Kind: Developmental epigenomics Back to research group The Kind group uses a combination of single-cell genomics and microscopy methods to study the role of chromatin and epigenetics in gene-regulation control, with a focus on early mouse embryonic development and tumorigenesis. The aim of the lab is to understand the principles governing cellular decision-making. We are interested in how cells acquire new identities and traits in lineage specification events in mice and in cancer. To this end we employ and continuously develop novel single-cell technologies to delineate these processes with high sensitivity and accuracy. Key words: Single-cell genomics, epigenetics, spatial genome organization, bioinformatics, gene-regulation, embryogenesis, tumorigenesis Technology development We have developed scDam&T, a method to simultaneously capture protein-DNA interactions and transcriptomes in the same cell (Rooijers et al, 2019, Nature Biotechnology) (Figure 1). This method enables linking transcriptional variability to epigenetic heterogeneity. In addition, scDam&T allows determining cellular identity of cells in complex tissues -such as embryos or tumors- based on transcriptomics, in direct relation to their epigenetic regulatory landscapes (DamID). We are employing scDam&T to obtain a better understanding of the epigenetic mechanisms underlying lineage decisions in early embryogenesis, to identify the epigenetic trajectories of single cells that acquire malignant identities in tumorigenesis and to obtain insight into the role of chromatin in DNA double-strand repair. Furthermore, as we consider technology development a core business of the group, we are continuously exploring ways to obtain richer single-cell datasets. Figure 1: scDamID&T enables capturing protein-DNA interactions and transcriptomics from the same cell. With this approach dynamic processes can be studied, such as linking heterogeneity in spatial genome positioning (LADs) to variations in gene-expression patterns and the epigenetic principles that govern random X-inactivation. Credit: Jop Kind, copyright Hubrecht Institute. Mechanisms underlying cell-fate choice in early embryogenesis A major interest in the group is how gene-expression control is achieved in early embryogenesis. An important contributor to the regulation of gene expression is the spatial positioning of genes within the 3-dimensional space of the nucleus. Chromosomal regions that associate with a thin filamentous lamina layer at the nuclear periphery are generally in an inactive transcriptional state (Guerreiro and Kind, 2019, Curr.Opin.Gen.Dev.). We profiled these genome-lamina interactions in single cells to obtain better understanding in gene-regulation control in pre- and post-implantation embryos (Borsos et al., 2019, Nature) (Figure 2). In addition, we profile the epigenetic landscapes, chromatin accessibility and transcriptome in single cells. We use this approach to understand how gene-expression is controlled and to obtain insight into the regulatory mechanisms that govern cellular decision making in mouse early embryogenesis. Figure 2: Examples of single-cell interaction profiles of the genome with the nuclear lamina (LADs) in three 2-cell stage daughter pairs. These data are obtained by mRNA microinjections of DamID constructs. Credit: Isabel Guerreiro and Jop Kind, copyright Hubrecht Institute. Cell-fate decisions and the role of epigenetics in DNA double-strand repair Accurate repair of DNA damage is critical for the maintenance of genomic integrity. Despite extensive research into the molecular pathways that sense and repair DNA double-strand breaks (DSBs), comparatively little is known about management of breaks within different genomic and chromatin contexts. This is particularly difficult to study because DSBs occur randomly in the genome, thus every single cell harbors a different DSB-landscape. To study DSB-repair in single cells, we have adapted scDam&T to generate maps of DSB-repair sites in single cells. Further, in the same cells, we also profile genome-wide chromatin states with a chromatin immunocleavage (ChIC) approach to study the relationships between DSB-repair and the local chromatin environment (Figure 3). Because DamID-signals are stable and can be carried-over for multiple generations, we are also looking into ways to exploit this property of DamID to study DSB-repair in the context of cellular decision making. Figure 3: Examples of combined single-cell profiles of RAD51 (marking DNA double-strand break (DSB) repair sites) and H3K36me3 (marking active gene regions). The DSBs are obtained by temporal induction of the restriction enzyme AsiSI. Credit: Kim de Luca, copyright Hubrecht Institute. Tumor-cell trajectories in organoid and mouse models Tumorigenesis involves the continuous selective adaptation of cells from healthy tissue, to adenoma, carcinoma and eventually metastasis. To obtain detailed insight into this trajectory, it is essential to capture all the stages with single-cell accuracy. We are particularly interested in understanding the role of epigenetics and spatial genome organization in this process. We employ our single-cell genomic methods to study cellular trajectories on route towards malignant metastatic identities. To achieve this, we make use of organoid and mouse models that capture tumorigenesis all the way from the initiating stages towards metastasis.