittal craniosynostosis, show elevated activity cranial neural crest cells, are enriched for processes related to skeletal development, and are shared with the face and brain. We present supporting evidence of regional localization for several of the identified genes based on expression patterns in the cranial vault bones of E15.5 mice. Overall, our study provides a comprehensive overview of the genetics underlying normal-range cranial vault shape and its relevance for understanding modern human craniofacial diversity and the etiology of congenital malformations.

Systematic differences in discovery of genetic effects on gene expression and complex traits.

Most signals in genome-wide association studies (GWAS) of complex traits implicate noncoding genetic variants with putative gene regulatory effects. However, currently identified regulatory variants, notably expression quantitative trait loci (eQTLs), explain only a small fraction of GWAS signals. Here, we show that GWAS and cis-eQTL hits are systematically different: eQTLs cluster strongly near transcription start sites, whereas GWAS hits do not. Genes near GWAS hits are enriched in key functional annotations, are under strong selective constraint and have complex regulatory landscapes across different tissue/cell types, whereas genes near eQTLs are depleted of most functional annotations, show relaxed constraint, and have simpler regulatory landscapes. We describe a model to understand these observations, including how natural selection on complex traits hinders discovery of functionally relevant eQTLs. Our results imply that GWAS and eQTL studies are systematically biased toward different types of variant, and support the use of complementary functional approaches alongside the next generation of eQTL studies.

A genome-wide genetic screen uncovers determinants of human pigmentation.

Skin color, one of the most diverse human traits, is determined by the quantity, type, and distribution of melanin. In this study, we leveraged the light-scattering properties of melanin to conduct a genome-wide screen for regulators of melanogenesis. We identified 169 functionally diverse genes that converge on melanosome biogenesis, endosomal transport, and gene regulation, of which 135 represented previously unknown associations with pigmentation. In agreement with their melanin-promoting function, the majority of screen hits were up-regulated in melanocytes from darkly pigmented individuals. We further unraveled functions of KLF6 as a transcription factor that regulates melanosome maturation and pigmentation in vivo, and of the endosomal trafficking protein COMMD3 in modulating melanosomal pH. Our study reveals a plethora of melanin-promoting genes, with broad implications for human variation, cell biology, and medicine.

DNA-guided transcription factor cooperativity shapes face and limb mesenchyme.

Transcription factors (TFs) can define distinct cellular identities despite nearly identical DNA-binding specificities. One mechanism for achieving regulatory specificity is DNA-guided TF cooperativity. Although in vitro studies suggest it may be common, examples of such cooperativity remain scarce in cellular contexts. Here, we demonstrate how 'Coordinator', a long DNA motif comprised of common motifs bound by many basic helix-loop-helix (bHLH) and homeodomain (HD) TFs, uniquely defines regulatory regions of embryonic face and limb mesenchyme. Coordinator guides cooperative and selective binding between the bHLH family mesenchymal regulator TWIST1 and a collective of HD factors associated with regional identities in the face and limb. TWIST1 is required for HD binding and open chromatin at Coordinator sites, while HD factors stabilize TWIST1 occupancy at Coordinator and titrate it away from HD-independent sites. This cooperativity results in shared regulation of genes involved in cell-type and positional identities, and ultimately shapes facial morphology and evolution.

Precise modulation of transcription factor levels identifies features underlying dosage sensitivity.

Transcriptional regulation exhibits extensive robustness, but human genetics indicates sensitivity to transcription factor (TF) dosage. Reconciling such observations requires quantitative studies of TF dosage effects at trait-relevant ranges, largely lacking so far. TFs play central roles in both normal-range and disease-associated variation in craniofacial morphology; we therefore developed an approach to precisely modulate TF levels in human facial progenitor cells and applied it to SOX9, a TF associated with craniofacial variation and disease (Pierre Robin sequence (PRS)). Most SOX9-dependent regulatory elements (REs) are buffered against small decreases in SOX9 dosage, but REs directly and primarily regulated by SOX9 show heightened sensitivity to SOX9 dosage; these RE responses partially predict gene expression responses. Sensitive REs and genes preferentially affect functional chondrogenesis and PRS-like craniofacial shape variation. We propose that such REs and genes underlie the sensitivity of specific phenotypes to TF dosage, while buffering of other genes leads to robust, nonlinear dosage-to-phenotype relationships.

Systematic discovery and perturbation of regulatory genes in human T cells reveals the architecture of immune networks.

Gene regulatory networks ensure that important genes are expressed at precise levels. When gene expression is sufficiently perturbed, it can lead to disease. To understand how gene expression disruptions percolate through a network, we must first map connections between regulatory genes and their downstream targets. However, we lack comprehensive knowledge of the upstream regulators of most genes. Here, we developed an approach for systematic discovery of upstream regulators of critical immune factors-IL2RA, IL-2 and CTLA4-in primary human T cells. Then, we mapped the network of the target genes of these regulators and putative cis-regulatory elements using CRISPR perturbations, RNA-seq and ATAC-seq. These regulators form densely interconnected networks with extensive feedback loops. Furthermore, this network is enriched for immune-associated disease variants and genes. These results provide insight into how immune-associated disease genes are regulated in T cells and broader principles about the structure of human gene regulatory networks.

Decoding the Human Face: Progress and Challenges in Understanding the Genetics of Craniofacial Morphology.

Variations in the form of the human face, which plays a role in our individual identities and societal interactions, have fascinated scientists and artists alike. Here, we review our current understanding of the genetics underlying variation in craniofacial morphology and disease-associated dysmorphology, synthesizing decades of progress on Mendelian syndromes in addition to more recent results from genome-wide association studies of human facial shape and disease risk. We also discuss the various approaches used to phenotype and quantify facial shape, which are of particular importance due to the complex, multipartite nature of the craniofacial form. We close by discussing how experimental studies have contributed and will further contribute to our understanding of human genetic variation and then proposing future directions and applications for the field.

Genome scans of facial features in East Africans and cross-population comparisons reveal novel associations.

Facial morphology is highly variable, both within and among human populations, and a sizable portion of this variation is attributable to genetics. Previous genome scans have revealed more than 100 genetic loci associated with different aspects of normal-range facial variation. Most of these loci have been detected in Europeans, with few studies focusing on other ancestral groups. Consequently, the degree to which facial traits share a common genetic basis across diverse sets of humans remains largely unknown. We therefore investigated the genetic basis of facial morphology in an East African cohort. We applied an open-ended data-driven phenotyping approach to a sample of 2,595 3D facial images collected on Tanzanian children. This approach segments the face into hierarchically arranged, multivariate features that capture the shape variation after adjusting for age, sex, height, weight, facial size and population stratification. Genome scans of these multivariate shape phenotypes revealed significant (p < 2.5 × 10-8) signals at 20 loci, which were enriched for active chromatin elements in human cranial neural crest cells and embryonic craniofacial tissue, consistent with an early developmental origin of the facial variation. Two of these associations were in highly conserved regions showing craniofacial-specific enhancer activity during embryological development (5q31.1 and 12q21.31). Six of the 20 loci surpassed a stricter threshold accounting for multiple phenotypes with study-wide significance (p < 6.25 × 10-10). Cross-population comparisons indicated 10 association signals were shared with Europeans (seven sharing the same associated SNP), and facilitated fine-mapping of causal variants at previously reported loci. Taken together, these results may point to both shared and population-specific components to the genetic architecture of facial variation.

3D facial phenotyping by biometric sibling matching used in contemporary genomic methodologies.

The analysis of contemporary genomic data typically operates on one-dimensional phenotypic measurements (e.g. standing height). Here we report on a data-driven, family-informed strategy to facial phenotyping that searches for biologically relevant traits and reduces multivariate 3D facial shape variability into amendable univariate measurements, while preserving its structurally complex nature. We performed a biometric identification of siblings in a sample of 424 children, defining 1,048 sib-shared facial traits. Subsequent quantification and analyses in an independent European cohort (n = 8,246) demonstrated significant heritability for a subset of traits (0.17-0.53) and highlighted 218 genome-wide significant loci (38 also study-wide) associated with facial variation shared by siblings. These loci showed preferential enrichment for active chromatin marks in cranial neural crest cells and embryonic craniofacial tissues and several regions harbor putative craniofacial genes, thereby enhancing our knowledge on the genetic architecture of normal-range facial variation.

Shared heritability of human face and brain shape.

Evidence from model organisms and clinical genetics suggests coordination between the developing brain and face, but the role of this link in common genetic variation remains unknown. We performed a multivariate genome-wide association study of cortical surface morphology in 19,644 individuals of European ancestry, identifying 472 genomic loci influencing brain shape, of which 76 are also linked to face shape. Shared loci include transcription factors involved in craniofacial development, as well as members of signaling pathways implicated in brain-face cross-talk. Brain shape heritability is equivalently enriched near regulatory regions active in either forebrain organoids or facial progenitors. However, we do not detect significant overlap between shared brain-face genome-wide association study signals and variants affecting behavioral-cognitive traits. These results suggest that early in embryogenesis, the face and brain mutually shape each other through both structural effects and paracrine signaling, but this interplay may not impact later brain development associated with cognitive function.

Human-chimpanzee fused cells reveal cis-regulatory divergence underlying skeletal evolution.

Gene regulatory divergence is thought to play a central role in determining human-specific traits. However, our ability to link divergent regulation to divergent phenotypes is limited. Here, we utilized human-chimpanzee hybrid induced pluripotent stem cells to study gene expression separating these species. The tetraploid hybrid cells allowed us to separate cis- from trans-regulatory effects, and to control for nongenetic confounding factors. We differentiated these cells into cranial neural crest cells, the primary cell type giving rise to the face. We discovered evidence of lineage-specific selection on the hedgehog signaling pathway, including a human-specific sixfold down-regulation of EVC2 (LIMBIN), a key hedgehog gene. Inducing a similar down-regulation of EVC2 substantially reduced hedgehog signaling output. Mice and humans lacking functional EVC2 show striking phenotypic parallels to human-chimpanzee craniofacial differences, suggesting that the regulatory divergence of hedgehog signaling may have contributed to the unique craniofacial morphology of humans.

The Intersection of the Genetic Architectures of Orofacial Clefts and Normal Facial Variation.

Unaffected relatives of individuals with non-syndromic cleft lip with or without cleft palate (NSCL/P) show distinctive facial features. The presence of this facial endophenotype is potentially an expression of underlying genetic susceptibility to NSCL/P in the larger unselected population. To explore this hypothesis, we first partitioned the face into 63 partially overlapping regions representing global-to-local facial morphology and then defined endophenotypic traits by contrasting the 3D facial images from 264 unaffected parents of individuals with NSCL/P versus 3,171 controls. We observed distinct facial features between parents and controls across 59 global-to-local facial segments at nominal significance (p ≤ 0.05) and 52 segments at Bonferroni corrected significance (p < 1.2 × 10-3), respectively. Next, we quantified these distinct facial features as univariate traits in another dataset of 8,246 unaffected European individuals and performed a genome-wide association study. We identified 29 independent genetic loci that were associated (p < 5 × 10-8) with at least one of the tested endophenotypic traits, and nine genetic loci also passed the study-wide threshold (p < 8.47 × 10-10). Of the 29 loci, 22 were in proximity of loci previously associated with normal facial variation, 18 were near genes that show strong evidence in orofacial clefting (OFC), and another 10 showed some evidence in OFC. Additionally, polygenic risk scores for NSCL/P showed associations with the endophenotypic traits. This study thus supports the hypothesis of a shared genetic architecture of normal facial development and OFC.

GWAS of three molecular traits highlights core genes and pathways alongside a highly polygenic background.

Genome-wide association studies (GWAS) have been used to study the genetic basis of a wide variety of complex diseases and other traits. We describe UK Biobank GWAS results for three molecular traits-urate, IGF-1, and testosterone-with better-understood biology than most other complex traits. We find that many of the most significant hits are readily interpretable. We observe huge enrichment of associations near genes involved in the relevant biosynthesis, transport, or signaling pathways. We show how GWAS data illuminate the biology of each trait, including differences in testosterone regulation between females and males. At the same time, even these molecular traits are highly polygenic, with many thousands of variants spread across the genome contributing to trait variance. In summary, for these three molecular traits we identify strong enrichment of signal in putative core gene sets, even while most of the SNP-based heritability is driven by a massively polygenic background.

Insights into the genetic architecture of the human face.

The human face is complex and multipartite, and characterization of its genetic architecture remains challenging. Using a multivariate genome-wide association study meta-analysis of 8,246 European individuals, we identified 203 genome-wide-significant signals (120 also study-wide significant) associated with normal-range facial variation. Follow-up analyses indicate that the regions surrounding these signals are enriched for enhancer activity in cranial neural crest cells and craniofacial tissues, several regions harbor multiple signals with associations to different facial phenotypes, and there is evidence for potential coordinated actions of variants. In summary, our analyses provide insights into the understanding of how complex morphological traits are shaped by both individual and coordinated genetic actions.

Quantitative analysis of Y-Chromosome gene expression across 36 human tissues.

Little is known about how human Y-Chromosome gene expression directly contributes to differences between XX (female) and XY (male) individuals in nonreproductive tissues. Here, we analyzed quantitative profiles of Y-Chromosome gene expression across 36 human tissues from hundreds of individuals. Although it is often said that Y-Chromosome genes are lowly expressed outside the testis, we report many instances of elevated Y-Chromosome gene expression in a nonreproductive tissue. A notable example is EIF1AY, which encodes eukaryotic translation initiation factor 1A Y-linked, together with its X-linked homolog EIF1AX Evolutionary loss of a Y-linked microRNA target site enabled up-regulation of EIF1AY, but not of EIF1AX, in the heart. Consequently, this essential translation initiation factor is nearly twice as abundant in male as in female heart tissue at the protein level. Divergence between the X and Y Chromosomes in regulatory sequence can therefore lead to tissue-specific Y-Chromosome-driven sex biases in expression of critical, dosage-sensitive regulatory genes.

Mammalian germ cells are determined after PGC colonization of the nascent gonad.

Mammalian primordial germ cells (PGCs) are induced in the embryonic epiblast, before migrating to the nascent gonads. In fish, frogs, and birds, the germline segregates even earlier, through the action of maternally inherited germ plasm. Across vertebrates, migrating PGCs retain a broad developmental potential, regardless of whether they were induced or maternally segregated. In mammals, this potential is indicated by expression of pluripotency factors, and the ability to generate teratomas and pluripotent cell lines. How the germline loses this developmental potential remains unknown. Our genome-wide analyses of embryonic human and mouse germlines reveal a conserved transcriptional program, initiated in PGCs after gonadal colonization, that differentiates germ cells from their germline precursors and from somatic lineages. Through genetic studies in mice and pigs, we demonstrate that one such gonad-induced factor, the RNA-binding protein DAZL, is necessary in vivo to restrict the developmental potential of the germline; DAZL's absence prolongs expression of a Nanog pluripotency reporter, facilitates derivation of pluripotent cell lines, and causes spontaneous gonadal teratomas. Based on these observations in humans, mice, and pigs, we propose that germ cells are determined after gonadal colonization in mammals. We suggest that germ cell determination was induced late in embryogenesis-after organogenesis has begun-in the common ancestor of all vertebrates, as in modern mammals, where this transition is induced by somatic cells of the gonad. We suggest that failure of this process of germ cell determination likely accounts for the origin of human testis cancer.

Conservation, acquisition, and functional impact of sex-biased gene expression in mammals.

Sex differences abound in human health and disease, as they do in other mammals used as models. The extent to which sex differences are conserved at the molecular level across species and tissues is unknown. We surveyed sex differences in gene expression in human, macaque, mouse, rat, and dog, across 12 tissues. In each tissue, we identified hundreds of genes with conserved sex-biased expression-findings that, combined with genomic analyses of human height, explain ~12% of the difference in height between females and males. We surmise that conserved sex biases in expression of genes otherwise operating equivalently in females and males contribute to sex differences in traits. However, most sex-biased expression arose during the mammalian radiation, which suggests that careful attention to interspecies divergence is needed when modeling human sex differences.