Long-term expandable mouse and human-induced nephron progenitor cells enable kidney organoid maturation and modeling of plasticity and disease.

Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here, manipulation of p38 and YAP activity allowed for long-term clonal expansion of primary mouse and human NPCs and induced NPCs (iNPCs) from human pluripotent stem cells (hPSCs). Molecular analyses demonstrated that cultured iNPCs closely resemble primary human NPCs. iNPCs generated nephron organoids with minimal off-target cell types and enhanced maturation of podocytes relative to published human kidney organoid protocols. Surprisingly, the NPC culture medium uncovered plasticity in human podocyte programs, enabling podocyte reprogramming to an NPC-like state. Scalability and ease of genome editing facilitated genome-wide CRISPR screening in NPC culture, uncovering genes associated with kidney development and disease. Further, NPC-directed modeling of autosomal-dominant polycystic kidney disease (ADPKD) identified a small-molecule inhibitor of cystogenesis. These findings highlight a broad application for the reported iNPC platform in the study of kidney development, disease, plasticity, and regeneration.

Quiescence enables unrestricted cell fate in naive embryonic stem cells.

Quiescence in stem cells is traditionally considered as a state of inactive dormancy or with poised potential. Naive mouse embryonic stem cells (ESCs) can enter quiescence spontaneously or upon inhibition of MYC or fatty acid oxidation, mimicking embryonic diapause in vivo. The molecular underpinning and developmental potential of quiescent ESCs (qESCs) are relatively unexplored. Here we show that qESCs possess an expanded or unrestricted cell fate, capable of generating both embryonic and extraembryonic cell types (e.g., trophoblast stem cells). These cells have a divergent metabolic landscape comparing to the cycling ESCs, with a notable decrease of the one-carbon metabolite S-adenosylmethionine. The metabolic changes are accompanied by a global reduction of H3K27me3, an increase of chromatin accessibility, as well as the de-repression of endogenous retrovirus MERVL and trophoblast master regulators. Depletion of methionine adenosyltransferase Mat2a or deletion of Eed in the polycomb repressive complex 2 results in removal of the developmental constraints towards the extraembryonic lineages. Our findings suggest that quiescent ESCs are not dormant but rather undergo an active transition towards an unrestricted cell fate.

Non-canonical MLL1 activity regulates centromeric phase separation and genome stability.

Epigenetic dysregulation is a prominent feature in cancer, as exemplified by frequent mutations in chromatin regulators, including the MLL/KMT2 family of histone methyltransferases. Although MLL1/KMT2A activity on H3K4 methylation is well documented, their non-canonical activities remain mostly unexplored. Here we show that MLL1/KMT2A methylates Borealin K143 in the intrinsically disordered region essential for liquid-liquid phase separation of the chromosome passenger complex (CPC). The co-crystal structure highlights the distinct binding mode of the MLL1 SET domain with Borealin K143. Inhibiting MLL1 activity or mutating Borealin K143 to arginine perturbs CPC phase separation, reduces Aurora kinase B activity, and impairs the resolution of erroneous kinetochore-microtubule attachments and sister-chromatid cohesion. They significantly increase chromosome instability and aneuploidy in a subset of hepatocellular carcinoma, resulting in growth inhibition. These results demonstrate a non-redundant function of MLL1 in regulating inner centromere liquid condensates and genome stability via a non-canonical enzymatic activity.

KMT2D links TGF-ß signaling to noncanonical activin pathway and regulates pancreatic cancer cell plasticity.

Although KMT2D, also known as MLL2, is known to play an essential role in development, differentiation, and tumor suppression, its role in pancreatic cancer development is not well understood. Here, we discovered a novel signaling axis mediated by KMT2D, which links TGF-ß to the activin A pathway. We found that TGF-ß upregulates a microRNA, miR-147b, which in turn leads to post-transcriptional silencing of KMT2D. Loss of KMT2D induces the expression and secretion of activin A, which activates a noncanonical p38 MAPK-mediated pathway to modulate cancer cell plasticity, promote a mesenchymal phenotype, and enhance tumor invasion and metastasis in mice. We observed a decreased KMT2D expression in human primary and metastatic pancreatic cancer. Furthermore, inhibition or knockdown of activin A reversed the protumoral role of KMT2D loss. These findings support a tumor-suppressive role of KMT2D in pancreatic cancer and identify miR-147b and activin A as novel therapeutic targets.

Structural insights on the KMT2-NCP interaction.

The MLL/KMT2 family enzymes are frequently mutated in human cancers and congenital diseases. They deposit the majority of histone 3 lysine 4 (H3K4) mono-, di-, or tri-methylation in mammals and are tightly associated with gene activation. Structural and biochemical studies in recent years provide in-depth understanding of how the MLL1 and homologous yeast SET1 complexes interact with the nucleosome core particle (NCP) and how their activities for H3K4 methylation are regulated by the conserved core components. Here, we will discuss the recent single molecule cryo-EM studies on the MLL1 and ySET1 complexes bound on the NCP. These studies highlight the dynamic regulation of the MLL/SET1 family lysine methyltransferases with unique features as compared with other histone lysine methyltransferases. These studies provide insights for loci-specific regulation of H3K4 methylation states in cells. The mechanistic studies on the MLL1 complex have already led to the development of the MLL1 inhibitors that show efficacy in acute leukemia and metastatic breast cancers. Future studies on the MLL/SET1 family enzymes will continue to bring to light potential therapeutic opportunities.

Loss of Wdr5 attenuates MLL-rearranged leukemogenesis by suppressing Myc targets.

WD repeat domain 5 (WDR5) is a prominent target for pharmacological inhibition in cancer through its scaffolding role with various oncogenic partners such as MLL and MYC. WDR5-related drug discovery efforts center on blocking these binding interfaces or degradation have been devoted to developing small-molecule inhibitors or degraders of WDR5 for cancer treatment. Nevertheless, the precise role of WDR5 in these cancer cells has not been well elucidated genetically. Here, by using an MLL-AF9 murine leukemia model, we found that genetically deletion of Wdr5 impairs cell growth and colony forming ability of MLL-AF9 leukemia cells in vitro or ex vivo and attenuates the leukemogenesis in vivo as well, which acts through direct regulation of ribosomal genes. Pharmacological inhibition of Wdr5 recapitulates genetic study results in the same model. In conclusion, our current study demonstrated the first genetic evidence for the indispensable role of Wdr5 in MLL-r leukemogenesis in vivo, which supports therapeutically targeting WDR5 in MLL-rearranged leukemia by strengthening its disease linkage genetically and deepening insights into its mechanism of action.

FOXQ1 recruits the MLL complex to activate transcription of EMT and promote breast cancer metastasis.

Aberrant expression of the Forkhead box transcription factor, FOXQ1, is a prevalent mechanism of epithelial-mesenchymal transition (EMT) and metastasis in multiple carcinoma types. However, it remains unknown how FOXQ1 regulates gene expression. Here, we report that FOXQ1 initiates EMT by recruiting the MLL/KMT2 histone methyltransferase complex as a transcriptional coactivator. We first establish that FOXQ1 promoter recognition precedes MLL complex assembly and histone-3 lysine-4 trimethylation within the promoter regions of critical genes in the EMT program. Mechanistically, we identify that the Forkhead box in FOXQ1 functions as a transactivation domain directly binding the MLL core complex subunit RbBP5 without interrupting FOXQ1 DNA binding activity. Moreover, genetic disruption of the FOXQ1-RbBP5 interaction or pharmacologic targeting of KMT2/MLL recruitment inhibits FOXQ1-dependent gene expression, EMT, and in vivo tumor progression. Our study suggests that targeting the FOXQ1-MLL epigenetic axis could be a promising strategy to combat triple-negative breast cancer metastatic progression.

Human WDR5 promotes breast cancer growth and metastasis via KMT2-independent translation regulation.

Metastatic breast cancer remains a major cause of cancer-related deaths in women, and there are few effective therapies against this advanced disease. Emerging evidence suggests that key steps of tumor progression and metastasis are controlled by reversible epigenetic mechanisms. Using an in vivo genetic screen, we identified WDR5 as an actionable epigenetic regulator that is required for metastatic progression in models of triple-negative breast cancer. We found that knockdown of WDR5 in breast cancer cells independently impaired their tumorigenic as well as metastatic capabilities. Mechanistically, WDR5 promotes cell growth by increasing ribosomal gene expression and translation efficiency in a KMT2-independent manner. Consistently, pharmacological inhibition or degradation of WDR5 impedes cellular translation rate and the clonogenic ability of breast cancer cells. Furthermore, a combination of WDR5 targeting with mTOR inhibitors leads to potent suppression of translation and proliferation of breast cancer cells. These results reveal novel therapeutic strategies to treat metastatic breast cancer.

Bistable Photoswitch Allows in Vivo Control of Hematopoiesis.

Optical control has enabled functional modulation in cell culture with unparalleled spatiotemporal resolution. However, current tools for in vivo manipulation are scarce. Here, we design and implement a genuine on-off optochemical probe capable of achieving hematopoietic control in zebrafish. Our photopharmacological approach first developed conformationally strained visible light photoswitches (CS-VIPs) as inhibitors of the histone methyltransferase MLL1 (KMT2A). In blood homeostasis MLL1 plays a crucial yet controversial role. CS-VIP 8 optimally fulfils the requirements of a true bistable functional system in vivo under visible-light irradiation, and with unprecedented stability. These properties are exemplified via hematopoiesis photoinhibition with a single isomer in zebrafish. The present interdisciplinary study uncovers the mechanism of action of CS-VIPs. Upon WDR5 binding, CS-VIP 8 causes MLL1 release with concomitant allosteric rearrangements in the WDR5/RbBP5 interface. Since our tool provides on-demand reversible control without genetic intervention or continuous irradiation, it will foster hematopathology and epigenetic investigations. Furthermore, our workflow will enable exquisite photocontrol over other targets inhibited by macrocycles.

KDM6A Regulates Cell Plasticity and Pancreatic Cancer Progression by Noncanonical Activin Pathway.

BACKGROUND & AIMS: Inactivating mutations of KDM6A, a histone demethylase, were frequently found in pancreatic ductal adenocarcinoma (PDAC). We investigated the role of KDM6A (lysine demethylase 6A) in PDAC development. METHODS: We performed a pancreatic tissue microarray analysis of KDM6A protein levels. We used human PDAC cell lines for KDM6A knockout and knockdown experiments. We performed bromouridine sequencing analysis to elucidate the effects of KDM6A loss on global transcription. We performed studies with Ptf1aCre; LSL-KrasG12D; Trp53R172H/+; Kdm6afl/fl or fl/Y, Ptf1aCre; Kdm6afl/fl or fl/Y, and orthotopic xenograft mice to investigate the impacts of Kdm6a deficiency on pancreatic tumorigenesis and pancreatitis. RESULTS: Loss of KDM6A was associated with metastasis in PDAC patients. Bromouridine sequencing analysis showed up-regulation of the epithelial-mesenchymal transition pathway in PDAC cells deficient in KDM6A. Loss of KDM6A promoted mesenchymal morphology, migration, and invasion in PDAC cells in vitro. Mechanistically, activin A and subsequent p38 activation likely mediated the role of KDM6A loss. Inhibiting either activin A or p38 reversed the effect. Pancreas-specific Kdm6a-knockout mice pancreata showed accelerated PDAC progression, developed a more aggressive undifferentiated type of PDAC, and increased metastases in the background of Kras and p53 mutations. Kdm6a-deficient pancreata in a pancreatitis model had a delayed recovery with increased PDAC precursor lesions compared with wild-type pancreata. CONCLUSIONS: Loss of KDM6A accelerates PDAC progression and metastasis, most likely by a noncanonical p38-dependent activin A pathway. KDM6A also promotes pancreatic tissue recovery from pancreatitis. Activin A might be used as a therapeutic target for KDM6A-deficient PDACs.

Regulation of MLL1 Methyltransferase Activity in Two Distinct Nucleosome Binding Modes.

Cryo-EM structures of the KMT2A/MLL1 core complex bound on nucleosome core particles (NCPs) suggest unusual rotational dynamics of the MLL1 complex approaching its physiological substrate. However, the functional implication of such dynamics remains unclear. Here, we show that the MLL1 core complex also shows high rotational dynamics bound on the NCP carrying the catalytically inert histone H3 lysine 4 to methionine (K4M) mutation. There are two major binding modes of the MLL1 complex on the NCPK4M. Importantly, disruption of only one of the binding modes compromised the overall MLL1 activity in an NCP-specific manner. We propose that the MLL1 core complex probably exists in an equilibrium of poised and active binding modes. The high rotational dynamics of the MLL1 complex on the NCP is a feature that can be exploited for loci-specific regulation of H3K4 methylation in higher eukaryotes.

p53 inactivation unmasks histone methylation-independent WDR5 functions that drive self-renewal and differentiation of pluripotent stem cells.

p53 alterations occur during culture of pluripotent stem cells (PSCs), but the significance of these events on epigenetic control of PSC fate determination remains poorly understood. Wdr5 deletion in p53-null (DKO) mouse ESCs (mESCs) leads to impaired self-renewal, defective retinal neuroectoderm differentiation, and de-repression of germ cell/meiosis (GCM)-specific genes. Re-introduction of a WDR5 mutant with defective H3K4 methylation activity into DKO ESCs restored self-renewal and suppressed GCM gene expression but failed to induce retinal neuroectoderm differentiation. Mechanistically, mutant WDR5 targets chromatin that is largely devoid of H3K4me3 and regulates gene expression in p53-null mESCs. Furthermore, MAX and WDR5 co-target lineage-specifying chromatin and regulate chromatin accessibility of GCM-related genes. Importantly, MAX and WDR5 are core subunits of a non-canonical polycomb repressor complex 1 responsible for gene silencing. This function, together with canonical, pro-transcriptional WDR5-dependent MLL complex H3K4 methyltransferase activity, highlight how WDR5 mediates crosstalk between transcription and repression during mESC fate choice.

Mechanism for DPY30 and ASH2L intrinsically disordered regions to modulate the MLL/SET1 activity on chromatin.

Recent cryo-EM structures show the highly dynamic nature of the MLL1-NCP (nucleosome core particle) interaction. Functional implication and regulation of such dynamics remain unclear. Here we show that DPY30 and the intrinsically disordered regions (IDRs) of ASH2L work together in restricting the rotational dynamics of the MLL1 complex on the NCP. We show that DPY30 binding to ASH2L leads to stabilization and integration of ASH2L IDRs into the MLL1 complex and establishes new ASH2L-NCP contacts. The significance of ASH2L-DPY30 interactions is demonstrated by requirement of both ASH2L IDRs and DPY30 for dramatic increase of processivity and activity of the MLL1 complex. This DPY30 and ASH2L-IDR dependent regulation is NCP-specific and applies to all members of the MLL/SET1 family of enzymes. We further show that DPY30 is causal for de novo establishment of H3K4me3 in ESCs. Our study provides a paradigm of how H3K4me3 is regulated on chromatin and how H3K4me3 heterogeneity can be modulated by ASH2L IDR interacting proteins.

Discovery of first-in-class inhibitors of ASH1L histone methyltransferase with anti-leukemic activity.

ASH1L histone methyltransferase plays a crucial role in the pathogenesis of different diseases, including acute leukemia. While ASH1L represents an attractive drug target, developing ASH1L inhibitors is challenging, as the catalytic SET domain adapts an inactive conformation with autoinhibitory loop blocking the access to the active site. Here, by applying fragment-based screening followed by medicinal chemistry and a structure-based design, we developed first-in-class small molecule inhibitors of the ASH1L SET domain. The crystal structures of ASH1L-inhibitor complexes reveal compound binding to the autoinhibitory loop region in the SET domain. When tested in MLL leukemia models, our lead compound, AS-99, blocks cell proliferation, induces apoptosis and differentiation, downregulates MLL fusion target genes, and reduces the leukemia burden in vivo. This work validates the ASH1L SET domain as a druggable target and provides a chemical probe to further study the biological functions of ASH1L as well as to develop therapeutic agents.

Dysregulation of intercellular signaling by MOF deletion leads to liver injury.

Epigenetic mechanisms that alter heritable gene expression and chromatin structure play an essential role in many biological processes, including liver function. Human MOF (males absent on the first) is a histone acetyltransferase that is globally downregulated in human steatohepatitis. However, the function of MOF in the liver remains unclear. Here, we report that MOF plays an essential role in adult liver. Genetic deletion of Mof by Mx1-Cre in the liver leads to acute liver injury, with increase of lipid deposition and fibrosis akin to human steatohepatitis. Surprisingly, hepatocyte-specific Mof deletion had no overt liver abnormality. Using the in vitro coculturing experiment, we show that Mof deletion-induced liver injury requires coordinated changes and reciprocal signaling between hepatocytes and Kupffer cells, which enables feedforward regulation to augment inflammation and apoptotic responses. At the molecular level, Mof deletion induced characteristic changes in metabolic gene programs, which bore noticeable similarity to the molecular signature of human steatohepatitis. Simultaneous deletion of Mof in both hepatocytes and macrophages results in enhanced expression of inflammatory genes and NO signaling in vitro. These changes, in turn, lead to apoptosis of hepatocytes and lipotoxicity. Our work highlights the importance of histone acetyltransferase MOF in maintaining metabolic liver homeostasis and sheds light on the epigenetic dysregulation in liver pathogenesis.

ER-associated degradation preserves hematopoietic stem cell quiescence and self-renewal by restricting mTOR activity.

Hematopoietic stem cells (HSC) self-renew to sustain stem cell pools and differentiate to generate all types of blood cells. HSCs remain in quiescence to sustain their long-term self-renewal potential. It remains unclear whether protein quality control is required for stem cells in quiescence when RNA content, protein synthesis, and metabolic activities are profoundly reduced. Here, we report that protein quality control via endoplasmic reticulum-associated degradation (ERAD) governs the function of quiescent HSCs. The Sel1L/Hrd1 ERAD genes are enriched in the quiescent and inactive HSCs, and conditional knockout of Sel1L in hematopoietic tissues drives HSCs to hyperproliferation, which leads to complete loss of HSC self-renewal and HSC depletion. Mechanistically, ERAD deficiency via Sel1L knockout leads to activation of mammalian target of rapamycin (mTOR) signaling. Furthermore, we identify Ras homolog enriched in brain (Rheb), an activator of mTOR, as a novel protein substrate of Sel1L/Hrd1 ERAD, which accumulates upon Sel1L deletion and HSC activation. Importantly, inhibition of mTOR, or Rheb, rescues HSC defects in Sel1L knockout mice. Protein quality control via ERAD is, therefore, a critical checkpoint that governs HSC quiescence and self-renewal by Rheb-mediated restriction of mTOR activity.

Mutually suppressive roles of KMT2A and KDM5C in behaviour, neuronal structure, and histone H3K4 methylation.

Histone H3 lysine 4 methylation (H3K4me) is extensively regulated by numerous writer and eraser enzymes in mammals. Nine H3K4me enzymes are associated with neurodevelopmental disorders to date, indicating their important roles in the brain. However, interplay among H3K4me enzymes during brain development remains largely unknown. Here, we show functional interactions of a writer-eraser duo, KMT2A and KDM5C, which are responsible for Wiedemann-Steiner Syndrome (WDSTS), and mental retardation X-linked syndromic Claes-Jensen type (MRXSCJ), respectively. Despite opposite enzymatic activities, the two mouse models deficient for either Kmt2a or Kdm5c shared reduced dendritic spines and increased aggression. Double mutation of Kmt2a and Kdm5c clearly reversed dendritic morphology, key behavioral traits including aggression, and partially corrected altered transcriptomes and H3K4me landscapes. Thus, our study uncovers common yet mutually suppressive aspects of the WDSTS and MRXSCJ models and provides a proof of principle for balancing a single writer-eraser pair to ameliorate their associated disorders.

Discovery of Potent Small-Molecule Inhibitors of MLL Methyltransferase.

The mixed-lineage leukemia (MLL) protein, also known as MLL1, is a lysine methyltransferase specifically responsible for methylation of histone 3 lysine 4. MLL has been pursued as an attractive therapeutic target for the treatment of acute leukemia carrying the MLL fusion gene or MLL leukemia. Herein, we report the design, synthesis, and evaluation of an S-adenosylmethionine-based focused chemical library which led to the discovery of potent small-molecule inhibitors directly targeting the MLL SET domain. Determination of cocrystal structures for a number of these MLL inhibitors reveals that they adopt a unique binding mode that locks the MLL SET domain in an open, inactive conformation.

Hepatic HuR modulates lipid homeostasis in response to high-fat diet.

Lipid transport and ATP synthesis are critical for the progression of non-alcoholic fatty liver disease (NAFLD), but the underlying mechanisms are largely unknown. Here, we report that the RNA-binding protein HuR (ELAVL1) forms complexes with NAFLD-relevant transcripts. It associates with intron 24 of Apob pre-mRNA, with the 3'UTR of Uqcrb, and with the 5'UTR of Ndufb6 mRNA, thereby regulating the splicing of Apob mRNA and the translation of UQCRB and NDUFB6. Hepatocyte-specific HuR knockout reduces the expression of APOB, UQCRB, and NDUFB6 in mice, reducing liver lipid transport and ATP synthesis, and aggravating high-fat diet (HFD)-induced NAFLD. Adenovirus-mediated re-expression of HuR in hepatocytes rescues the effect of HuR knockout in HFD-induced NAFLD. Our findings highlight a critical role of HuR in regulating lipid transport and ATP synthesis.

Insights on the regulation of the MLL/SET1 family histone methyltransferases.

In eukaryotes, histone H3K4 methylation by the MLL/SET1 family histone methyltransferases is enriched at transcription regulatory elements including gene promoters and enhancers. The level of H3K4 methylation is highly correlated with transcription activation and is one of the most frequently used histone post-translational modifications to predict transcriptional outcome. Recently, it has been shown that rearrangement of the cellular landscape of H3K4 mono-methylation at distal enhancers precedes cell fate transition and is used for identification of novel regulatory elements for development and disease progression. Similarly, broad H3K4 tri-methylation regions have also been used to predict intrinsic tumor suppression properties of regulator regions in a variety of cellular models. Understanding the regulation for how H3K4 methylation is deposited and regulated is of paramount importance. In this review, we will discuss new findings on how the MLL/SET1 family enzymes are regulated on chromatin and their potential functional and regulatory implications. This article is part of a Special Issue entitled: The MLL family of proteins in normal development and disease edited by Thomas A Milne.