Cell Fate Determination Research Articles

Anchoring super-enhancer-driven oncogenic lncRNAs for anti-tumor therapy in hepatocellular carcinoma

Super-enhancer (SE) plays a vital role in the determination of cell identity and fate. Up-regulated expression of coding genes is frequently associated with SE. However, the transcription dysregulation driven by SE, from the viewpoint of long non-coding RNA (lncRNA), remains unclear. Here, SE-associated lncRNAs in HCC are comprehensively outlined for the first time. This study integrally screens and identifies several novel SE-associated lncRNAs that are highly abundant and sensitive to JQ1. Especially, HSAL3 is identified as an uncharacterized SE-driven oncogenic lncRNA, which is activated by transcription factors HCFC1 and HSF1 via its super-enhancer. HSAL3 interference negatively regulates NOTCH signaling, implying the potential mechanism of its tumor-promoting role. The expression of HSAL3 is increased in HCC samples, and higher HSAL3 expression indicates an inferior overall survival of HCC patients. Furthermore, siHSAL3 loaded nanoparticles exert anti-tumor effect on HCC invitro and invivo. In conclusion, this is the first comprehensive survey of SE-associated lncRNAs in HCC. HSAL3 is a novel SE-driven oncogenic lncRNA, and siHSAL3 loaded nanoparticles are therapeutic candidates for HCC. This work sheds lights on the merit of anchoring SE-driven oncogenic lncRNAs for HCC treatment.

Precise modulation of embryonic development through optogenetics.

The past decade has witnessed enormous progress in optogenetics, which uses photo-sensitive proteins to control signal transduction in live cells and animals. The ever-increasing amount of optogenetic tools, however, could overwhelm the selection of appropriate optogenetic strategies. In this work, we summarize recent progress in this emerging field and highlight the application of opsin-free optogenetics in studying embryonic development, focusing on new insights gained into optical induction of morphogenesis, cell polarity, cell fate determination, tissue differentiation, neuronal regeneration, synaptic plasticity, and removal of cells during development.

Functional hepatobiliary organoids recapitulate liver development and reveal essential drivers of hepatobiliary cell fate determination

AbstractDuring liver development, hepatocytes, and cholangiocytes are concurrently differentiated from common liver progenitor cells and are assembled into hepatobiliary architecture to perform proper hepatic function. However, the generation of functional hepatobiliary architecture from hepatocytes in vitro is still challenging, and the exact molecular drivers of hepatobiliary cell lineage determination is largely unknown. In this study, functional hepatobiliary organoids (HBOs) are generated from hepatocytes. These HBOs contain a bile duct network surrounded by mature hepatocytes and stably maintain hepatic characteristics and function in vitro and upon transplantation in vivo. Morphological transition and expression profile of hepatocyte-derived organoids recapitulate the process of liver development. Gene regulation landscape of hepatocyte-derived organoids reveal that Tead4 and Ddit3 promote the cell fate commitment of liver progenitors to functional cholangiocytes and hepatocytes, respectively. Liver cell fate determination is reversed by inhibiting Tead4 or increasing Ddit3 expression both in vitro and upon transplantation in vivo. Collectively, hepatocyte-derived HBOs reveal the essential transcription drivers of liver hepatobiliary cell lineage determination and represent powerful models for liver development and regeneration.

Regulation of RNA localization during oocyte maturation by dynamic RNA-ER association and remodeling of the ER

Asymmetric localization of mRNAs is crucial for cell polarity and cell fate determination. By performing fractionation RNA-seq, we report here that a large number of maternal RNAs are associated with the ER in Xenopus oocytes but are released into the cytosol after oocyte maturation. We provide evidence that the majority of ER-associated RNA-binding proteins (RBPs) remain associated with the ER after oocyte maturation. However, all ER-associated RBPs analyzed exhibit reduced binding to some of their target RNAs after oocyte maturation. Our results further show that the ER is remodeled massively during oocyte maturation, leading to the formation of a widespread tubular ER network in the animal hemisphere that is required for the asymmetric localization of mRNAs in mature eggs. Thus, our findings demonstrate that dynamic regulation of RNA-ER association and remodeling of the ER are important for the asymmetric localization of RNAs during development.

Interactions between Major Bioactive Polyphenols of Sugarcane Top: Effects on Human Neural Stem Cell Differentiation and Astrocytic Maturation

Sugarcane (Saccharum officinarum L.) is a tropical plant grown for sugar production. We recently showed that sugarcane top (ST) ameliorates cognitive decline in a mouse model of accelerated aging via promoting neuronal differentiation and neuronal energy metabolism and extending the length of the astrocytic process in vitro. Since the crude extract consists of multicomponent mixtures, it is crucial to identify bioactive compounds of interest and the affected molecular targets. In the present study, we investigated the bioactivities of major polyphenols of ST, namely 3-O-caffeoylquinic acid (3CQA), 5-O-caffeoylquinic acid (5CQA), 3-O-feruloylquinic acid (3FQA), and Isoorientin (ISO), in human fetal neural stem cells (hNSCs)- an in vitro model system for studying neural development. We found that multiple polyphenols of ST contributed synergistically to stimulate neuronal differentiation of hNSCs and induce mitochondrial activity in immature astrocytes. Mono-CQAs (3CQA and 5CQA) regulated the expression of cyclins related to G1 cell cycle arrest, whereas ISO regulated basic helix-loop-helix transcription factors related to cell fate determination. Additionally, mono-CQAs activated p38 and ISO inactivated GSK3β. In hNSC-derived immature astrocytes, the compounds upregulated mRNA expression of PGC-1α, a master regulator of astrocytic mitochondrial biogenesis. Altogether, our findings suggest that synergistic interactions between major polyphenols of ST contribute to its potential for neuronal differentiation and astrocytic maturation.

Elongator and the role of its subcomplexes in human diseases.

The Elongator complex was initially identified in yeast, and a variety of distinct cellular functions have been assigned to the complex. In the last decade, several research groups focussed on dissecting its structure, tRNA modification activity and role in translation regulation. Recently, Elongator emerged as a crucial factor for various human diseases, and its involvement has triggered a strong interest in the complex from numerous clinical groups. The Elongator complex is highly conserved among eukaryotes, with all six subunits (Elp1-6) contributing to its stability and function. Yet, recent studies have shown that the two subcomplexes, namely the catalytic Elp123 and accessory Elp456, may have distinct roles in the development of different neuronal subtypes. This Commentary aims to provide a brief overview and new perspectives for more systematic efforts to explore the functions of the Elongator in health and disease.

Insights into Regulators of p53 Acetylation

The tumor suppressor p53 is a transcription factor that regulates the expression of dozens of target genes and diverse physiological processes. To precisely regulate the p53 network, p53 undergoes various post-translational modifications and alters the selectivity of target genes. Acetylation plays an essential role in cell fate determination through the activation of p53. Although the acetylation of p53 has been examined, the underlying regulatory mechanisms remain unclear and, thus, have attracted the interest of researchers. We herein discuss the role of acetylation in the p53 pathway, with a focus on p53 acetyltransferases and deacetylases. We also review recent findings on the regulators of these enzymes to understand the mode of p53 acetylation from a broader perspective.

Subcellular-Level Mitochondrial Energy Metabolism Response in the Fat Body of the German Cockroach Fed Abamectin.

Mitochondria are the leading organelle for energy metabolism. The toxic effects of environmental toxicants on mitochondrial morphology, energy metabolism, and their determination of cell fate have already been broadly studied. However, minimal research exists on effects of environmental toxicants such as pesticides on mitochondrial energy metabolism at in vitro subcellular level, particularly from an omics perspectives (e.g., metabolomics). Here, German cockroach (Blattella germanica) was fed diets with (0.01 and 0.001 mg/mL) and without abamectin, and highly purified fat body mitochondria were isolated. Swelling measurement confirmed abnormal mitochondrial swelling caused by abamectin stress. The activity of two key mitochondrial energy metabolism-related enzymes, namely succinic dehydrogenase and isocitrate dehydrogenase, was significantly affected. The metabolomic responses of the isolated mitochondria to abamectin were analyzed via untargeted liquid chromatography/mass spectrometry metabolomics technology. Fifty-two differential metabolites (DMs) were identified in the mitochondria between the 0.001 mg/mL abamectin-fed and the control groups. Many of these DMs were significantly enriched in pathways involved in ATP production and energy consumption (e.g., oxidative phosphorylation, TCA cycle, and pentose phosphate pathway). Nineteen of the DMs were typically related to energy metabolism. This study is valuable for further understanding mitochondrial toxicology under environmental toxicants, particularly its subcellular level.

Single-cell multiomics reveals the complexity of TGFβ signalling to chromatin in iPSC-derived kidney organoids

TGFβ1 plays a regulatory role in the determination of renal cell fate and the progression of renal fibrosis. Here we show an association between SMAD3 and the histone methyltransferase, EZH2, during cell differentiation; ChIP-seq revealed that SMAD3 and EZH2 co-occupy the genome in iPSCs and in iPSC-derived nephron progenitors. Through integration of single cell gene expression and epigenome profiling, we identified de novo ACTA2+ve/POSTN+ve myofibroblasts in kidney organoids treated with TGFβ1, characterised by increased SMAD3-dependent cis chromatin accessibility and gene expression associated with fibroblast activation. We have identified fibrosis-associated regulons characterised by enrichment of SMAD3, AP1, the ETS family of transcription factors, and NUAK1, CREB3L1, and RARG, corresponding to enriched motifs at accessible loci identified by scATACseq. Treatment with the EZH2 specific inhibitor GSK343, blocked SMAD3-dependent cis co-accessibility and inhibited myofibroblast activation. This mechanism, through which TGFβ signals directly to chromatin, represents a critical determinant of fibrotic, differentiated states.

E3 ligase Cul2 mediates Drosophila early germ cell differentiation through targeting Bam

Drosophila ovary has been one of the most mature and excellent systems for studying the in vivo regulatory mechanisms of stem cell fate determination. It has been well-known that the bone morphogenetic protein (BMP) signaling released by the niche cells promotes the maintenance of germline stem cells (GSCs) through inhibiting the transcription of the bag-of-marbles (bam) gene, which encodes a key factor for GSC differentiation. However, whether Bam is regulated at the post-translational level remains largely unknown. Here we show that the E3 ligase Cullin-2 (Cul2) is involved in modulating Bam ubiquitination, which occurs probably at multiple lysine residues of Bam's C-terminal region. Genetic evidence further supports the notion that Cul2-mediated Bam ubiquitination and turnover are essential for GSC maintenance and proper germline development. Collectively, our data not only uncovers a novel regulatory mechanism by which Bam is controlled at the post-translational level, but also provides new insights into how Cullin family protein determines the differentiation fate of early germ cells.

Characterization of Retinal Development in 13-Lined Ground Squirrels.

The cone-dominant, 13-lined ground squirrel (13-LGS) retina mimics the human central retina, but a thorough examination of retinal development in this species has not been reported. Here, the embryonic and postnatal development of the 13-LGS retina was studied to further characterize 13-LGS as a practical alternative animal model for investigating cone-based vision in health and disease. The spatiotemporal expression of key progenitor and cell type markers was examined in retinas from defined embryonic and postnatal stages using immunohistochemistry. Postnatal gene expression changes were validated by quantitative PCR. The 13-LGS neuroblastic layer expressed key progenitor markers (Sox2, Vsx2, Pax6, and Lhx2) at E18. Sequential cell fate determination evidenced by the first appearance of cell-type-specific marker labeling was at embryonic stage 18 (E18) with ganglion cells (Brn-3A, HuC/D) and microglia (Iba1); at E22.5 with photoreceptor progenitors (Otx2, recoverin) followed shortly by horizontal and amacrine cells (Lhx1, Oc1) at E24 to E25.5; and at postnatal stage 15 (P15) with bipolar cells (Vsx1, CaBP5) and Müller glia cells (GS, Rlbp1). Photoreceptor maturation indicated by opsin-positive outer segments and peanut agglutinin (PNA) labeling of cone sheaths was completed at the time of eye opening (P21-P24). The timeline and order of retinal cell development in the 13-LGS generally matches that recorded from other mammalian models but with a stark variation in the proportion of various cell types due to cone-dense photoreceptors. This thorough examination of an emerging translationally relevant cone-dominant specie provides a baseline for future disease modeling and stem cell approach studies of human vision.

Physical Cue‐Based Strategies on Peripheral Nerve Regeneration

AbstractPeripheral nerve injury (PNI) remains an intractable challenge in regenerative medicine. Recently, physical cue‐based strategies (e.g., electrical neurostimulation, acoustic radiation, electromagnetic bioregulation, as well as directional fiber guiding, etc.) have drawn increasing attention not only as a stimulator for cell functions modulation and fate determination, but also as a morphology‐index for modulating cell phenotype, proliferation, and differentiation, especially for nerve cells. More importantly, the advanced percutaneous power transmission technology, self‐power nanotechnology that leverages piezoelectrical/triboelectricity materials, and focused ultrasound and pulsed electromagnetic field technology exhibit the appealing practice potential for achieving low‐invasive, wireless, and battery‐free neuromodulation. In this review, recent advances of physical cue‐based strategies including electrical, acoustic, magnetic, and morphology for PNI are systematically overviewed, and the open challenges for realizing scalable clinical/commercial transformation and future perspectives of these strategies for PNI are concluded.

Exposure to bisphenol A induced oxidative stress, cell death and impaired epithelial homeostasis in the adult Drosophila melanogaster midgut

Recently, the chemical compound Bisphenol A (BPA) has been attracting worldwide attention due to its various toxic effects in animals, including reprotoxicity, neurotoxicity, hepatoxicity, and nephrotoxicity. Here, the midgut of adult Drosophila melanogaster (D. melanogaster), an invertebrate model organism, was employed to investigate the gastrointestinal toxicity of BPA in D. melanogaster and explore its underlying mechanisms of action in insects. As a result, exposure of flies to 0.5 mM BPA resulted in a dramatic morphological alteration of D. melanogaster midgut and decrease in survival rates and climbing ability of flies. Further study indicated that BPA induced high levels of oxidative stress in D. melanogaster midgut due to the imbalance between the production of reactive oxygen species and the activities of cellular antioxidant enzymes, including glutathione-S-transferase, catalase and superoxide dismutase. Oxidative stress induced by BPA then caused intestinal epithelial cell death and gut barrier dysfunction and elevated gut permeability, leading to oxidative injury of midgut epithelium. Antioxidant vitamin E alleviated midgut injury induced by BPA. Subsequently, BPA-induced oxidative injury of midgut further stimulated the proliferation of intestinal stem cell (ISC) and ISC-mediated midgut regeneration, but did not alter cell fate determination of ISCs in Drosophila midgut. Meanwhile, activation of Jun N-terminal kinase signal pathway was found to be required for BPA-induced cell death and tissue regeneration in midgut. Collectively, the present study provided additional evidence from an invertebrate model organism that BPA exposure induced gastrointestinal toxicity in D. melanogaster and further extended our understanding of the molecular mechanisms mediating BPA toxicity in insects.

CTR9 drives osteochondral lineage differentiation of human mesenchymal stem cells via epigenetic regulation of BMP-2 signaling.

Cell fate determination of human mesenchymal stem/stromal cells (hMSCs) is precisely regulated by lineage-specific transcription factors and epigenetic enzymes. We found that CTR9, a key scaffold subunit of polymerase-associated factor complex (PAFc), selectively regulates hMSC differentiation to osteoblasts and chondrocytes, but not to adipocytes. An in vivo ectopic osteogenesis assay confirmed the essentiality of CTR9 in hMSC-derived bone formation. CTR9 counteracts the activity of Enhancer Of Zeste 2 (EZH2), the epigenetic enzyme that deposits H3K27me3, in hMSCs. Accordingly, CTR9 knockdown (KD) hMSCs gain H3K27me3 mark, and the osteogenic differentiation defects of CTR9 KD hMSCs can be partially rescued by treatment with EZH2 inhibitors. Transcriptome analyses identified bone morphology protein-2 (BMP-2) as a downstream effector of CTR9. BMP-2 secretion, membrane anchorage, and the BMP-SMAD pathway were impaired in CTR9 KD MSCs, and the effects were rescued by BMP-2 supplementation. This study uncovers an epigenetic mechanism engaging the CTR9-H3K27me3-BMP-2 axis to regulate the osteochondral lineage differentiation of hMSCs.

Loss of Cell Fate Determinant LLGL1 Impairs Leukemia Development in Acute Myeloid Leukemia

Cell fate determinants and polarity regulators influence the fine balance between self-renewal and differentiation in HSC and leukemic stem cells (LSC). Previously our group provided first evidence that inactivation of the cell fate regulator Llgl1 in murine HSCs leads to alterations in self-renewal, proliferative capacity and differentiation (Heidel et al., 2013). The impact of Llgl1 on malignant transformation and especially acute myeloid leukemia (AML) development had not been investigated in detail. Inactivation of LLGL1 in several human AML cell lines (MOLM13, HEL, MV4;11, HL-60) by CRISPR/Cas9 resulted in decreased proliferation of leukemic cells. Consistently, xenograft studies using LLGL1-depleted human AML cells resulted in delayed disease development compared to non-targeting control (median survival NT-control: 37.5 days vs. LLGL1-KD: 92 days; p=0.0002). Genetic inactivation of LLGL1 in primary AML patient cells resulted in reduced colony formation in vitro and improved survival of recipient mice in a patient-derived xenograft model of AML. To recapitulate these findings in defined model systems of murine AML, we used a conditional Llgl1 knockout mouse model (Klezovitch et al, 2004) and intercrossed it with AML-models harboring different driver mutations: MllAF9 (Cerral et al., 1995) or Flt3ITD/ITD (Lee et al., 2006). Conditional deletion of Llgl1 in MllAF9 knockin mice using an Mx1-Cre-recombinase (Cre+) resulted in significant delay of leukemia development (median survival +/+: 174 days; -/-: 203 days; p=0.0361). To confirm these findings, we investigated a second oncogenic model and induced deletion of Llgl1 in Flt3ITD/ITD knockin mice. Leukemic Flt3ITD/ITD; Llgl1+/+; Cre+ animals (n=12) showed rapid onset of leukemia development, whereas the Flt3ITD/ITD; Llgl1-/-; Cre+ animals (n=25) showed significant delay in leukemia progression (median survival +/+: 38 days; -/-: 51 days; p=0.0005). Llgl1-/- animals show reduced peripheral blast counts (WBC +/+: 332.558 Gpt/l; -/-: 141.083 Gpt/l; p=0.0012) and decreased organ infiltration. Importantly, immunophenotypic analysis of bone marrow (BM) compartments indicated a shift of immature cells towards a more differentiated state. The overall abundance of leukemic Lin-Kit+Sca-1+ (LSK) cells was reduced in Flt3ITD/ITD; Llgl1-/- animals compared to Flt3ITD/ITD; Llgl1+/+ controls (LSK +/+: 13099±3144/106 BMC; -/-: 8117±2810 /106 BMC; p=0.0227). In contrast, we found increased numbers of Lin-Kit+Sca-1+FcgR+CD34+ of leukemic granulocyte-macrophage progenitors (LGMPs: +/+: 8306±1963/106 BMC; -/-: 14755±4783/106 BMC; p=0.0141) upon deletion of Llgl1 in Flt3ITD/ITD animals. These findings indicate that genetic inactivation of Llgl1 results in loss of leukemia stemness and shifts the leukemia stem cell phenotype from HSC-like to GMP-like LSCs. To test for this hypothesis, we performed global transcriptome analysis of sorted LSK cells of Flt3ITD/ITD mice early after genetic deletion of Llgl1. Among the significantly downregulated genes, we found several members of the HoxA-cluster, consistent with the immunophenotypic loss of stemness. Gene-set-enrichment analysis revealed downregulation of leukemia stem cell signatures (Gal et al., 2006) and enrichment of a gene set associated with LGMPs (compared to leukemic-HSC) signatures (Krivtsov et al., 2006). To validate whether loss of Hox-gene expression is relevant for impaired leukemia development, we performed rescue experiments in vivo. LSK cells derived from Flt3ITD/ITD; Llgl1+/+ or Flt3ITD/ITD; Llgl1-/- animals were retrovirally transduced with HoxA9 or empty vector control (eV). When transplanted into sublethally irradiated recipient mice, overexpression of HoxA9 (OE) in Llgl1-/-; Flt3ITD/ITD cells rescued the disease phenotype comparable to Flt3ITD/ITD; Llgl1+/+ cells (median survival +/+;eV: 62.50%; -/-; OE: 55.56%). In contrast, empty vector control Flt3ITD/ITD; Llgl1-/- recipients showed no disease development (median survival -/-; eV: 100%). Taken together our results may provide first evidence for a functional role of Llgl1 in models of AML. In contrast to its function in normal HSCs, deletion of Llgl1 appears to reduce stemness of leukemic stem cells and impair leukemia development in vitro and in vivo. Experiments to assess for the effects of Llgl1 on LSC polarity and cell fate decisions are currently under way.

The PRC2 Co-Factor JARID2 Regulates Hematopoiesis through Non-Canonical Mechanisms

Epigenetic mechanisms play an important role in the determination of hematopoietic stem and progenitor cell fate through the modification of chromatin architecture. Jarid2 is a co-factor of the polycomb repressive complex 2 (PRC2) that recruits this complex to different sites in the genome to catalyze the repressive epigenetic mark of Histone 3 Lysine 27 tri-methylation (H3K27me3). Previous studies from our lab have demonstrated that Jarid2 functions as a tumor suppressor in chronic myeloid neoplasms by recruiting PCR2 to silence self-renewal gene expression programs in committed hematopoietic progenitor cells. In the absence of Jarid2, mouse multipotent progenitor (MPP) cells gain ectopic self-renewal in normal hematopoiesis, and neoplastic transformation is accelerated from JAK2V617F-mutant MPN. However, our previous studies showed that in Jarid2-null MPPs, only about half of the upregulated genes were associated with a loss of H3K27me3, suggesting that Jarid2 might have important non-canonical (PCR2-independent) functions in hematopoiesis, which forms the scientific premise of the current study. When bound to PRC2, the lysine 116 (K116) residue of Jarid2 becomes methylated by the catalytic subunit Ezh2. Jarid2K116 methylation was shown to be indispensable for the regulation of the PRC2 catalytic activity through competing with the H3K27me3 binding to PRC2. Here, we created a separation of function mouse model to study the non-canonical functions of Jarid2 in hematopoiesis. Through CRISPR/Cas9 gene targeting, we created a genetic knock in mouse model by mutating lysine 116 to alanine (K116A), a point mutation that cannot be methylated by PRC2. We generated a Vav-cre:Jarid2fl/K116A(K116A) mouse model, that express one K116A mutated copy of Jarid2, while the other floxed copy will be conditionally knocked out by Vav-cre recombinase system in hematopoietic stem and progenitor cells (HSPCs). We hypothesized if Jarid2K116A HSPCs functionally phenocopy Vav-Cre:Jarid2fl/fl (Jarid2-KO) HSPCs then the function of Jarid2 in these cells is largely PCR2-dependent. However, if the output of Jarid2-K116A HSPCs resembles that of Vav-Cre:Jarid2fl/+ (Jarid2-HET) HSPCs, then this suggests Jarid2 has previously undescribed non-canonical functions in hematopoiesis. To characterize the functional effects of this mutation, we performed competitive transplantation of stem and progenitor subsets. Transplantation of MPP1 (Lineage-/c-Kit+/Sca1+/CD135-/CD48-/CD150-) cells revealed that Jarid2-K116A MPPs behaved similarly to Jarid2-KO MPPs. Recipients of both Jarid2-K116A and Jarid2-KO MPPs displayed higher peripheral blood (53% and 60%, respectively) and bone marrow (33% and 43%, respectively) engraftment compared to the control (12% and 3%, respectively) and Jarid2-HET (37.5% and 11%, respectively) MPPs. Additionally, Jarid2-K116A mutation enhanced donor MPP1 chimerism over recipient cells and promoted MPP1 self-renewal, similarly to what we previously described for Jarid2-KO MPP1 cells. These results suggest that Jarid2 function in MPP1 cells is largely dependent on PRC2. However, competitive transplantation of Jarid2-K116A HSCs (HSCs; Lineage-/c-Kit+/Sca1+/CD135-/CD48-/CD150+) revealed a striking phenotype. Jarid2-K116A HSCs showed a neomorphic phenotype compared to control, Jarid2-HET and Jarid2-KO HSCs in both primary and secondary transplantations. Jarid2-K116A mutation significantly enhanced the peripheral blood and bone marrow myeloid engraftment (both 40% increase). Furthermore, Jarid2-K116A enhanced HSC fitness and self-renewal with these mutant HSCs able to gain increased chimerism in the HSC pool and increased donor-derived HSC number in the bone marrow of recipient mice (Figure 1). These results suggest that the unmethylated version of Jarid2-K116 exerts a neomorphic effect on HSC function confirming the importance of the post-translational modification at the K116 residue of Jarid2 in hematopoiesis. Thus far, our studies suggest that Jarid2 function is dependent on PRC2 in MPPs, but may act independently of PRC2 in HSCs. Ongoing studies are focused on examining the importance of PCR2-related Jarid2 function in malignant hematopoiesis, and determining if the ratio of methylated to unmethylated JARID2 influences leukemic transformation of human chronic myeloid disorders. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cellular Plasticity As Mechanisms to Escape from NOTCH1-Inhibition in T-ALL

Cellular plasticity has recently emerged as an underlying mechanism for treatment refraction in cancer. Hence, characterizing cell states and the transcriptional changes required for state transitions is critical for the development of efficient targeted therapies. Acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy in children and adolescents that is associated with high rates of treatment failure and early relapse. T-ALL patients frequently harbor NOTCH1 activating mutations as the driving oncogene in this disease. Using T-ALL patient-derived xenograft (PDX) models carrying activating NOTCH1 mutations, we aim to u First, to determine how cell state diversity drives treatment refraction in T-ALL, we performed full-length single-cell transcriptome analysis on 3188 immune cells collected from 2 sensitive and 3 refractory PDX models. PDX models were treated with either with NOTCH inhibitor DBZ (Dipenzazepine; 10 μM/kg every other day intravenously) or vehicle. Profiled cells were collected at both a short (5 days) and a late time point (x-150 days) post-treatment to assess the effects of DBZ. Analysis of early hematopoietic and thymic precursor signatures demonstrate expression of immature hematopoietic signatures and acquisition of alternative lineage identities coexisting within the same cell, predominantly in refractory models. Analyses of pre-treatment samples indicated that disruption of the normal developmental hierarchy was associated with blockade at an early stage of T-cell commitment and can predict response to NOTCH inhibition in vivo. Upon treatment, cells underwent dramatic transcriptional reprogramming resulting in expansion of 2 distinct populations that greatly differ in both differentiation stage and developmental trajectory. One population expresses lymphoid differentiation programs, whereas the other population is enriched in genes associated with RNA processing and more immature hematopoietic signatures. Cell fate trajectories assessment using Monocle 2, revealed that consistent with their immature hematopoietic signature, refractory cells map at the start point of the differentiation trajectory, while sensitive cells clustered at the endpoint. We next sought to characterize the differentiation hierarchies that define the immature states. Developmental potential as assessed by CytoTRACE was higher in refractory cells when compared to their sensitive counterpart. To identify determinants of cell fate in a functionally relevant manner, we inferred gene regulatory network configurations using SCENIC and defined state specific regulon activity. Among the most prominently expressed regulons present in mature states, we identified transcriptional regulators involved in T-cell differentiation such as IKAROS, GATA3, and SOX5, while in immature states we observed high transcriptional regulon activity in transcription factors associated with renewal of hematopoietic progenitors such as ATF4, BCLAF1, and MYC in addition to the chromatin remodelers SMARCA4 and EZH2. To assess whether transcriptional rewiring in refractory cells results from greater chromatin accessibility, we performed single-cell ATAC sequencing (10x Genomics) on splenic cells from 1 sensitive and 1 refractory T-ALL PDX model treated with DBZ or vehicle. After integration with corresponding single-cell RNA-sequencing data, annotated ATAC profiles demonstrated greater chromatin accessibility, predominantly in active promotor and enhancer regions, in the refractory model compared to the sensitive model. Peak to gene linkage analysis revealed widespread alterations in cis-regulatory regions near genes associated with immature hematopoietic precursor signatures such as CD33. Furthermore, greater chromatin accessibility in refractory cells was associated with an increased number of enhancer loops per expressed gene as determined by H3K27ac HiChIP analysis. In conclusion, we demonstrated that presence of highly plastic cellular states defined by aberrant differentiation trajectories, distinct transcriptional circuitries, and remodeled chromatin architecture results in treatment escape in T-ALL.

The bHLH Transcription Factors in Neural Development and Therapeutic Applications for Neurodegenerative Diseases.

The development of functional neural circuits in the central nervous system (CNS) requires the production of sufficient numbers of various types of neurons and glial cells, such as astrocytes and oligodendrocytes, at the appropriate periods and regions. Hence, severe neuronal loss of the circuits can cause neurodegenerative diseases such as Huntington's disease (HD), Parkinson's disease (PD), Alzheimer's disease (AD), and Amyotrophic Lateral Sclerosis (ALS). Treatment of such neurodegenerative diseases caused by neuronal loss includes some strategies of cell therapy employing stem cells (such as neural progenitor cells (NPCs)) and gene therapy through cell fate conversion. In this report, we review how bHLH acts as a regulator in neuronal differentiation, reprogramming, and cell fate determination. Moreover, several different researchers are conducting studies to determine the importance of bHLH factors to direct neuronal and glial cell fate specification and differentiation. Therefore, we also investigated the limitations and future directions of conversion or transdifferentiation using bHLH factors.

Blockage of PPARγ T166 phosphorylation enhances the inducibility of beige adipocytes and improves metabolic dysfunctions

Beige adipocytes in mammalian white adipose tissue (WAT) can reinforce fat catabolism and energy expenditure. Promoting beige adipocyte biogenesis is a tantalizing tactic for combating obesity and its associated metabolic disorders. Here, we report that a previously unidentified phosphorylation pattern (Thr166) in the DNA-binding domain of PPARγ regulates the inducibility of beige adipocytes. This unique posttranslational modification (PTM) pattern influences allosteric communication between PPARγ and DNA or coactivators, which impedes the PPARγ-mediated transactivation of beige cell-related gene expression in WAT. The genetic mutation mimicking T166 phosphorylation (p-T166) hinders the inducibility of beige adipocytes. In contrast, genetic or chemical intervention in this PTM pattern favors beige cell formation. Moreover, inhibition of p-T166 attenuates metabolic dysfunction in obese mice. Our results uncover a mechanism involved in beige cell fate determination. Moreover, our discoveries provide a promising strategy for guiding the development of novel PPARγ agonists for the treatment of obesity and related metabolic disorders.

Spatial dynamic metabolomics identifies metabolic cell fate trajectories in human kidney differentiation.

Accumulating evidence demonstrates important roles for metabolism in cell fate determination. However, it is a challenge to assess metabolism at a spatial resolution that acknowledges both heterogeneity and cellular dynamics in its tissue microenvironment. Using a multi-omics platform to study cell-type-specific dynamics in metabolism in complex tissues, we describe the metabolic trajectories during nephrogenesis in the developing human kidney. Exploiting in situ analysis of isotopic labeling, a shift from glycolysis toward fatty acid β-oxidation was observed during the differentiation from the renal vesicle toward the S-shaped body and the proximal tubules. In addition, we show that hiPSC-derived kidney organoids are characterized by a metabolic immature phenotype that fails to use mitochondrial long-chain fatty acids for energy metabolism. Furthermore, supplementation of butyrate enhances tubular epithelial differentiation and maturation in cultured kidney organoids. Our findings highlight the relevance of understanding metabolic trajectories to efficiently guide stem cell differentiation.