Bertie Gottgens

Cell of Origin in AML: Susceptibility to MN1-Induced Transformation Is Regulated by the MEIS1/AbdB-like HOX Protein Complex

Pathways defining susceptibility of normal cells to oncogenic transformation may be valuable therapeutic targets. We characterized the cell of origin and its critical pathways in MN1-induced leukemias. Common myeloid (CMP) but not granulocyte-macrophage progenitors (GMP) could be transformed by MN1. Complementation studies of CMP-signature genes in GMPs demonstrated that MN1-leukemogenicity required the MEIS1/AbdB-like HOX-protein complex. ChIP-sequencing identified common target genes of MN1 and MEIS1 and demonstrated identical binding sites for a large proportion of their chromatin targets. Transcriptional repression of MEIS1 targets in established MN1 leukemias demonstrated antileukemic activity. As MN1 relies on but cannot activate expression of MEIS1/AbdB-like HOX proteins, transcriptional activity of these genes determines cellular susceptibility to MN1-induced transformation and may represent a promising therapeutic target.

Growth Factor Independence 1 Antagonizes a p53-Induced DNA Damage Response Pathway in Lymphoblastic Leukemia

Most patients with acute lymphoblastic leukemia (ALL) fail current treatments highlighting the need for better therapies. Because oncogenic signaling activates a p53-dependent DNA damage response and apoptosis, leukemic cells must devise appropriate countermeasures. We show here that growth factor independence 1 (Gfi1) can serve such a function because Gfi1 ablation exacerbates p53 responses and lowers the threshold for p53-induced cell death. Specifically, Gfi1 restricts p53 activity and expression of proapoptotic p53 targets such as Bax, Noxa (Pmaip1), and Puma (Bbc3). Subsequently, Gfi1 ablation cures mice from leukemia and limits the expansion of primary human T-ALL xenografts in mice. This suggests that targeting Gfi1 could improve the prognosis of patients with T-ALL or other lymphoid leukemias.

RUNX1 regulates the CD34 gene in haematopoietic stem cells by mediating interactions with a distal regulatory element

The transcription factor RUNX1 is essential to establish the haematopoietic gene expression programme; however, the mechanism of how it activates transcription of haematopoietic stem cell (HSC) genes is still elusive. Here, we obtained novel insights into RUNX1 function by studying regulation of the human CD34 gene, which is expressed in HSCs. Using transgenic mice carrying human CD34 PAC constructs, we identified a novel downstream regulatory element (DRE), which is bound by RUNX1 and is necessary for human CD34 expression in long‐term (LT)‐HSCs. Conditional deletion of Runx1 in mice harbouring human CD34 promoter–DRE constructs abrogates human CD34 expression. We demonstrate by chromosome conformation capture assays in LT‐HSCs that the DRE physically interacts with the human CD34 promoter. Targeted mutagenesis of RUNX binding sites leads to perturbation of this interaction and decreased human CD34 expression in LT‐HSCs. Overall, our in vivo data provide novel evidence about the role of RUNX1 in mediating interactions between distal and proximal elements of the HSC gene CD34.

Graph abstraction reconciles clustering with trajectory inference through a topology preserving map of single cells

Single-cell RNA-seq quantifies biological heterogeneity across both discrete cell types and continuous cell transitions. Partition-based graph abstraction (PAGA) provides an interpretable graph-like map of the arising data manifold, based on estimating connectivity of manifold partitions (https://github.com/theislab/paga). PAGA maps provide interpretable discrete and continuous latent coordinates for both disconnected and continuous structure in data, preserve the global topology of data, allow analyzing data at different resolutions and result in much higher computational efficiency of the typical exploratory data analysis workflow — one million cells take on the order of a minute, a speedup of 130 times compared to UMAP. We demonstrate the method by inferring structure-rich cell maps with consistent topology across four hematopoietic datasets, confirm the reconstruction of lineage relations of adult planaria and the zebrafish embryo, benchmark computational performance on a neuronal dataset and detect a biological trajectory in one deep-learning processed image dataset.

Establishment In Culture Of Expanded Potential Stem Cells

Mouse embryonic stem cells are derived from in vitro explantation of blastocyst epiblasts1,2 and contribute to both the somatic lineage and germline when returned to the blastocyst3 but are normally excluded from the trophoblast lineage and primitive endoderm4–6. Here, we report that cultures of expanded potential stem cells (EPSCs) can be established from individual blastomeres, by direct conversion of mouse embryonic stem cells (ESCs) and by genetically reprogramming somatic cells. Remarkably, a single EPSC contributes to the embryo proper and placenta trophoblasts in chimeras. Critically, culturing EPSCs in a trophoblast stem cell (TSC) culture condition permits direct establishment of TSC lines without genetic modification. Molecular analyses including single cell RNA-seq reveal that EPSCs share cardinal pluripotency features with ESCs but have an enriched blastomere transcriptomic signature and a dynamic DNA methylome. These proof-of-concept results open up the possibility of establishing cultures of similar stem cells in other mammalian species.

211 Characterising functional heterogeneity in human epicardium

Introduction The epicardium is a heterogeneous cell layer covering the mammalian heart. During embryogenesis, the epicardial lineage is essential to heart and vascular development, yielding cardiac fibroblasts and coronary vascular smooth muscle cells. The epicardium also has a trophic effect on developing cardiomyocytes. It is quiescent in adulthood but reactivates post-injury to a limited degree to yield cardiac fibroblasts, which allows fast healing, yet also causes fibrosis. Epicardial functional heterogeneity remains incompletely characterised. Methods The Sinha group has derived a robust model of human epicardium (hpsc-epi) from human pluripotent stem cells; this was used for single-cell RNA sequencing (scRNA-seq). Immunohistochemistry and immunocytochemistry were used to validate scRNA-seq results in primary human foetal tissue. The candidate gene BNC1 has been investigated by siRNA-mediated knockdown studies in vitro. Results Single-cell RNA-seq identified two main epicardial subpopulations in hpsc-epi: WT1high/BNC1high/TCF21low and WT1low/BNC1low/TCF21high. Here we show validation of our scRNA-seq data in human foetal epicardium by immunohistochemistry in cryosections and human foetal epicardial explants, confirming our hpsc-epi model is representative of the in vivo situation. We show preliminary data from siRNA-mediated knockdown of BNC1, which indicate this gene may play a role in epicardial function, possibly in regulating cell migration in a model of epithelial-to-mesenchymal transition. Implications Improved understanding of developmental epicardial regulation could pave the way towards harnessing epicardial potential in prospective strategies to aid revascularisation and regeneration of the injured heart.

Integrated analyses of chromatin accessibility and gene expression data for elucidating the transcriptional regulatory mechanisms during early hematopoietic development in mouse

The regulation of gene expression is a key factor influencing cell fate decisions during differentiation. The molecular mechanisms underlying gene regulation are multi-layered, with regulatory inputs traversing both within and across different layers, thus providing a very difficult system to decipher. Although the gene expression patterns are cell-type specific, the regulatory inputs follow similar fundamental guidelines. One basic and easily interpretable transcriptional control layer is chromatin accessibility which is a consequence of combinatorial binding of transcription factors (TFs) and various chromatin modifications. Hematopoiesis is a well-characterized system for understanding transcriptional control mechanisms. The differentiation of hemogenic endothelium (HE) to hematopoietic progenitor cells (HP) forms a crucial step in hematopoiesis as it marks the exclusive commitment of bi-potent stem cells capable of following both hematopoietic and endothelial lineages, to hematopoietic lineages. In this work, we have performed a genome-wide analysis of chromatin accessibility dynamics in the differentiation of HE to HP and subsequently correlated them to gene expression dynamics for comprehending the basic regulatory themes. Chromatin accessibility was examined by global DNasel Hypersensitivity Sites (DHS) mapping during the differentiation, in addition to measuring gene expression by RNA-Seq for HE and HP. Accessible regions unique to HE and HP, and common peaks were identified. In general, there was a direct correlation between the DHS fold change and the expression fold change of the nearest genes for the corresponding peaks, with peaks near promoter regions showing a considerably higher degree of correlation than the rest of the peak populations. In further analyses, we applied a range of bioinformatics approaches such as peak overlap analysis, de novo motif discovery, and gene ontology analysis with the ultimate aim to identify the factors influencing lineage commitment. To this end, we have formulated a novel cluster-based approach for studying motif co-occurrence to obtain potential combinatorial TF binding regions, and by correlating those with the expression of the TFs we have systematically explored the relationship between the changes in chromatin accessibility and gene expression dynamics. We are presently developing mathematical models that integrate chromatin accessibility, chromatin modifications and transcription factors to predict gene expression patterns specific for a given cellular differentiation state.

System-wide studies of the transcriptional programming of chromatin during early hematopoietic development

Materials and methods Here we use in vitro differentiation of pluripotent mouse embryonic stem (ES) cells to macrophages as a model to identify molecular mechanisms and dynamics of transcriptional programs that define the individual cellular identities. We analyze the chromatin landscape and gene expression of the following five successive cell types that are purified by FACS according to their specific surface marker profiles: mesoderm, hemangioblast, hemogenic endothelium, hematopoietic precursors and macrophages. Genome-wide transcriptional activity of the individual cell populations was assessed by RNA sequencing and the distribution of histone modifications associated with either active or repressed regulatory elements was investigated by ChIP sequencing. Moreover, general DNA accessibility, i.e. open chromatin structure, was detected by DNase 1 digestion and subsequent genomewide sequencing.