Polycomb group (PcG) proteins are major determinants of gene silencing and epigenetic memory in higher eukaryotes. Here, we systematically mapped the human PcG complexome using a robust affinity purification mass spectrometry approach. Our high-density protein interaction network uncovered a diverse range of PcG complexes. Moreover, our analysis identified PcG interactors linking them to the PcG system, thus providing insight into the molecular function of PcG complexes and mechanisms of recruitment to target genes. We identified two human PRC2 complexes and two PR-DUB deubiquitination complexes, which contain the O-linked N-acetylglucosamine transferase OGT1 and several transcription factors. Finally, genome-wide profiling of PR-DUB components indicated that the human PR-DUB and PRC1 complexes bind distinct sets of target genes, suggesting differential impact on cellular processes in mammals.

Regeneration of fragmented Drosophila imaginal discs occurs in an epimorphic manner involving local cell proliferation at the wound site. After disc fragmentation, cells at the wound site activate a restoration program through wound healing, regenerative cell proliferation, and repatterning of the tissue. However, the interplay of signaling cascades driving these early reprogramming steps is not well-understood. Here, we profiled the transcriptome of regenerating cells in the early phase within 24 h after wounding. We found that JAK/STAT signaling becomes activated at the wound site and promotes regenerative cell proliferation in cooperation with Wingless (Wg) signaling. In addition, we showed that the expression of Drosophila insulin-like peptide 8 (dilp8), which encodes a paracrine peptide to delay the onset of pupariation, is controlled by JAK/STAT signaling in early regenerating discs. Our findings suggest that JAK/STAT signaling plays a pivotal role in coordinating regenerative disc growth with organismal developmental timing.

In Drosophila, the sine oculis (so) gene is important for the development of the entire visual system, including Bolwig's organ, compound eyes and ocelli. Together with twin of eyeless, eyeless, eyes absent and dachshund, sobelongs to a network of genes that by complex interactions initiate eye development. Although much is known about the genetic interactions of the genes belonging to this retinal determination network, only a few such regulatory interactions have been analysed down to the level of DNA-protein interactions. Previous work in our laboratory identified an eye/ocellus specific enhancer of the sine oculis gene that is directly regulated by eyeless and twin of eyeless. We further characterized this regulatory element and identified a minimal enhancer fragment of so that sets up an autoregulatory feedback loop crucial for proper ocelli development. By systematic analysis of the DNA-binding specificity of so we identified the most important nucleotides for this interaction. Using the emerging consensus sequence for SO-DNA binding we performed a genome-wide search and have thereby been able to identify eyeless as well as the signalling gene hedgehog as putative targets of so. Our results strengthen the general assumption that feedback loops among the genes of the retinal determination network are crucial for proper development of eyes and ocelli.

Self-organizing neural organoids grown from pluripotent stem cells1–3 combined with single-cell genomic technologies provide opportunities to examine gene regulatory networks underlying human brain development. Here we acquire single-cell transcriptome and accessible chromatin data over a dense time course in human organoids covering neuroepithelial formation, patterning, brain regionalization and neurogenesis, and identify temporally dynamic and brain-region-specific regulatory regions. We developed Pando—a flexible framework that incorporates multi-omic data and predictions of transcription-factor-binding sites to infer a global gene regulatory network describing organoid development. We use pooled genetic perturbation with single-cell transcriptome readout to assess transcription factor requirement for cell fate and state regulation in organoids. We find that certain factors regulate the abundance of cell fates, whereas other factors affect neuronal cell states after differentiation. We show that the transcription factor GLI3 is required for cortical fate establishment in humans, recapitulating previous research performed in mammalian model systems. We measure transcriptome and chromatin accessibility in normal or GLI3-perturbed cells and identify two distinct GLI3 regulomes that are central to telencephalic fate decisions: one regulating dorsoventral patterning with HES4/5 as direct GLI3 targets, and one controlling ganglionic eminence diversification later in development. Together, we provide a framework for how human model systems and single-cell technologies can be leveraged to reconstruct human developmental biology.