key: cord-103864-4kec8re4 authors: Miao, Zhen; Balzer, Michael S.; Ma, Ziyuan; Liu, Hongbo; Wu, Junnan; Shrestha, Rojesh; Aranyi, Tamas; Kwan, Amy; Kondo, Ayano; Pontoglio, Marco; Kim, Junhyong; Li, Mingyao; Kaestner, Klaus H.; Susztak, Katalin title: Single cell resolution regulatory landscape of the mouse kidney highlights cellular differentiation programs and renal disease targets date: 2020-06-04 journal: bioRxiv DOI: 10.1101/2020.05.24.113910 sha: doc_id: 103864 cord_uid: 4kec8re4 Determining the epigenetic program that generates unique cell types in the kidney is critical for understanding cell-type heterogeneity during tissue homeostasis and injury response. Here, we profiled open chromatin and gene expression in developing and adult mouse kidneys at single cell resolution. We show critical reliance of gene expression on distal regulatory elements (enhancers). We define key cell type-specific transcription factors and major gene-regulatory circuits for kidney cells. Dynamic chromatin and expression changes during nephron progenitor differentiation demonstrated that podocyte commitment occurs early and is associated with sustained Foxl1 expression. Renal tubule cells followed a more complex differentiation, where Hfn4a was associated with proximal and Tfap2b with distal fate. Mapping single nucleotide variants associated with human kidney disease identified critical cell types, developmental stages, genes, and regulatory mechanisms. We provide a global single cell resolution view of chromatin accessibility of kidney development. The dataset is available via interactive public websites. the true cell type specificity of these enhancers is critically important. Here, we reasoned that 474 single cell accessible chromatin information could be extremely useful to identify the cell type-475 specific enhancer regions and thereby the target cell type for the GWAS hits, however, such maps 476 have not been generated for the human kidney. We combined three recent kidney disease GWAS 477 Discussion 532 533 In summary, here we present the first cellular resolution open chromatin map for the developing 534 and adult mouse kidney. Using this dataset, we identified key cell type-specific regulatory 535 networks for kidney cells, defined the cellular differentiation trajectory, characterized regulatory 536 dynamics and identified key driving TFs for nephron development, especially for the terminal 537 differentiation of epithelial cells. Furthermore, our results shed light on the cell types and target 538 genes for genetic variants associated with kidney disease development. 539 540 By performing massively parallel single cell profiling of chromatin state, we were able to define 541 the key regulatory logic for each kidney cell type by investigating cis-regulatory elements and TF-542 target gene interaction. We found that most cell type-specific open chromatin regions are within 543 distal regulatory elements and intronic regions. Our studies identified a massive amount of highly 544 dynamic co-regulated peaks indicating the important correlation between distal regulatory 545 elements and gene expression. Future studies will examine the relative contribution of promoters 546 and enhancer openness in gene expression regulation. However, these studies highlight that both 547 chromatin opening and looping are critical for gene regulation. 548 549 We also observed that the single cell open chromatin atlas was able to define more distinct cell 550 types even in the developing kidney compared to scRNA-seq analysis. Given the continuous nature 551 of RNA expression, it has been exceedingly difficult to dissect specific cell types in the developing 552 kidney 9,10,13 . In addition, it has been difficult to resolve the cell type origin of lowly expressed 553 transcripts in scRNA-seq data. However, this is not the case for snATAC-seq data, which were 554 able to capture the chromatin state irrespective of gene expression magnitude. There were several 555 examples where accessible peaks were identified in specific cell types even for lowly expressed 556 genes such as Shroom3. 557 558 We identified critical cell type-specific TFs by integrating multiple computational analyses. TF 559 identification is challenging in scRNA-seq data since the expression of several cell type-specific 560 TFs is low and some of them do not show a high degree of cell type-specificity 52 . By extracting 561 motif information, snATAC-seq data provides additional information for TF identification. 562 20/50 Together with regulon analysis, as implemented in SCENIC, we have identified several TFs as 563 well as their target genes that are important for kidney development. Leveraging this newly 564 identified cell type-specific regulatory network will be essential for future studies of cellular 565 reprogramming of precursors into specific kidney cell types and for better understanding 566 homeostatic and maladaptive regeneration. 567 568 Our studies revealed dynamic chromatin accessibility that tracks with renal cell differentiation. 569 These states may reveal mechanisms governing the establishment of cell fate during development, 570 in particular those underlying the emergence of specific cell types. We found a consistent and 571 coherent pattern between gene expression and open chromatin information, where the nephron 572 progenitors differentiated into two branches representing podocytes and tubule cells 53 . We found 573 that podocytes commitment occurred earlier, while tubule differentiation and segmentation 574 appeared to be more complex. This podocyte specification correlated with the maintenance of 575 expression of Foxc2 and Foxl1 expression in podocytes. While Foxc2 has been known to play a 576 role in nephron progenitors and podocytes, this is the first description of Foxl1 in kidney and 577 podocyte development. Our studies are consistent with recent observations from organoid models 578 that recapitulated podocyte differentiation better than tubule cell differentiation 54 . Our study also 579 sheds light on tubule differentiation and segmentation. We confirmed the key role of Hnf4a in 580 proximal tubules. We have identified a large number of new transcriptional regulators such as 581 Tfap2a that seem to be critical for the distal portion of the nephron. open exclusively in nephron progenitors, whereas chromatin becomes inaccessible as 611 differentiation progresses during later stages, such as Shroom3 and Uncx. This is an interesting 612 and important novel mechanism, indicating that the altered expression of this gene might play a 613 role in the development rewiring of the kidney. This mechanism is similar to genes associated with 614 autism that are known to be expressed in the fetal but not in the adult stages 59 and highlights the 615 critical role of understanding chromatin accessibility at multiple stages of differentiation. 616 617 While we have generated a large amount of high-quality data, this information will need further 618 experimental validation, which is beyond the scope of the current manuscript. In addition, one 619 needs to be aware of the limitations when interpreting different computational analyses, for 620 example, the motif enrichment analyses such as implemented by HOMER, SCENIC, and 621 chromVAR, are not able to distinguish between TFs with similar binding sites. 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