Discrete bladder cancer molecular subtypes exhibit differential clinical aggressiveness and therapeutic response, which may have significant implications for identifying novel treatments for this common malignancy. However, research is hindered by the lack of suitable models to study each subtype. To address this limitation, we classified bladder cancer cell lines into molecular subtypes using publically available data in the Cancer Cell Line Encyclopedia (CCLE), guided by genomic characterization of bladder cancer by The Cancer Genome Atlas (TCGA). This identified a panel of bladder cancer cell lines which exhibit genetic alterations and gene expression patterns consistent with luminal and basal molecular subtypes of human disease. A subset of bladder cancer cell lines exhibit in vivo histomorphologic patterns consistent with luminal and basal subtypes, including papillary architecture and squamous differentiation. Using the molecular subtype assignments, and our own RNA-seq analysis, we found overexpression of GATA3 and FOXA1 cooperate with PPARɣ activation to drive transdifferentiation of a basal bladder cancer cells to a luminial phenotype. In summary, our analysis identified a set of human cell lines suitable for the study of molecular subtypes in bladder cancer, and furthermore indicates a cooperative regulatory network consisting of GATA3, FOXA1, and PPARɣ drive luminal cell fate.

Examining kidney fibrosis is crucial for mechanistic understanding and developing targeted strategies against chronic kidney disease (CKD). Persistent fibroblast activation and tubular epithelial cell (TEC) injury are key CKD contributors. However, cellular and transcriptional landscapes of CKD and specific activated kidney fibroblast clusters remain elusive. Here, we analyzed single cell transcriptomic profiles of two clinically relevant kidney fibrosis models which induced robust kidney parenchymal remodeling. We dissected the molecular and cellular landscapes of kidney stroma and newly identified three distinctive fibroblast clusters with "secretory", "contractile" and "vascular" transcriptional enrichments. Also, both injuries generated failed repair TECs (frTECs) characterized by decline of mature epithelial markers and elevation of stromal and injury markers. Notably, frTECs shared transcriptional identity with distal nephron segments of the embryonic kidney. Moreover, we identified that both models exhibited robust and previously unrecognized distal spatial pattern of TEC injury, outlined by persistent elevation of renal TEC injury markers including Krt8 and Vcam1, while the surviving proximal tubules (PTs) showed restored transcriptional signature. We also found that long-term kidney injuries activated a prominent nephrogenic signature, including Sox4 and Hox gene elevation, which prevailed in the distal tubular segments. Our findings might advance understanding of and targeted intervention in fibrotic kidney disease.

The Tead family transcription factors are the major intracellular mediators of the Hippo-Yap pathway. Despite the importance of Hippo signaling in tumorigenesis, Tead-dependent downstream oncogenic programs and target genes in cancer cells remain poorly understood. Here, we characterize Tead4-mediated transcriptional networks in a diverse range of cancer cells, including neuroblastoma, colorectal, lung, and endometrial carcinomas. By intersecting genome-wide chromatin occupancy analyses of Tead4, JunD, and Fra1/2, we find that Tead4 cooperates with AP1 transcription factors to coordinate target gene transcription. We find that Tead-AP1 interaction is JNK independent but engages the SRC1-3 co-activators to promote downstream transcription. Furthermore, we show that Tead-AP1 cooperation regulates the activity of the Dock-Rac/CDC42 module and drives the expression of a unique core set of target genes, thereby directing cell migration and invasion. Together, our data unveil a critical regulatory mechanism underlying Tead- and AP1-controlled transcriptional and functional outputs in cancer cells.

YAP/TAZ are the major mediators of mammalian Hippo signaling; however, their precise function in the gastrointestinal tract remains poorly understood. Here we dissect the distinct roles of YAP/TAZ in endodermal epithelium and mesenchyme and find that, although dispensable for gastrointestinal epithelial development and homeostasis, YAP/TAZ function as the critical molecular switch to coordinate growth and patterning in gut mesenchyme. Our genetic analyses reveal that Lats1/2 kinases suppress expansion of the primitive mesenchymal progenitors, where YAP activation also prevents induction of the smooth muscle lineage through transcriptional repression of Myocardin. During later development, zone-restricted downregulation of YAP/TAZ provides the positional cue and allows smooth muscle cell differentiation induced by Hedgehog signaling. Taken together, our studies identify the mesenchymal requirement of YAP/TAZ in the gastrointestinal tract and highlight the functional interplays between Hippo and Hedgehog signaling underlying temporal and spatial control of tissue growth and specification in developing gut.

Dysregulation of Wnt signaling is closely associated with human liver tumorigenesis. However, liver cancer-specific Wnt transcriptional programs and downstream effectors remain poorly understood. Here, we identify tribbles homolog 2 (TRIB2) as a direct target of Wnt/TCF in liver cancer and demonstrate that transcription of Wnt target genes, including TRIB2, is coordinated by the TCF and FoxA transcription factors in liver cancer cells. We show that Wnt-TRIB2 activation is critical for cancer cell survival and transformation. Mechanistically, TRIB2 promotes protein stabilization of the YAP transcription coactivator through interaction with the βTrCP ubiquitin ligase. Furthermore, we find that TRIB2 relieves the liver tumor suppressor protein C/EBPα-mediated inhibition of YAP/TEAD transcriptional activation in liver cancer cells. Altogether, our study uncovers a regulatory mechanism underlying liver cancer-specific Wnt transcriptional output, and suggests that TRIB2 functions as a signaling nexus to integrate the Wnt/β-catenin, Hippo/YAP, and C/EBPα pathways in cancer cells.

We used a single cell RNA-seq strategy to create an atlas of gene expression patterns in the developing kidney. At several stages of kidney development, histologically uniform populations of cells give rise to multiple distinct lineages. We performed single cell RNA-seq analysis of total mouse kidneys at E11.5 and E12.5, as well as the renal vesicles at P4. We define an early stage of progenitor cell induction driven primarily by gene repression. Surprising stochastic expression of marker genes associated with differentiated cell types was observed in E11.5 progenitors. We provide a global view of the polarized gene expression already present in the renal vesicle, the first epithelial precursor of the nephron. We show that Hox gene read-through transcripts can be spliced to produce intergenic homeobox swaps. We also identify a surprising number of genes with partially degraded noncoding RNA. Perhaps most interesting, at early developmental times single cells often expressed genes related to several developmental pathways. This provides powerful evidence that initial organogenesis involves a process of multilineage priming. This is followed by a combination of gene repression, which turns off the genes associated with most possible lineages, and the activation of increasing numbers of genes driving the chosen developmental direction.

The transcription factor atonal homolog 1 (ATOH1) controls the fate of intestinal progenitors downstream of the Notch signaling pathway. Intestinal progenitors that escape Notch activation express high levels of ATOH1 and commit to a secretory lineage fate, implicating ATOH1 as a gatekeeper for differentiation of intestinal epithelial cells. Although some transcription factors downstream of ATOH1, such as SPDEF, have been identified to specify differentiation and maturation of specific cell types, the bona fide transcriptional targets of ATOH1 still largely are unknown. Here, we aimed to identify ATOH1 targets and to identify transcription factors that are likely to co-regulate gene expression with ATOH1.

Progenitor self-renewal and differentiation is often regulated by spatially restricted cues within a tissue microenvironment. Here, we examine how progenitor cell migration impacts regionally induced commitment within the nephrogenic niche in mice. We identify a subset of cells that express Wnt4, an early marker of nephron commitment, but migrate back into the progenitor population where they accumulate over time. Single cell RNA-seq and computational modelling of returning cells reveals that nephron progenitors can traverse the transcriptional hierarchy between self-renewal and commitment in either direction. This plasticity may enable robust regulation of nephrogenesis as niches remodel and grow during organogenesis.

The nature and role of global transcriptional deregulations in cancers are not fully understood. We report that a large proportion of cancers have widespread defects in mRNA transcription elongation (TE). Cancers with TE defects (TEdeff) display spurious transcription and defective mRNA processing of genes characterized by long genomic length, poised promoters and inducible expression. Signaling pathways regulated by such genes, such as pro-inflammatory response pathways, are consistently suppressed in TEdeff tumors. Remarkably, TEdeff correlates with the poor response and outcome in immunotherapy, but not chemo- or targeted therapy, -treated renal cell carcinoma and metastatic melanoma patients. Forced pharmacologic or genetic induction of TEdeff in tumor cells impairs pro-inflammatory response signaling, and imposes resistance to the innate and adaptive anti-tumor immune responses and checkpoint inhibitor therapy in vivo. Therefore, defective TE is a previously unknown mechanism of tumor immune resistance, and should be assessed in cancer patients undergoing immunotherapy.

Mesenchymal nephron progenitors (mNPs) give rise to all nephron tubules in the mammalian kidney. Since premature depletion of these cells leads to low nephron numbers, high blood pressure, and various renal diseases, it is critical to understand how mNPs are maintained. While Fgf, Bmp, and Wnt signaling pathways are known to be required for the maintenance of these cells, it is unclear if any other signaling pathways also play roles.

A balance between Six2-dependent self-renewal and canonical Wnt signaling-directed commitment regulates mammalian nephrogenesis. Intersectional studies using chromatin immunoprecipitation and transcriptional profiling identified direct target genes shared by each pathway within nephron progenitors. Wnt4 and Fgf8 are essential for progenitor commitment; cis-regulatory modules flanking each gene are cobound by Six2 and β-catenin and are dependent on conserved Lef/Tcf binding sites for activity. In vitro and in vivo analyses suggest that Six2 and Lef/Tcf factors form a regulatory complex that promotes progenitor maintenance while entry of β-catenin into this complex promotes nephrogenesis. Alternative transcriptional responses associated with Six2 and β-catenin cobinding events occur through non-Lef/Tcf DNA binding mechanisms, highlighting the regulatory complexity downstream of Wnt signaling in the developing mammalian kidney.

Mammalian kidney organogenesis involves reciprocal epithelial-mesenchymal interactions that drive iterative cycles of nephron formation. Recent studies have demonstrated that the Six2 transcription factor acts cell autonomously to maintain nephron progenitor cells, whereas canonical Wnt signaling induces nephron differentiation. How Six2 maintains the nephron progenitor cells against Wnt-directed commitment is not well understood, however. We report here that Six2 is required to maintain expression of Osr1, a homolog of the Drosophila odd-skipped zinc-finger transcription factor, in the undifferentiated cap mesenchyme. Tissue-specific inactivation of Osr1 in the cap mesenchyme caused premature depletion of nephron progenitor cells and severe renal hypoplasia. We show that Osr1 and Six2 act synergistically to prevent premature differentiation of the cap mesenchyme. Furthermore, although both Six2 and Osr1 could form protein interaction complexes with TCF proteins, Osr1, but not Six2, enhances TCF interaction with the Groucho family transcriptional co-repressors. Moreover, we demonstrate that loss of Osr1 results in β-catenin/TCF-mediated ectopic activation of Wnt4 enhancer-driven reporter gene expression in the undifferentiated nephron progenitor cells in vivo. Together, these data indicate that Osr1 plays crucial roles in Six2-dependent maintenance of nephron progenitors during mammalian nephrogenesis by stabilizing TCF-Groucho transcriptional repressor complexes to antagonize Wnt-directed nephrogenic differentiation.

Nephron progenitor number determines nephron endowment; a reduced nephron count is linked to the onset of kidney disease. Several transcriptional regulators including Six2, Wt1, Osr1, Sall1, Eya1, Pax2, and Hox11 paralogues are required for specification and/or maintenance of nephron progenitors. However, little is known about the regulatory intersection of these players. Here, we have mapped nephron progenitor-specific transcriptional networks of Six2, Hoxd11, Osr1, and Wt1. We identified 373 multi-factor associated 'regulatory hotspots' around genes closely associated with progenitor programs. To examine their functional significance, we deleted 'hotspot' enhancer elements for Six2 and Wnt4. Removal of the distal enhancer for Six2 leads to a ~40% reduction in Six2 expression. When combined with a Six2 null allele, progeny display a premature depletion of nephron progenitors. Loss of the Wnt4 enhancer led to a significant reduction of Wnt4 expression in renal vesicles and a mildly hypoplastic kidney, a phenotype also enhanced in combination with a Wnt4 null mutation. To explore the regulatory landscape that supports proper target gene expression, we performed CTCF ChIP-seq to identify insulator-boundary regions. One such putative boundary lies between the Six2 and Six3 loci. Evidence for the functional significance of this boundary was obtained by deep sequencing of the radiation-induced Brachyrrhine (Br) mutant allele. We identified an inversion of the Six2/Six3 locus around the CTCF-bound boundary, removing Six2 from its distal enhancer regulation, but placed next to Six3 enhancer elements which support ectopic Six2 expression in the lens where Six3 is normally expressed. Six3 is now predicted to fall under control of the Six2 distal enhancer. Consistent with this view, we observed ectopic Six3 in nephron progenitors. 4C-seq supports the model for Six2 distal enhancer interactions in wild-type and Br/+ mouse kidneys. Together, these data expand our view of the regulatory genome and regulatory landscape underpinning mammalian nephrogenesis.

The nephron is composed of distinct segments that perform unique physiological functions. Little is known about how multipotent nephron progenitor cells differentiate into different nephron segments. It is well known that β-catenin signaling regulates the maintenance and commitment of mesenchymal nephron progenitors during kidney development. However, it is not fully understood how it regulates nephron segmentation after nephron progenitors undergo mesenchymal-to-epithelial transition. To address this, we performed β-catenin loss-of-function and gain-of-function studies in epithelial nephron progenitors in the mouse kidney. Consistent with a previous report, the formation of the renal corpuscle was defective in the absence of β-catenin. Interestingly, we found that epithelial nephron progenitors lacking β-catenin were able to form presumptive proximal tubules but that they failed to further develop into differentiated proximal tubules, suggesting that β-catenin signaling plays a critical role in proximal tubule development. We also found that epithelial nephron progenitors lacking β-catenin failed to form the distal tubules. Expression of a stable form of β-catenin in epithelial nephron progenitors blocked the proper formation of all nephron segments, suggesting tight regulation of β-catenin signaling during nephron segmentation. This work shows that β-catenin regulates the formation of multiple nephron segments along the proximo-distal axis of the mammalian nephron.