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.

The homeobox gene orthodenticle (otd) controls the process of regional specification that takes place in the Drosophila eye-antennal disc during ocelli development. Mutations that reduce or abolish otd expression in the ocelli primordium give rise to ocelliless flies. We have identified the cis-regulatory sequence (ocelliless enhancer) that controls otd expression during ocelli development and studied its regulation at the molecular level. The ocelliless enhancer is initially activated by the combined action of Wingless (Wg) and Hedgehog (Hh) signaling pathways. Later, a positive autoregulatory feedback loop sets in to maintain otd expression. Moreover, we have analyzed the role of otd during ocelli primordium development and determined its involvement in the expression of the retinal determination gene eyes absent (eya). otd indirectly regulates eya in ocellar precursor cells through the inhibition of wg, an eya repressor, and the maintenance of hh expression in the ocelli primordium. Hh signaling is necessary for eya activation in ocellar precursor cells and this activation is mediated by the full-length activator form of the transcription factor Cubitus interruptus.

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.

Pax6 genes encode transcription factors with two DNA-binding domains that are highly conserved during evolution. In Drosophila,two Pax6 genes function in a pathway in which twin of eyeless (toy) directly regulates eyeless (ey),which is necessary for initiating the eye developmental pathway. To investigate the gene duplication of Pax6 that occurred in holometabolous insects like Drosophila and silkworm, we used different truncated forms of toy and small eyes(sey), and tested their capacity to induce ectopic eye development in an ey-independent manner. Even though the Paired domains of TOY and SEY have DNA-binding properties that differ from those of the Paired domain of EY, they all are capable of inducing ectopic eye development in an eymutant background. We also show that one of the main functional differences between toy and ey lies in the C-terminal region of their protein products, implying differences in their transactivation potential. Furthermore, we show that only the homeodomain (HD) of EY is able to downregulate the expression of Distal-less (Dll), a feature that is required during endogenous eye development. These results suggest distinct functions of the two DNA-binding domains of TOY and EY, and significant evolutionary divergence between the two Drosophila Pax6genes.

The porifera represent the most primitive phylum of the metazoa. We identified three homeoboxcontaining genes in the freshwater sponge (Ephydatia jluviatilis). Genomic DNA of the sponge was subjected to amplification by PCR with two primers that corresponded to the helix-1 and helix-3 regions of the homeodomain. Using the amplified products as probes, we isolated two homeobox genes, designated proxl and prox2. The amino acid sequences of the homeodomains of proxl and prox2 were 72% and 62% identical to those of the NK-3 and Om(1 D) genes of Drosophita, respectively. Screening of a sponge genomic library with degenerate oligonucleotides that corresponded to helix 3 further revealed the presence of one more homeobox gene, prox3. The amino acid sequence of the homeodomain of the p o x 3 product was 77% identical to that of the msh gene product of human. These results indicate that, when the metazoa appeared during the course of evolution, the multiple and distinct classes of homeobox-containing genes that have been identified in higher organisms already existedThe homeobox was first identified as a conserved sequence that was observed in several homeotic and segmental genes in Drosophila melanogaster [l, 21. It was later found that the homeodomains encoded by homeoboxes are one of the major types of structural motif that can bind to DNA in a sequence-specific manner and that they are present in large numbers of transcription factors from various organisms [3, 41. From their similarities in sequence, the homeodomains have been divided into more than 20 classes [3, 51. Since, in each class of homeodomain, the homeodomains of Drosophila and mammals are more than 80% identical in terms of amino acid residues, it is quite likely that representatives of each class of homeodomains were established before the divergence of arthropods and vertebrates in evolution [3, Two kinds of method have been developed to identify homeobox genes in lower classes of metazoa. Burglin et al.[ 121 synthesized 500 -2000-fold degenerate oligonucleotides that corresponded to a set of well-conserved sequences of eight amino acid residues from the helix-3 region of homeodomains. They screened Caenorhabditis elegans genomic libraries with these probes and identified 49 putative homeobox genes. This approach has been successfully utilized for identification of homeobox genes in more primitive animals, such as the planarian, hydra and flatworm [13-1.51. Another approach that involved the polymerase chain reaction (PCR) was also adopted to identify homeobox genes in primitive animals [16, 171. In result in alterations in leaf development, was the first to be shown to contain a homeobox [18]. Several groups [19-211 isolated homeobox genes from the plant Arabidopsis thaliana using the same set of degenerate oligonucleotide probes as those used by Burglin et al. [12]. Bellmann and Werr [22] isolated a novel maize homeobox gene using an expression vector and a control element. In fungi including yeast, several regulatory proteins involved in the reg...

Polycomb group (PcG) proteins are major determinants of gene silencing and epigenetic 1 memory in higher eukaryotes. Here, we used a robust affinity purification mass spec-2 trometry (AP-MS) approach to systematically map the human PcG protein interactome, 3 uncovering an unprecedented breadth of PcG complexes. The obtained high density 4 protein interaction data identified new modes of combinatorial PcG complex formation 5 with proteins previously not associated with the PcG system, thus providing new insights 6 into their molecular function and recruitment mechanisms to target genes. Importantly, 7 we identified two human PR-DUB de-ubiquitination complexes, which comprise the O-8 linked N-acetylglucosamine transferase OGT1 and a number of transcription factors. By 9further mapping chromatin binding of PR-DUB components genome-wide, we conclude 10 that the human PR-DUB and PRC1 complexes bind distinct sets of target genes and 11 impact on different cellular processes in mammals. 12 159 WD40 domain proteins DCAF7 and WDR5 are central scaffold-160 ing proteins for PRC1.3/PRC1.5 and PRC1.6 161The WD40 domain protein DCAF7 has been implicated in skin development and cell 162 proliferation by interacting with DIAP1 and the dual-specificity tyrosine phosphorylation-163 regulated kinase DYR1A 53,54 . Intriguingly, DCAF7 co-purified with CBX4/6/8, RING1/2, 164 RYBP/YAF2 and PCGF3/5/6, indicating that the protein is deeply embedded in the 165