Combinatorial effects on gene expression at the Lbx1/Fgf8 locus resolve split-hand/foot malformation type 3

Split-Hand/Foot Malformation type 3 (SHFM3) is a congenital limb malformation associated with tandem duplications at the LBX1/FGF8 locus. Yet, the disease patho-mechanism remains unsolved. Here we investigate the functional consequences of SHFM3-associated rearrangements on chromatin conformation and gene expression in vivo in transgenic mice. We show that the Lbx1/Fgf8 locus consists of two separate, but interacting, regulatory domains. Re-engineering of a SHFM3-associated duplication and a newly reported inversion in mice results in restructuring of the chromatin architecture. This leads to ectopic activation of the Lbx1 and Btrc genes in the apical ectodermal ridge (AER) in an Fgf8-like pattern induced by AER-specific enhancers of Fgf8. We provide evidence that the SHFM3 phenotype is the result of a combinatorial effect on gene misexpression in the developing limb. Our results reveal insights into the molecular mechanism underlying SHFM3 and provide conceptual framework for how genomic rearrangements can cause gene misexpression and disease.This study was supported by grants from the Deutsche Forschungsgemeinschaft (MU 880/16-1, MU 880/20-1) to S.M. We thank the transgenic unit, sequencing core and animal facility of Max Planck Institute for Molecular Genetics for technical assistance, Ute Fischer for technical support and Norbert Brieske for help with whole mount in situ hybridizations and image processing

Position effects at the FGF8 locus are associated with femoral hypoplasia

Copy-number variations (CNVs) are a common cause of congenital limb malformations and are interpreted primarily on the basis of their effect on gene dosage. However, recent studies show that CNVs also influence the 3D genome chromatin organization. The functional interpretation of whether a phenotype is the result of gene dosage or a regulatory position effect remains challenging. Here, we report on two unrelated families with individuals affected by bilateral hypoplasia of the femoral bones, both harboring de novo duplications on chromosome 10q24.32. The ∼0.5 Mb duplications include FGF8, a key regulator of limb development and several limb enhancer elements. To functionally characterize these variants, we analyzed the local chromatin architecture in the affected individuals’ cells and re-engineered the duplications in mice by using CRISPR-Cas9 genome editing. We found that the duplications were associated with ectopic chromatin contacts and increased FGF8 expression. Transgenic mice carrying the heterozygous tandem duplication including Fgf8 exhibited proximal shortening of the limbs, resembling the human phenotype. To evaluate whether the phenotype was a result of gene dosage, we generated another transgenic mice line, carrying the duplication on one allele and a concurrent Fgf8 deletion on the other allele, as a control. Surprisingly, the same malformations were observed. Capture Hi-C experiments revealed ectopic interaction with the duplicated region and Fgf8, indicating a position effect. In summary, we show that duplications at the FGF8 locus are associated with femoral hypoplasia and that the phenotype is most likely the result of position effects altering FGF8 expression rather than gene dosage effects.M.S. and A.S.-S. were supported by the Polish National Science Centre (UMO-2016/23/N/NZ2/02362 to M.S. and UMO-2016/21/D/NZ5/00064 to A.S.-S.). A.S.-S. was also supported by the Polish National Science Centre scholarship for PhD students (UMO-2013/08/T/NZ2/00027). C.L. is supported by postdoctoral Beatriu de Pinós from Secretaria d’Universitats I Recerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya and by the Marie Sklodowska-Curie COFUND program from H2020 (2018-BP-00055). A.J. was supported by the Polish National Science Centre (UMO-2016/22/E/NZ5/00270) as well as the Polish National Centre for Research and Development (LIDER/008/431/L-4/12/NCBR/2013). M.S. is supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (SP1532/3-1, SP1532/4-1, and SP1532/5-1), the Max Planck Foundation, and the Deutsches Zentrum für Luft- und Raumfahrt (DLR 01GM1925)

Repression and 3D-restructuring resolves regulatory conflicts in evolutionarily rearranged genomes

Regulatory landscapes drive complex developmental gene expression, but it remains unclear how their integrity is maintained when incorporating novel genes and functions during evolution. Here, we investigated how a placental mammal-specific gene, Zfp42, emerged in an ancient vertebrate topologically associated domain (TAD) without adopting or disrupting the conserved expression of its gene, Fat1. In ESCs, physical TAD partitioning separates Zfp42 and Fat1 with distinct local enhancers that drive their independent expression. This separation is driven by chromatin activity and not CTCF/cohesin. In contrast, in embryonic limbs, inactive Zfp42 shares Fat1’s intact TAD without responding to active Fat1 enhancers. However, neither Fat1 enhancer-incompatibility nor nuclear envelope-attachment account for Zfp42’s unresponsiveness. Rather, Zfp42’s promoter is rendered inert to enhancers by context-dependent DNA methylation. Thus, diverse mechanisms enabled the integration of independent Zfp42 regulation in the Fat1 locus. Critically, such regulatory complexity appears common in evolution as, genome wide, most TADs contain multiple independently expressed genes.We thank the Montpellier Ressources Imagerie facility (BioCampus Montpellier, Centre National de la Recherche Scientifique [CNRS], INSERM, University of Montpellier) and for computer resources from CINECA (ISCRA grant thanks to computer resources from INFN and CINECA [ISCRA Grant HP10C8JWU7]). G.C., Q.S., and F.B. were supported by a grant from the European Research Council (Advanced Grant 3DEpi, 788972) and by the CNRS. This work was funded by EMBO and the Wellcome Trust (ALTF1554-2016 and 206475/Z/17/Z; to M.I.R.) as well as the Deutsche Forschungsgemeinschaft (KR3985/7-3 and MU 880/16-1 to S.M.)

Role of the chromatin landscape and sequence in determining cell type-specific genomic glucocorticoid receptor binding and gene regulation

International audienceThe genomic loci bound by the glucocorticoid receptor (GR), a hormone-activated transcription factor, show little overlap between cell types. To study the role of chromatin and sequence in specifying where GR binds, we used Bayesian modeling within the universe of accessible chromatin. Taken together, our results uncovered that although GR preferentially binds accessible chromatin, its binding is biased against accessible chromatin located at promoter regions. This bias can only be explained partially by the presence of fewer GR recognition sequences, arguing for the existence of additional mechanisms that interfere with GR binding at promoters. Therefore, we tested the role of H3K9ac, the chromatin feature with the strongest negative association with GR binding, but found that this correlation does not reflect a causative link. Finally, we find a higher percentage of promoter-proximal GR binding for genes regulated by GR across cell types than for cell typespecific target genes. Given that GR almost exclusively binds accessible chromatin, we propose that cell type-specific regulation by GR preferentially occurs via distal enhancers, whose chromatin accessibility is typically cell type-specific, whereas ubiquitous target gene regulation is more likely to result from binding to promoter regions, which are often accessible regardless of cell type examined

Role of the chromatin landscape and sequence in determining cell type-specific genomic glucocorticoid receptor binding and gene regulation.

The genomic loci bound by the glucocorticoid receptor (GR), a hormone-activated transcription factor, show little overlap between cell types. To study the role of chromatin and sequence in specifying where GR binds, we used Bayesian modeling within the universe of accessible chromatin. Taken together, our results uncovered that although GR preferentially binds accessible chromatin, its binding is biased against accessible chromatin located at promoter regions. This bias can only be explained partially by the presence of fewer GR recognition sequences, arguing for the existence of additional mechanisms that interfere with GR binding at promoters. Therefore, we tested the role of H3K9ac, the chromatin feature with the strongest negative association with GR binding, but found that this correlation does not reflect a causative link. Finally, we find a higher percentage of promoter-proximal GR binding for genes regulated by GR across cell types than for cell type-specific target genes. Given that GR almost exclusively binds accessible chromatin, we propose that cell type-specific regulation by GR preferentially occurs via distal enhancers, whose chromatin accessibility is typically cell type-specific, whereas ubiquitous target gene regulation is more likely to result from binding to promoter regions, which are often accessible regardless of cell type examined

Preformed chromatin topology assists transcriptional robustness of Shh during limb development

Long-range gene regulation involves physical proximity between enhancers and promoters to generate precise patterns of gene expression in space and time. However, in some cases, proximity coincides with gene activation, whereas, in others, preformed topologies already exist before activation. In this study, we investigate the preformed configuration underlying the regulation of the Shh gene by its unique limb enhancer, the ZRS, in vivo during mouse development. Abrogating the constitutive transcription covering the ZRS region led to a shift within the Shh-ZRS contacts and a moderate reduction in Shh transcription. Deletion of the CTCF binding sites around the ZRS resulted in the loss of the Shh-ZRS preformed interaction and a 50% decrease in Shh expression but no phenotype, suggesting an additional, CTCF-independent mechanism of promoter-enhancer communication. This residual activity, however, was diminished by combining the loss of CTCF binding with a hypomorphic ZRS allele, resulting in severe Shh loss of function and digit agenesis. Our results indicate that the preformed chromatin structure of the Shh locus is sustained by multiple components and acts to reinforce enhancer-promoter communication for robust transcription