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

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.)

Caractérisation fonctionnelle du locus Liz/Zdbf2 soumis à empreinte chez la souris : de l'embryon précoce à la physiologie adulte

Genomic imprinting refers to the epigenetic mechanism by which approximately 120 genes are expressed in a parent-of-origin manner. This parental asymmetry in gene expression is mediated through differential profiles of DNA methylation established in the oocyte and the sperm and maintained after fertilization in the developing individual. In mammals, imprinted genes are essential for normal embryo development as well as behavioral and physiological functions after birth. Clarifying the regulation and the function of those genes is thus fundamental in the field of developmental biology and health. During my PhD, I functionally characterized the imprinted Zdbf2 locus in mice. Zdbf2 is a paternally expressed gene, conserved from mouse to human, whose biological function was unknown. I revealed that Zdbf2 activation in the post-natal brain requires an indelible epigenetic signal that is established during the first days of embryogenesis. Additionally, I provided in vivo evidence that early programming of Zdbf2 is essential for proper growth after birth. By generating multiple CRISPR-mediated genetic mutants with varied doses of Zdbf2 in the hypothalamo-pituitary axis, I finally demonstrated that Zdbf2 is a growth-promoting gene, with a dose-sensitive effect and acting independently of its parental origin. Altogether, my work shed light onto the crucial function of a mammalian imprinted gene, from its regulation in the early embryo to its role in adult physiology.L’empreinte parentale est un mécanisme de régulation épigénétique qui réduit l’expression d’environ 120 gènes à une seule dose parentale. L’expression monoallélique dépend de marques différentielles de méthylation de l’ADN, établies dans l’ovocyte et le spermatozoïde et maintenues après fécondation dans l’individu en développement. Chez les mammifères, les gènes soumis à empreinte sont essentiels au développement embryonnaire et à certaines fonctions comportementales et physiologiques après la naissance. Définir les mécanismes de régulation et la fonction des gènes soumis à empreinte est une question cruciale en biologie du développement et en pathologie. Mon travail de thèse a concerné l’étude fonctionnelle du locus Zdbf2 chez la souris. Zdbf2 est un gène exprimé paternellement, conservé chez l’humain, mais dont la fonction était inconnue. J’ai pu démontrer que l’activation de Zdbf2 dans le cerveau de souris pré-pubères dépend d’un signal épigénétique indélébile mis en place dès les premiers jours de développement embryonnaire. Cette programmation précoce de Zdbf2 assure une croissance normale du nouveau-né. Mes résultats indiquent de plus que la dose, mais pas l’origine parentale de Zdbf2 est essentielle. Ces découvertes ont été possibles par la création de divers modèles mutants avec des variations de la dose de Zdbf2 dans l’axe hypothalamo-hypophysaire. Ce travail met en lumière la fonction cruciale d’un gène soumis à empreinte, de sa régulation dans l’embryon précoce à son rôle sur la physiologie adulte

Continuous enzymatic stirred tank reactor cascade with unconventional medium yielding high concentrations of (S)-2-hydroxyphenyl propanone and its derivatives

The implementation of biocatalysis in flow chemistry offers synergistic synthesis advantages in line with green chemistry principles. Yet, the conversion of high substrate concentrations is in many cases hindered by insolubility issues or substrate toxicity. Here, the continuous synthesis of (S)-2-hydroxyphenyl propanone (2-HPP) from inexpensive benzaldehyde and acetaldehyde in a methyl tert-butyl ether based organic reaction environment, namely micro-aqueous reaction system, has been established. Kinetic parameters of the applied whole cell catalyst were identified to design a continuous process for (S)-2-HPP synthesis. This revealed a necessity to distribute acetaldehyde over a spatial coordinate to remain below a toxic concentration threshold. Hence, three continuous stirred tank reactors (cSTR) were conjugated in a technical cascade with an additional influx of acetaldehyde into each unit. The catalytic behaviour of this reaction setup was described based on mass balances and a kinetic model. Enzyme deactivation was described by a novel staged model and compared to a simple generic model. The optimized continuous setup yielded 190 mM (S)-HPP with an ee > 98% over 8 h. The product was easily recovered from the organic reaction environment by crystallization with an isolated yield of 68% and a purity of >99%. Further, the substrate range of the applied catalyst Pseudomonas putida benzoylformate decarboxylase variant L461A was analysed. This revealed numerous halogenated, methoxylated and nitro-derivatives in ortho, meta, and para position, which can in principle be gained by the established process. As an example, the applied cSTR concept was transferred to p-methoxy benzaldehyde with good results even without further optimization

Lese- und schreibdidaktisches Wissen: Empirische Modellierung sowie Identifikation möglicher Prädiktoren.

H. Lohse-Bossenz, M. Rehm, M. Friesen, M. Seidenfuß, J. Rutsch, M. Vogel, T. Dörfler (Hrsg.), Professionalisierung in der Lehrerbildung. Erkenntnisse und Perspektiven des interdisziplinären Forschungsprogramms ""Effektive Kompetenzdiagose in der Lehrerbildung"

Lese- und schreibdidaktisches Wissen: Empirische Modellierung sowie Identifikation möglicher Prädiktoren.

H. Lohse-Bossenz, M. Rehm, M. Friesen, M. Seidenfuß, J. Rutsch, M. Vogel, T. Dörfler (Hrsg.), Professionalisierung in der Lehrerbildung. Erkenntnisse und Perspektiven des interdisziplinären Forschungsprogramms ""Effektive Kompetenzdiagose in der Lehrerbildung"