key: cord-0315629-5f171dyg authors: Li, Kun; Wu, Yang; Li, Young; Yu, Qiaoni; Tian, Zhigang; Wei, Haiming; Qu, Kun title: Landscape and dynamics of the transcriptional regulatory network during natural killer cell differentiation date: 2019-03-10 journal: bioRxiv DOI: 10.1101/572768 sha: 829ba7eef269c1e5ff6dbf72bfe5a218fc30644e doc_id: 315629 cord_uid: 5f171dyg Natural killer (NK) cells are essential in controlling cancer and infection. However, little is known about the dynamics of the transcriptional regulatory machinery during NK cell differentiation. In this study, we applied assay of transposase accessible chromatin with sequencing (ATAC-seq) technique in a self-developed in vitro NK cell differentiation system. Analysis of ATAC-seq data illustrated two distinct transcription factor (TF) clusters that dynamically regulate NK cell differentiation. Moreover, two TFs from the second cluster, FOSL2 and EGR2, were identified as novel essential TFs that control NK cell maturation and function. Knocking down either of these two TFs significantly impacted NK cell transformation. Finally, we constructed a genome-wide transcriptional regulatory network that provides an understanding of the regulatory dynamics during NK cell differentiation. Introduction 47 significance ( Figure 2B ,10 -8 1.5) (Figure S7) , 306 and defined them as the nodes of the regulatory network. The connections (edges) between any 2 TFs were defined as follows: If the TF A motif is on the promoter of TF B, then we say TF A 308 regulates TF B and draw an arrow from TF A to TF B. Here, only TFs that were expressed at the 309 specific time point were consideration [58] . Using this method, we constructed the transcriptional 310 regulatory network at each time points with both the enrichment (P-value) and expression 311 information for all the relevant TFs (Figure 5A-E) . Interestingly, day 7 specific TFs were densely 312 interconnected at the beginning, and quickly vanished after two weeks ( Figure 5A ). In contrast, 313 the day 35 specific network gradually grew out through the induction of relevant TFs. Many known 314 regulators, such as EOMES, TBX21, ETS1, PRDM1, and GATA3 and also FOLS2 and EGR2 315 were increasingly enriched on the network (Figure 5E ). Similar phenomena were also observed 316 on other networks (Figure 5B-D) . We believe the dynamics of the transcriptional regulatory 317 network explain the increase in the proportion and the differentiation of NK cells. In short, this study provides an epigenomic landscape and dynamics of NK cell differentiation and 328 presents foundational profiles for studying the relationship between chromatin accessibility, gene 329 expression and cell growth during this process (Figure 2) . 330 TFs bind to their motifs and are often obligate nucleosome evictors and the creators of 331 accessible DNA sites, and therefore we can use ATAC-seq to predict critical regulators in NK cell 332 differentiation [26] . By motif analysis in HOMER, we found that several known TFs were enriched 333 at different stages during NK cell differentiation. Similar results were also obtained using 334 Genomica ( Figure 3B) . The discovery of known regulators strongly suggested the reliability of 335 our analysis Furthermore, by integrating results from HOMER, Genomica and motif footprint 336 analysis, we identified two novel TFs, FOSL2 and EGR2 that were essential for NK cell maturation. 337 Knockdown of either of these two TFs significantly inhibited NK cell transformation in the in vitro 338 NK cell differentiation system. Module map analysis suggested these two TFs may regulate NK 339 cell through the JAK-STAT pathway, and therefore further studies of this pathway may facilitate 340 the generation of NK cells and thus promote the NK cell-based immunotherapy. Overall, this study 341 also provided a framework to identify new regulators from chromatin accessible data for NK cell 342 TFs do not usually function alone, they always interact with other molecules to fulfill their 344 unique roles. Hence, we depicted the transcriptional regulatory networks at different stages during 345 NK cell differentiation. In order to construct a stable transcriptional regulatory network, we 346 performed a rigorous screening of TFs to avoid stochastic fluctuations, and integrated both the 347 enrichment (P-value) and expression information for all the relevant TFs. Therefore, even a change 348 of either the enrichment or gene expression cutoff may result in different networks, the most 349 critical TFs to the regulatory process still remain. However, since the screening of TFs mainly rely 350 on the gene microarray, which is not as accurate as RNA-seq, the structure of these predicted 351 network may not as robust as well. 352 From our previous study, we noticed that with a minimal cytokine cocktail, we can generate 353 sufficient number of functional NK cells that express the cytokines necessary for NK cells and and then, the lentivirus particles were concentrated by ultracentrifugation at 50000 g for 2 h at 4°C. 396 Finally, the virus particles were gently resuspended in HBSS and stored at -80°C. After UCB 397 CD34 + cells were cultured with multiple cytokines for 14-18 d, we incubated the lentivirus and the 398 cultured cells with polybrene (5 µg/ml) and centrifuged them at 1000 rpm for 70 min at 10°C. values less than 0.05 were considered statistically significant. 401 Gene Expression Array. The signal values of the samples were normalized using RMA. 403 Differential analysis was performed using Student's t-test as previously described [19] . The Stage-specific peaks: Each stage (e.g., day 7) consists of genes that were induced (>1.5-fold 417 change) at that stage compared with all the other stages. We defined peaks that were accessible 418 only in one stage as stage-specific peaks, and those that were accessible at all stages as 419 conservative peaks. TFs that regulates NK cell development, the pink box indicates the new TF whose function will 672 be experimentally tested later in Figure 4 . TCF3 TTK TGIF1 THRA TGIF2 ZNF711 MYCN IRF4 MAZ NFATC1 MYC MEF2C TAL1 SOX4 PBX1 RBFOX2 NFE2 SPI1 TGIF2 ERG TGIF1 GFI1B MEF2C THRA MYC TTK MAZ IRF4 ZNF711 TCF3 TAL1 SOX4 MYCN PBX1 SPI1 NFATC1 NFE2 TCF4 RBFOX2 MAZ MEF2C TGIF1 THRA IRF4 MYC ERG TTK ZNF711 GFI1B TGIF2 NFE2 SOX4 TCF4 TAL1 PBX1 NFATC1 SPI1 TCF3 MYCN RBFOX2 MAZ TGIF1 ZNF711 MYC THRA TTK GFI1B IRF4 MEF2C ERG TGIF2 PBX1 NFATC1 TCF4 SOX4 TAL1 SPI1 NFE2 TCF3 MYCN RBFOX2 PBX1 SOX4 TCF4 TAL1 NFATC1 RBFOX2 SPI1 MYCN TCF3 NFE2 ERG TGIF2 MAZ ZNF711 GFI1B THRA TGIF1 TTK MEF2C MYC IRF4 NK 505 cells differentiated from bone marrow, cord blood and peripheral blood stem cells exhibit similar 506 phenotype and functions Enhancement of 508 human cord blood CD34+ cell-derived NK cell cytotoxicity by dendritic cells Combined IL-15 and IL-12 drives the generation of CD34(+)-derived natural killer cells with 512 superior maturation and alloreactivity potential following adoptive transfer Programmed differentiated natural killer 515 cells kill leukemia cells by engaging SLAM family receptors Differential requirement 517 for the transcription factor PU.1 in the generation of natural killer cells versus B and T cells The transcription factors T-bet 520 and Eomes control key checkpoints of natural killer cell maturation Transposition of native 522 chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins 523 and nucleosome position The epigenetic 525 landscape of T cell exhaustion Epigenetic 527 stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade Nfib Promotes 530 Metastasis through a Widespread Increase in Chromatin Accessibility Individuality and variation of 532 personal regulomes in primary human T cells Chromatin Accessibility 534 Landscape of Cutaneous T Cell Lymphoma and Dynamic Response to HDAC Inhibitors Epigenetic control 537 of innate and adaptive immune memory Progress report on Safe 539 VISITOR: approaching a practical instrument for terahertz security screening. Passive Millimeter-540 Wave Imaging Technology Xiii Location and cellular stages of natural killer cell development Human 544 NK cell receptors/markers: a tool to analyze NK cell development, subsets and function Molecular portraits 547 of human breast tumours GREAT improves 549 functional interpretation of cis-regulatory regions Heterochromatin and epigenetic control of gene expression Runx proteins are involved 553 in regulation of CD122, Ly49 family and IFN-gamma expression during NK cell differentiation CCAAT/enhancer binding proteins are critical components of the transcriptional regulation of 557 hematopoiesis (Review) 1 expression 559 delineates heterogeneity in primary Th2 cells PU.1 regulates the commitment 561 of adult hematopoietic progenitors and restricts granulopoiesis Dynamic regulation of PU.1 expression in 563 multipotent hematopoietic progenitors The transcription factor 565 PU.1 controls dendritic cell development and Flt3 cytokine receptor expression in a dose-566 dependent manner Distinctive 568 and indispensable roles of PU.1 in maintenance of hematopoietic stem cells and their 569 differentiation Mature natural killer cell and lymphoid tissue-571 inducing cell development requires Id2-mediated suppression of E protein activity A novel Ncr1-Cre 574 mouse reveals the essential role of STAT5 for NK-cell survival and development RUNX proteins in transcription factor networks that 577 regulate T-cell lineage choice JASPAR 2016: a major 579 expansion and update of the open-access database of transcription factor binding profiles The JAK-STAT 582 pathway: impact on human disease and therapeutic intervention The IRF family transcription factors in 584 immunity and oncogenesis IRF family of transcription factors as 586 regulators of host defense Deficiency in 588 the transcription factor interferon regulatory factor (IRF)-2 leads to severely compromised 589 development of natural killer and T helper type 1 cells IFN Regulatory Factor-2 Deficiency 591 Revealed a Novel Checkpoint Critical for the Generation of Peripheral NK Cells A novel transcription 594 factor, T-bet, directs Th1 lineage commitment T-bet 596 regulates the terminal maturation and homeostasis of NK and V alpha 14i NKT cells Gene 599 deregulation and chronic activation in natural killer cells deficient in the transcription factor ETS1 GATA-3 602 promotes maturation, IFN-gamma production, and liver-specific homing of NK cells SOX4 is a direct target gene of FRA-605 2 and induces expression of HDAC8 in adult T-cell leukemia/lymphoma The transcription factors Egr2 608 and Egr3 are essential for the control of inflammation and antigen-induced proliferation of B and 609 T cells Roles of LAG3 and EGR2 in regulatory T 611 cells Densely interconnected transcriptional circuits control cell states in human hematopoiesis Emerging natural killer cell immunotherapies: large-scale ex vivo 616 production of highly potent anticancer effectors New aspects of natural-killer-cell surveillance 618 and therapy of cancer Expansion of highly 620 cytotoxic human natural killer cells for cancer cell therapy ATAC-pipe: general analysis of genome-wide 622 chromatin accessibility Differential expression analysis for sequence count data WGCNA: an R package for weighted correlation network analysis. 626 STRING: a web-server to retrieve and display the 628 repeatedly occurring neighbourhood of a gene Database Resources of the BIG Data Center The y axis represents the number of TFs that pass the fold change cutoff. 776 C: The ratio of known TFs versus all known TFs that qualify the different fold change cutoffs. 777Known TFs: TFs regulating NK cell development reported in the literature.