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Genome-wide Transcription Factor Binding Maps Reveal Cell-specific Changes in the Regulatory Architecture of Human HSPCs
Julie A.I. Thoms1,* , Shruthi Subramanian2,*, Yizhou Huang3, Paola Cornejo4, Forrest C. Koch5, Sebastien Jacquelin6, Sylvie Shen7, Emma Song7, Swapna Joshi2, Chris Brownlee8, Petter S. Woll9, Diego Chacon Fajardo3, Dominik Beck3, David J. Curtis10, Kenneth Yehson11, Vicki Antonenas11, Tracey O' Brien12, Annette Trickett7, Jason A. Powell13,14, Ian D. Lewis13, Stuart M. Pitson13, Maher K. Gandhi15, Steven W. Lane16, Fatemeh Vafaee5,17, Emily S. Wong4,5, Berthold Göttgens18, Hamid Alinejad Rokny19, Jason W.H Wong20 and John E. Pimanda1,2,211School of Biomedical Sciences, UNSW Sydney, Australia
2School of Clinical Medicine, UNSW Sydney, Australia
3Centre for Health Technologies and the School of Biomedical Engineering, University of Technology Sydney, Australia
4Victor Chang Cardiac Research Institute, Sydney, Australia
5School of Biotechnology and Biomolecular Sciences, Faculty of Science, UNSW Sydney, Australia
6Macrophage Biology Laboratory, Mater Research, Brisbane, Australia
7Bone Marrow Transplant Lab, NSW Health Pathology, Prince of Wales Hospital, Sydney, Australia
8Mark Wainwright Analytical Centre, UNSW Sydney, Australia
9Center for Hematology and Regenerative Medicine, Department of Medicine (MedH), Karolinska Institutet, Huddinge, Sweden
10Australian Centre for Blood Diseases, Monash University, Melbourne, Australia
11Blood Transplant and Cell Therapies Laboratory, NSW Health Pathology, Westmead, Australia
12Sydney Children's Hospital, Sydney, Australia
13Centre for Cancer Biology, SA Pathology, University of South Australia, Australia
14Adelaide Medical School, University of Adelaide, Australia
15Blood Cancer Research Group, Mater Research, University of Queensland, Australia
16Cancer Program, QIMR Berghofer Medical Research, Brisbane, Australia
17UNSW Data Science Hub, UNSW Sydney, Australia
18Wellcome - MRC Cambridge Stem Cell Institute, Cambridge, United Kingdom
19BioMedical Machine Learning Lab, Graduate School of Biomedical Engineering, UNSW Sydney, Australia
20School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region, China
21Haematology Department, Prince of Wales Hospital, Sydney, Australia
*Equal contributions
Background/Aims: Dynamic remodeling of gene regulatory networks (GRN) establishes and maintains gene expression and cell identity during hematopoiesis, and dysregulation of hematopoietic transcription factors (TFs) is a key mechanism in leukemia development. However, the sequential changes that occur as cells transit from multipotent stem cells to lineage restricted progenitors remain poorly understood. We previously described combinatorial binding of seven TFs (heptad: FLI1, ERG, GATA2, RUNX1, TAL1, LYL1 and LMO2) which form an autoregulatory network in bulk human CD34+ hematopoietic stem and progenitor cells (HSPC). However, whether heptad-centered regulatory networks are remodeled, and whether specific heptad-TF combinations have distinct roles across the differentiation trajectory, remained unknown.
Methods: To resolve these questions, we sorted apheresis-derived CD34+ LIN- cells into hematopoietic stem cells (HSC: CD38loCD45RA-), common myeloid progenitors (CMP: CD38hiCD45RA-CD123+), granulocyte monocyte progenitors (GMP: CD38hiCD45RA+CD123+) and megakaryocyte erythroid progenitors (MEP: CD38hiCD45RA-CD123-), then mapped genome-wide chromatin contacts (HiC, H3K27ac HiChIP), chromatin modifications (H3K4me3, H3K27ac, H3K27me3) and binding profiles of 10 TFs (heptad, PU.1, CTCF, and STAG2) in each cell type.
Results: Combinatorial binding of heptad TFs was observed in all cell types, with the most overrepresented pattern being co-binding of all seven factors. However, chromatin occupancy was highly variable across cell types and some TF combinations were observed in restricted cell types or associated with lineage specific gene promoters. Promoter looping patterns were dynamic across cell types, although high order genome architecture was mostly conserved, and we found multiple distal regions with specific patterns of TF binding connected to each heptad promoter. Many of these distal regions were previously undescribed and may constitute novel regulators of heptad genes. We next used HiChIP data to link promoters to candidate distal regulatory regions and focused on promoter-regulator pairs with differential heptad binding across the cell types. Distinct regulatory elements were enriched with specific heptad-TF combinations, including stem-cell-specific elements with ERG, and myeloid- and erythroid-specific elements with combinations of FLI1, RUNX1, GATA2, TAL1, LYL1, and LMO2. Furthermore, heptad occupancy in progenitors primed specific regulatory elements for subsequent binding of lineage defining TFs such as PU.1 and GATA1. Finally, we found that enhancers with cell-type-specific heptad occupancy shared a common grammar with respect to TF binding motifs, suggesting that combinatorial binding of specific TF complexes was at least partially regulated by features encoded in specific DNA sequence motifs. Taken together our data strongly supports a mechanistic link between differential heptad TF binding and cell type specific expression patterns and provides insight into how expression of multiple genes is coordinated during cell state transitions.
Conclusions: Overall, this study provides a comprehensive characterization of the gene regulatory landscape in rare subpopulations of human HSPCs and will be a key resource for understanding adult hematopoiesis. Importantly, our work also provides a framework for analyzing aberrant regulatory networks in leukemic cells and leveraging these to devise novel therapeutic strategies.
This work was supported by the Anthony Rothe Memorial Trust (JAIT, JEP), Wellcome #206328/Z/17/Z (BG), the National Health and Medical Research Council of Australia #GNT1139787, GNT2011627, MRF1200271 (JEP), and the Leukemia Lymphoma Society (LLS)-Snowdome Foundation-Leukemia Foundation #6620-21 (JEP).