René Buschow

Body size represents a complex phenotype driven by genetic variation and epigenetic regulation, with the molecular processes underlying this trait remaining a central challenge to disentangle. To elucidate these fundamental mechanisms, we apply a multi-omics approach that combines ROH-based selection mapping with growth plate epigenomics in pigs. Taking advantage of divergent selection that separates pigs into miniature and larger-sized groups, we target genomic regions under this intense selection for height, which harbour functional variants with pronounced effects. We assemble a multi-omics dataset, identifying homozygous alternative SNPs in Aachen Minipigs and Mini-LEWE predicted to affect cis-regulatory elements potentially interacting with differentially expressed genes that drive body size in breed-dependent ways. Our results point to an lncRNA ( ENSSSCG00000048200 ) near SDR16C5 and PLAG1, HPX and NET-related pathways, as central players in the growth plates. In summary, our study offers a multi-layered characterisation of regulatory mechanisms in the growth plates in the pig model.

Body size represents a complex phenotype driven by genetic variation and epigenetic regulation, with the molecular processes underlying this trait remaining a central challenge to disentangle. To elucidate these fundamental mechanisms, we applied a multi-omics approach that combines ROH-based selection mapping with growth plate epigenomics in pigs, a valuable model species for skeletal development. Taking advantage of divergent selection that separates pigs into miniature and larger-sized types, we targeted genomic regions under this intense selection for height, which harbour functional variants with pronounced effects. We assembled a multi-omics dataset, identifying 23 homozygous mutant variants in Aachen Minipigs and 13 distinct variants in Mini-LEWE predicted to affect cis-regulatory elements and potentially interact with differentially expressed genes that drive body size in breed-dependent ways. Our results pointed to an lncRNA (ENSSSCG00000048200) near SDR16C5 and PLAG1, HPX and NET-related pathways, as central players in impaired growth in the growth plates. Furthermore, network propagation across protein-protein interaction networks highlighted 16 proteins with consistent interaction network changes between miniature and larger-sized pig breeds. In summary, our study offers a multi-layered characterisation of regulatory mechanisms in the growth plates, using miniature pigs as a model to understand the genetic and epigenetic control of long-bone growth. *Nadia Khaveh and Jan Berghöfer contributed equally.

The developing mammalian embryo is guided by the continuously changing signals that it receives from maternal tissues and its microenvironment. The dynamic cell-cell and cell-environment interactions that together shape the embryo largely remained unexplorable until the advance of stem cell-based embryo models. These revealed the self-organizing properties of cells in response to endogenous and exogenous cues. Among the latter, restricted oxygen (hypoxia) emerged as a critical microenvironmental regulator that influences cell type diversification in multicellular systems. Here we built a modular ESC-based head-to-tail model of mouse embryogenesis by developing an a ntero- p osterior (AP) assembly strategy under hypoxia. These structures called HAP-gastruloids feature stage-appropriate anterior neural tissues that recapitulate the morphological organization and transcriptional identity of fore- and midbrain including spatial organizer regions such as the midbrain-hindbrain boundary. These anterior tissues develop in synchrony with posterior tissues such as the spinal cord, somites, and gut endoderm derivatives, ultimately yielding a unified structure. We show via genetic, environmental, and pharmacological perturbations that timed hypoxia is essential to boost anterior neural cell identities and their patterning through HIF1a and in part by modulating TGFβ activity. These results underline the key beneficial role of hypoxia in early development and offer a uniquely modular system to investigate antero-posterior phenotypes for basic discovery and translation.

Transcription factors (TFs) control specificity and activity of gene transcription, but whether a relationship between these two features exists is unclear. Here we provide evidence for an evolutionary trade-off between the activity and specificity in human TFs encoded as submaximal dispersion of aromatic residues in their intrinsically disordered protein regions. We identified approximately 500 human TFs that encode short periodic blocks of aromatic residues in their intrinsically disordered regions, resembling imperfect prion-like sequences. Mutation of periodic aromatic residues reduced transcriptional activity, whereas increasing the aromatic dispersion of multiple human TFs enhanced transcriptional activity and reprogramming efficiency, promoted liquid–liquid phase separation in vitro and more promiscuous DNA binding in cells. Together with recent work on enhancer elements, these results suggest an important evolutionary role of suboptimal features in transcriptional control. We propose that rational engineering of amino acid features that alter phase separation may be a strategy to optimize TF-dependent processes, including cellular reprogramming.

X-chromosome inactivation (XCI) balances gene expression between the sexes in female mammals. Shortly after fertilization, upregulation of Xist RNA from one X chromosome initiates XCI, leading to chromosome-wide gene silencing. XCI is maintained in all cell types, except the germ line and the pluripotent state where XCI is reversed. The mechanisms triggering Xist upregulation have remained elusive. Here we identify GATA transcription factors as potent activators of Xist. Through a pooled CRISPR activation screen in murine embryonic stem cells, we demonstrate that GATA1, as well as other GATA transcription factors can drive ectopic Xist expression. Moreover, we describe GATA-responsive regulatory elements in the Xist locus bound by different GATA factors. Finally, we show that GATA factors are essential for XCI induction in mouse preimplantation embryos. Deletion of GATA1/4/6 or GATA-responsive Xist enhancers in mouse zygotes effectively prevents Xist upregulation. We propose that the activity or complete absence of various GATA family members controls initial Xist upregulation, XCI maintenance in extra-embryonic lineages and XCI reversal in the epiblast.

Chromophobe renal cell carcinoma (chRCC) accounts for approximately 5% of all renal cancers and around 30% of chRCC cases have mutations in TP53. chRCC is poorly supported by microvessels and has markably lower glucose uptake than clear cell RCC and papillary RCC. Currently, the metabolic status and mechanisms by which this tumor adapts to nutrient-poor microenvironments remain to be investigated. In this study, we performed proteome and metabolome profiling of chRCC tumors and adjacent kidney tissues and identified major metabolic alterations in chRCC tumors, including the classical Warburg effect, the downregulation of gluconeogenesis and amino acid metabolism, and the upregulation of protein degradation and endocytosis. chRCC cells depended on extracellular macromolecules as an amino acid source by activating endocytosis to sustain cell proliferation and survival. Inhibition of the phospholipase C gamma 2 (PLCG2)/inositol 1,4,5-trisphosphate (IP3)/Ca2+/protein kinase C (PKC) pathway significantly impaired the activation of endocytosis for amino acid uptakes into chRCC cells. In chRCC, whole-exome sequencing revealed that TP53 mutations were not related to expression of PLCG2 and activation of endocytosis. Our study provides novel perspectives on metabolic rewiring in chRCC and identifies the PLCG2/IP3/Ca2+/PKC axis as a potential therapeutic target in patients with chRCC.Significance:This study reveals macropinocytosis as an important process utilized by chRCC to gain extracellular nutrients in a p53-independent manner.

Live-cell fluorescence imaging showed that the DQ-Red BSA accumulated in UOK276 cells over time, providing evidence that macropinocytosis is an established process in chRCC cells to uptake macromolecules as nutrient source.

Supplementary figures and figure legends

Somatic nonsynonymous SNV and indels identified in our corhort.

Proteome profiling and a subsequent GSEA analysis of UOK276 cells supplemented with 1% amino acids in the presence or absence of 2.5% BSA versus a complete medium.