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)

Prioritization of genes driving congenital phenotypes of patients with de novo genomic structural variants

Background:Genomic structural variants (SVs) can affect many genes and regulatory elements. Therefore, the molecular mechanisms driving the phenotypes of patients carrying de novo SVs are frequently unknown. Methods:We applied a combination of systematic experimental and bioinformatic methods to improve the molecular diagnosis of 39 patients with multiple congenital abnormalities and/or intellectual disability harboring apparent de novo SVs, most with an inconclusive diagnosis after regular genetic testing. Results: In 7 of these cases (18%), whole-genome sequencing analysis revealed disease-relevant complexities of the SVs missed in routine microarray-based analyses. We developed a computational tool to predict the effects on genes directly affected by SVs and on genes indirectly affected likely due to the changes in chromatin organization and impact on regulatory mechanisms. By combining these functional predictions with extensive phenotype information, candidate driver genes were identified in 16/39 (41%) patients. In 8 cases, evidence was found for the involvement of multiple candidate drivers contributing to different parts of the phenotypes. Subsequently, we applied this computational method to two cohorts containing a total of 379 patients with previously detected and classified de novo SVs and identified candidate driver genes in 189 cases (50%), including 40 cases whose SVs were previously not classified as pathogenic. Pathogenic position effects were predicted in 28% of all studied cases with balanced SVs and in 11% of the cases with copy number variants. Conclusions:These results demonstrate an integrated computational and experimental approach to predict driver genes based on analyses of WGS data with phenotype association and chromatin organization datasets. These analyses nominate new pathogenic loci and have strong potential to improve the molecular diagnosis of patients with de novo SVs

Spatial Genome Organization:From Development to Disease

Every living organism, from bacteria to humans, contains DNA encoding anything from a few hundred genes in intracellular parasites such as Mycoplasma, up to several tens of thousands in many higher organisms. The first observations indicating that the nucleus had some kind of organization were made over a hundred years ago. Understanding of its significance is both limited and aided by the development of techniques, in particular electron microscopy, fluorescence in situ hybridization, DamID and most recently HiC. As our knowledge about genome organization grows, it becomes apparent that the mechanisms are conserved in evolution, even if the individual players may vary. These mechanisms involve DNA binding proteins such as histones, and a number of architectural proteins, some of which are very much conserved, with some others having diversified and multiplied, acquiring specific regulatory functions. In this review we will look at the principles of genome organization in a hierarchical manner, from DNA packaging to higher order genome associations such as TADs, and the significance of radial positioning of genomic loci. We will then elaborate on the dynamics of genome organization during development, and how genome architecture plays an important role in cell fate determination. Finally, we will discuss how misregulation can be a factor in human disease

Eine dynamische Chromatinarchitektur moduliert die Pitx1 Genregulation in der Entwiklung von Extremitäten und Krankheiten

The tissue specific expression of developmental genes is encoded in enhancer elements often located hundreds of kb away from their cognate promoters. Physical chromatin interactions between these enhancers and target promoters are associated with active transcription and conventionally thought to be confined to topologically associating domains (TADs). However, little is known about the underlying nature and dynamics of this 3D-architecture during development and its perturbation in disease. In this work, the mouse embryonic limb bud was used as a paradigm to investigate the dynamics of gene regulation underlying the development of either arms or legs. Pitx1 is the one of few transcription factors shown to be expressed exclusively in hindlimbs and not in forelimbs. Yet, its regulation in mammals continues to be largely unknown. Here, we identified an unexpectedly complex regulatory basis of hindlimb- specific Pitx1 expression that expands the current model of enhancer sequences as the sole determinants of tissue specificity. We demonstrate that Pitx1 is regulated by the active fore- and hindlimb enhancer, Pen, that is required for normal expression of Pitx1 in hindlimbs, but does not activate Pitx1 expression in forelimbs. Investigation of the chromatin architecture of the Pitx1 locus in both fore-and hindlimb buds using cHi-C and derived 3D-models, revealed a modular regulatory landscape that is not confined to a TAD structure. Instead, Pitx1 is controlled by a Multi-Anchor Domain (MAD), which can assume distinct tissue-specific conformations. In the hindlimb, the locus forms an active MAD that enable Pen and Pitx1 interactions, producing a transcriptionally active pocket. Intriguingly, an alternate forelimb-specific MAD conformation prevents the promiscuous activity of Pen by physically separating it from Pitx1. Disruption of this segregated forelimb chromatin conformation in engineered mice, as well as in human Liebenberg syndrome patients, results in forelimb Pitx1 misexpression, and a partial transformation of forelimb morphology into a hindlimb-like. This work provides further understanding of gene regulation whereby unspecific enhancer activity is actively regulated by the dynamics in 3D-chromatin architecture, independent of TADs, to confer a tissue specific transcriptional output. Together our findings help build the groundwork for the interpretation of structural variants disrupting genome organisation, not only resulting in human disease, but also in the evolution of phenotypes in natural populations.Die gewebespezifische Expression von Entwicklungsgenen wird durch Enhancerelemente gesteuerte, welche häufig hunderte Kilobasen von ihren Zielpromotoren entfernt liegen können. Physische Chromatininteraktionen zwischen diesen Enhancern und ihren Zielpromotoren werden mit aktiver Transkription in Zusammenhang gebracht und ist in der Regel auf „topologically associating domains“ (TADs) begrenzt. Allerdings ist noch wenig über die dieser 3D-Architektur in der Entwicklung zugrunde liegenden Natur und Dynamik bekannt und wie diese in Krankheiten gestört wird. In dieser Arbeit wurde die embryonale Extremitätenentwicklung der Maus als Paradigma genutzt, um die komplexe Genregulation während der spezifischen Arm- und Beinentwicklung zu untersuchen. Pitx1 ist einer der wenigen Transkriptionsfaktoren, die ausschließlich in den hinteren Extremitäten, und nicht in den vorderen Extremitäten, exprimiert werden. Dessen Regulation in Säugetieren ist jedoch bislang noch größtenteils unbekannt. Hier, wurde eine unerwartet komplexe regulatorische Grundlage für die Beinentwicklung spezifische Pitx1 Expression identifiziert. Diese erweitert das aktuelle Modell, welches Enhancersequenzen noch als alleinige Determinanten der Gewebespezifität benennt. Wir zeigen, dass Pitx1 von einem in Vorder-und Hintergliedmaßen aktiven Enhancer, genannt Pen, reguliert wird. Dieser wird in den hinteren Extremitäten für die Pitx1 Expression benötigt, führt aber in der regulären Entwicklung der Vordergliedmaßen zu keiner Pitx1 Aktivierung. Untersuchungen der Chromatinarchitektur, des Pitx1 Lokus in Extremitätenknospen von sowohl Vorder-, als auch Hintergliedmaßen, mittels „capture Hi-C“ und abgeleiteten 3D-Modellen, ergaben eine modulare regulatorische Landschaft, welche nicht auf eine TAD Struktur begrenzt ist. Stattdessen, konnte nachgewiesen werden, dass Pitx1 von einer „Multi-Anchor Domain“ (MAD), die Gewebespezifische Konformationen annehmen kann, gelenkt wird. In den Hintergliedmaßen formt der Lokus eine aktive MAD, die eine Interaktion zwischen Pen und Pitx1 ermöglicht, wobei eine transkriptionell aktive Tasche erzeugt wird. Interessanterweise verhindert eine alternative Vordergliedmaßen-spezifische MAD Konformation unspezifische Pen Aktivitäten, indem Pen physisch von Pitx1 getrennt wird. Störungen dieser segregierten Chromatinkonformation, sowohl in genetisch manipulierten Mäusen, als auch in menschlichen Patienten mit Liebenberg Syndrom, führen zu einer Misexpression von Pitx1 in den vorderen Extremitäten, wobei eine partielle Transformation der Vordergliedmaßenmorpholgie in eine den Hintergliedmaßen ähnlichen Struktur entsteht. Diese Arbeit schafft neue Erkenntnisse über Genregulation, da nachgewiesen wurde, dass, unabhängig von TADs unspezifische Enhancer in einer aktiven Weise von den Dynamiken der 3D- Architektur des Chromatins reguliert werden können, um gewebespezifische transkriptionelle Aktivität zu ermöglichen. Gemeinsam helfen unsere Erkenntnisse Grundlagen für die Interpretation von Strukturvariationen, welche die 3D-Organisation des Genoms stören, und dadurch nicht nur menschliche Erkrankungen, sondern auch die Beeinflussung der Evolution von Phänotypen in natürlichen Populationen zur Folge haben können, zu bilden