Lineage-specific Gene Expression Programs Research Articles

Cellular development and evolution of the mammalian cerebellum

The expansion of the neocortex, a hallmark of mammalian evolution1,2, was accompanied by an increase in cerebellar neuron numbers3. However, little is known about the evolution of the cellular programmes underlying the development of the cerebellum in mammals. In this study we generated single-nucleus RNA-sequencing data for around 400,000 cells to trace the development of the cerebellum from early neurogenesis to adulthood in human, mouse and the marsupial opossum. We established a consensus classification of the cellular diversity in the developing mammalian cerebellum and validated it by spatial mapping in the fetal human cerebellum. Our cross-species analyses revealed largely conserved developmental dynamics of cell-type generation, except for Purkinje cells, for which we observed an expansion of early-born subtypes in the human lineage. Global transcriptome profiles, conserved cell-state markers and gene-expression trajectories across neuronal differentiation show that cerebellar cell-type-defining programmes have been overall preserved for at least 160 million years. However, we also identified many orthologous genes that gained or lost expression in cerebellar neural cell types in one of the species or evolved new expression trajectories during neuronal differentiation, indicating widespread gene repurposing at the cell-type level. In sum, our study unveils shared and lineage-specific gene-expression programmes governing the development of cerebellar cells and expands our understanding of mammalian brain evolution.

Single-cell analysis of prenatal and postnatal human cortical development.

We analyzed >700,000 single-nucleus RNA sequencing profiles from 106 donors during prenatal and postnatal developmental stages and identified lineage-specific programs that underlie the development of specific subtypes of excitatory cortical neurons, interneurons, glial cell types, and brain vasculature. By leveraging single-nucleus chromatin accessibility data, we delineated enhancer gene regulatory networks and transcription factors that control commitment of specific cortical lineages. By intersecting our results with genetic risk factors for human brain diseases, we identified the cortical cell types and lineages most vulnerable to genetic insults of different brain disorders, especially autism. We find that lineage-specific gene expression programs up-regulated in female cells are especially enriched for the genetic risk factors of autism. Our study captures the molecular progression of cortical lineages across human development.

ARID4B loss leads to activated STAT1-dependent interferon pathway in mouse embryonic stem cells and during meso/endodermal differentiation.

Proper deactivation of the pluripotency network and activation of a lineage-specific gene expression program are critical for mouse embryonic stem cell (mESC) differentiation. This is achieved by the coordinated action of transcription and chromatin factors. Our previous work identified ARID4B as a critical chromatin factor for mesoderm and endoderm differentiation. As part of a histone deacetylase complex, ARID4B plays a role in transcriptional suppression of its direct targets. Here, we investigated the mechanism of ARID4B function in mESC differentiation by focusing on genes and pathways that are upregulated in its absence. We analyzed transcriptomic results of wild-type and arid4bΔ endoderm or mesoderm differentiated cells through integrative genomics viewer and ingenuity pathway analysis. We performed real-time quantitative polymerase chain reaction for selected genes. To understand pathway activation, we performed Western blot for candidate proteins during the time-course of differentiation. We also analyzed H3K4me3, H3K27me3 and H3K27Ac ChIP-seq results to understand changes in the chromatin environment. Interferon-related genes were activated in arid4bΔ mESCs and endoderm or mesoderm directed cells. Consistent with this, higher phosphorylated STAT1 levels were found in arid4bΔ mESCs while a related phosphorylated STAT3 was unchanged. Finally, we observed a significant increase in H3K4me3 around interferon-related distal gene regulatory regions with a combination of either upregulation of H3K27Ac level or downregulation of H3K27me3 level. These results provide evidence that ARID4B is involved in the suppression of interferon-related genes in mESCs and during meso/endoderm differentiation through modulation, mainly of H3K4me3. This regulation might be important for successful mESC differentiation.

DNA dioxygenases Tet2/3 regulate gene promoter accessibility and chromatin topology in lineage-specific loci to control epithelial differentiation.

Execution of lineage-specific differentiation programs requires tight coordination between many regulators including Ten-eleven translocation (TET) family enzymes, catalyzing 5-methylcytosine oxidation in DNA. Here, by using Keratin 14-Cre-driven ablation of Tet genes in skin epithelial cells, we demonstrate that ablation of Tet2/Tet3 results in marked alterations of hair shape and length followed by hair loss. We show that, through DNA demethylation, Tet2/Tet3 control chromatin accessibility and Dlx3 binding and promoter activity of the Krt25 and Krt28 genes regulating hair shape, as well as regulate interactions between the Krt28 gene promoter and distal enhancer. Moreover, Tet2/Tet3 also control three-dimensional chromatin topology in Keratin type I/II gene loci via DNA methylation-independent mechanisms. These data demonstrate the essential roles for Tet2/3 in establishment of lineage-specific gene expression program and control of Dlx3/Krt25/Krt28 axis in hair follicle epithelial cells and implicate modulation of DNA methylation as a novel approach for hair growth control.

Detecting Cell Compartment-Specific PRC2-RNA Interactions via UV-RIP.

Upon cellular reprogramming, the activity of polycomb repressive complex 2 (PRC2), together with histone demethylases, is essential for the suppression of cell lineage-specific gene expression programs, for resetting of epigenetic memory and for the reacquisition of pluripotency.PRC2 requires interaction with RNAs for the correct protein complex assembly and recruitment on chromatin. Moreover, PRC2 components can be found in different cell compartments and their intracellular dynamics is part of their functional activity. Several loss-of-function studies revealed that many lncRNAs expressed upon reprogramming are essential for the silencing of lineage-specific genes and the function of chromatin modifiers. Compartment-specific UV-RIP technique is a method that will help understanding which is the nature of those interactions, with no interference from indirect interactions typical of methods involving the use of chemical cross-linkers or performed in native conditions with non-stringent buffers. This technique will shed lights on the specificity of lncRNA interaction and PRC2 stability/activity on chromatin and whether PRC2-lncRNA interaction occurs in specific cell compartments.

Mechanisms of Polycomb Group Protein-Mediated Fetal Hemoglobin Silencing

Reactivation of fetal hemoglobin (HbF) is beneficial for patients with β-globin gene disorders, such as sickle cell disease and β-thalassemia. Polycomb repressive complex 1 (PRC1) and 2 (PRC2) are multi-subunit protein complexes that function as transcriptional repressors to maintain lineage-specific gene expression programs. Prior works showed that genetic or pharmacologic manipulation of select PRC2 subunits can induce HbF expression in adult erythroid cells, and an inhibitor targeting the EED subunit of PRC2 has entered a clinical trial. Yet the molecular basis underlying HbF regulation by PRC1/2 complexes is unknown. Using a CRISPR-Cas9-based genetic screen, we identified BMI1, a subunit of the PRC1 complex as novel HbF repressor. Disrupting BMI1 in the adult-type erythroid cell line HUDEP2 or primary human erythroblast cells robustly increased HbF production. We failed to detect BMI1 binding at the β-globin gene locus by CUT&RUN, indicating that BMI1 functions via an indirect mechanism. RNA-seq analysis of BMI1-deficient HUDEP2 cells uncovered upregulation of two known HbF regulators: IGF2BP1 and LIN28B, both of which induce HbF when forcibly expressed in adult erythroid cells. Depletion of either IGF2BP1 or LIN28B partially restored HbF silencing in BMI1-deficient HUDEP2 cells, whereas simultaneous depletion of IGF2BP1 and LIN28B lowered HbF production virtually to basal levels in these cells. In primary erythroid cells, IGF2BP3, which is functionally related to IGF2BP1, also contributes to the effects of BMI1 depletion. Together, these data indicate that IGF2BP1, IGF2BP3, and LIN28B are the critical BMI1 targets in human erythroid cells. Since BMI1 functions in two distinct PRC1 complexes (cPRC1 and ncPRC1), we aimed to define which of these is critical for HbF regulation. cPRC1 is defined by the presence of CBX proteins 2, 4, 6, 7, and 8, while ncPRC1 contains RYBP or its homolog YAF2. ncPRC1 and cPRC1 deposit the histone mark H2AK119ub1, and facilitate chromatin compaction, respectively. Disruption of CBX2/4/7/8 proteins but not RYAP/YAF2 in HUDEP2 cells activated HbF to levels similar to those observed upon BMI1 depletion, implicating cPRC1 in HbF control. Accordingly, CUT&RUN analysis in adult erythroid cells revealed a strong genome-wide co-localization of BMI1 and CBX2/4/7/8, including at the IGF2BP1, IGF2BP3, and LIN28B genes. Finally, BMI1 depletion does not lead to significant changes in H2AK119ub1 at these genes, in line with the fact that the repressive activity of the cPRC1 complex does not rely on its H2A ubiquitination activity. Together these results indicate that BMI1 represses HbF via the cPRC1 complex in adult erythroid cells. The BMI1-containing cPRC1 complex is known to repress transcription in conjunction with the PRC2 complex which deposits the histone mark H3K27me3. CUT&RUN profiling studies revealed focal loss of H3K27me3 in BMI1-deficient HUDEP2 cells, including at the CG-rich regions near the promoters of the IGF2BP1 and LIN28B genes. Therefore, we tested whether cPRC1 and PRC2 complexes converge on HbF regulation by regulating IGF2BP1 and LIN28B. Disruption in HUDEP2 cells of EZH2, the catalytic component of PRC2, led to global depletion of H3K27me3 along with upregulation of IGF2BP1 and LIN28B. Co-depletion of IGF2BP1 and LIN28B largely restored the EZH2 depletion-induced HbF upregulation. In summary, our studies further define the nature of PRC complexes involved in HbF regulation and identify the IGF2BP1, IGF2BP3, and LIN28B genes as critical links between PcG activity and HbF silencing. Our results may guide the development of improved PRC inhibitors for the treatment of β-globin disorders.

Transcriptomics, regulatory syntax, and enhancer identification in mesoderm-induced ESCs at single-cell resolution.

Embryonic stem cells (ESCs) can adopt lineage-specific gene-expression programs by stepwise exposure to defined factors, resulting in the generation of functional cell types. Bulk and single-cell-based assays were employed to catalog gene expression, histone modifications, chromatin conformation, and accessibility transitions in ESC populations and individual cells acquiring a presomitic mesoderm fate and undergoing further specification toward myogenic and neurogenic lineages. These assays identified cis-regulatory regions and transcription factors presiding over gene-expression programs occurring at defined ESC transitions and revealed the presence of heterogeneous cell populations within discrete ESC developmental stages. The datasets were employed to identify previously unappreciated genomic elements directing the initial activation of Pax7 and myogenic and neurogenic gene-expression programs. This study provides a resource for the discovery of genomic and transcriptional features of pluripotent, mesoderm-induced ESCs and ESC-derived cell lineages.

723 DNA dioxygenases Tet2/3 regulate gene promoter accessibility and three-dimensional chromatin topology in lineage-specific loci to control hair growth

Lineage-specific and functionally-related genes frequently form conserved clusters or loci in the mammalian genomes. Execution of lineage-specific differentiation programs requires tight coordination between many regulators including Ten-eleven translocation (TET) family enzymes catalyzing DNA oxidation. However, the role of Tet genes in the control of cell differentiation at the levels of lineage-specific gene loci is unknown. Epithelial keratin genes are organized in the genome into two distinct (Keratin type I and II) loci on mouse chromosomes 11 and 15. Here, by using Keratin 14-driven ablation of Tet genes in skin epithelial cells, we demonstrate that Tet enzymes play essential roles in the control of gene expression in Keratin type I/II gene loci and execution of hair-specific differentiation program. Mice with Keratin 14-driven ablation of Tet2/Tet3 or all three Tet genes exhibit marked alterations of hair shape and length followed by hair loss. We show that through DNA demethylation, Tet2 and Tet3 control chromatin accessibility, Dlx3 binding and promoter activity of the Krt25 and Krt28 genes regulating hair shape, as well as regulate interactions between the Krt28 gene promoter and distal enhancer. However, Tet2/3 also control three-dimensional chromatin topology in Keratin type I/II gene loci via catalytic activity-independent mechanisms. These data demonstrate for the first time the essential roles for Tet2/3 in establishment of lineage-specific gene expression program and control of Dlx3/Krt25/Krt28 axis in hair follicle epithelial cells and implicate modulation of DNA methylation as a novel approach for hair growth control.

P1271: ELUCIDATING TUMOR SUPPRESSOR FUNCTIONS OF TET2 IN B CELL LYMPHOMAS

Background: Remodeling of the epigenetic landscape via DNA cytosine methylation directs cell lineage specification, establishing and maintaining gene expression programs that uniquely define given cell types. During tumorigenesis the epigenome becomes corrupted, impairing lineage-specific gene expression programs. TET2, an enzyme that contributes to the erasure of cytosine methylation via oxidation of the 5’ methyl group, is among the most frequent somatically mutated genes in human hematological tumors, commonly identified as loss-of-function versions. In mouse models, TET2 loss on its own is only weakly tumorigenic, but rather facilitates leukemic transformation driven by prototypic oncogenes such as AML-ETO or FLT3ITD. However, TET2 LOF is not limited to leukemias. In B cell malignancies, TET2 is found mutated in a fraction of diffuse large B cell lymphomas (DLBCL), which display a distinct gene expression profile. Aims: Having established a role for TET2 loss-of-function in enhancing MYC-driven lymphomas, our overall aim is to understand how TET2 loss conspires with MYC to overcome the barriers that maintain cell identity and function. Methods: Using pre-clinical mouse models and in vitro culture systems, we establish that TET2 loss boosts the formation of B cell lymphomas driven by the MYC oncoprotein. Results: Thus, (1) TET2 deficiency specifically in B cells facilitates MYC-mediated transformation in a dose dependent manner, and (2) TET2 deficiency early on in hematopoietic stem and progenitor cells enhances the transformative potential later on in antigen-activated B cells. Our data suggest that TET2 may exert tumor suppressor functions via controlling the expression of DNA repair genes, or via direct functions of oxidized cytosine species. In addition, TET2-deficient lymphomas select for high expression of the pro-survival BCL2 family protein BCLXL, potentially providing protection from MYC-driven apoptosis. Summary/Conclusion: Altogether, our data concerning the impact of TET2 loss-of-function on DNA repair and apoptosis mechanisms creates understanding of how TET2 deficiency nudges transformation, and reveals testable therapeutic vulnerabilities.

ARID4B is critical for mouse embryonic stem cell differentiation towards mesoderm and endoderm, linking epigenetics to pluripotency exit

Distinct cell types emerge from embryonic stem cells through a precise and coordinated execution of gene expression programs during lineage commitment. This is established by the action of lineage specific transcription factors along with chromatin complexes. Numerous studies have focused on epigenetic factors that affect embryonic stem cells (ESC) self-renewal and pluripotency. However, the contribution of chromatin to lineage decisions at the exit from pluripotency has not been as extensively studied. Using a pooled epigenetic shRNA screen strategy, we identified chromatin-related factors critical for differentiation toward mesodermal and endodermal lineages. Here we reveal a critical role for the chromatin protein, ARID4B. Arid4b-deficient mESCs are similar to WT mESCs in the expression of pluripotency factors and their self-renewal. However, ARID4B loss results in defects in up-regulation of the meso/endodermal gene expression program. It was previously shown that Arid4b resides in a complex with SIN3A and HDACS 1 and 2. We identified a physical and functional interaction of ARID4B with HDAC1 rather than HDAC2, suggesting functionally distinct Sin3a subcomplexes might regulate cell fate decisions Finally, we observed that ARID4B deficiency leads to increased H3K27me3 and a reduced H3K27Ac level in key developmental gene loci, whereas a subset of genomic regions gain H3K27Ac marks. Our results demonstrate that epigenetic control through ARID4B plays a key role in the execution of lineage-specific gene expression programs at pluripotency exit.

Dynamic changes in cis-regulatory occupancy by Six1 and its cooperative interactions with distinct cofactors drive lineage-specific gene expression programs during progressive differentiation of the auditory sensory epithelium.

The transcription factor Six1 is essential for induction of sensory cell fate and formation of auditory sensory epithelium, but how it activates gene expression programs to generate distinct cell-types remains unknown. Here, we perform genome-wide characterization of Six1 binding at different stages of auditory sensory epithelium development and find that Six1-binding to cis-regulatory elements changes dramatically at cell-state transitions. Intriguingly, Six1 pre-occupies enhancers of cell-type-specific regulators and effectors before their expression. We demonstrate in-vivo cell-type-specific activity of Six1-bound novel enhancers of Pbx1, Fgf8, Dusp6, Vangl2, the hair-cell master regulator Atoh1 and a cascade of Atoh1’s downstream factors, including Pou4f3 and Gfi1. A subset of Six1-bound sites carry consensus-sequences for its downstream factors, including Atoh1, Gfi1, Pou4f3, Gata3 and Pbx1, all of which physically interact with Six1. Motif analysis identifies RFX/X-box as one of the most significantly enriched motifs in Six1-bound sites, and we demonstrate that Six1-RFX proteins cooperatively regulate gene expression through binding to SIX:RFX-motifs. Six1 targets a wide range of hair-bundle regulators and late Six1 deletion disrupts hair-bundle polarity. This study provides a mechanistic understanding of how Six1 cooperates with distinct cofactors in feedforward loops to control lineage-specific gene expression programs during progressive differentiation of the auditory sensory epithelium.

NUR77 HAS A CELL INTRINSIC ROLE IN NATURAL KILLER T CELL DEVELOPMENT AND FUNCTION

Abstract Natural killer T (NKT) cells are innate-like lymphocytes that respond to self and foreign glycolipids under inflammatory conditions. NKT cells have constitutively high levels of Nur77, an orphan nuclear receptor family transcription factor that is induced very early during their ontogeny. Nur77 function in NKT cells has not been explored, however. Nur77 is known to transcriptionally control negative selection of conventional T cells, development of regulatory T cells and differentiation of effector CD8+ T cells. Thus, we hypothesized that Nur77 controls a lineage-specific gene expression program essential for the proper development of functional NKT cells. We found that, akin to conventional T cells, NKT cells poorly developed in transgenic mice that overexpressed wild type Nur77 (wtNur77tg) in T lineage only. Introgression of the rearranged Va14i a-chain gene into wtNur77tg mice (wtNur77tg; Va14tg mice) rescued NKT cell development but was arrested at the precursor stage 0. NKT cells in wtNur77tg; Va14tg mice expressed lower levels of PLZF when compared to NKT cells in non-transgenic mice. So also, NKT cells in wtNur77tg; Va14tg mice do not respond to glycolipid agonists. These findings are consistent with differences in the transcripomes of stage 0 NKT cells isolated from Va14tg versus wtNur77tg; Va14tg mice. Collectively, our data suggest that Nur77 induction initiates a gene expression program in developing NKT cells that are critical for developmental progression and function of NKT cells.

RHOA activity in expanding blastocysts is essential to regulate HIPPO-YAP signaling and to maintain the trophectoderm-specific gene expression program in a ROCK/actin filament-independent manner.

What molecular signals are required to maintain the functional trophectoderm (TE) during blastocyst expansion of the late stage of preimplantation development? The activity of ras homology family member A (RHOA) GTPases is necessary to retain the expanded blastocyst cavity and also to sustain the gene expression program specific to TE. At the early stages of preimplantation development, the precursor of the TE lineage is generated through the molecular signals that integrate RHOA, RHO-associated coiled-coil containing protein kinase (ROCK), the apicobasal cell polarity, and the HIPPO-Yes-associated protein (YAP) signaling pathway. By contrast, molecular mechanisms regulating the maintenance of the TE characteristics at the later stage, which is crucial for blastocyst hatching and implantation, are scarcely understood. Expanding mouse blastocysts, obtained from crosses of the F1 (C57BL6 × DBA/2) strain, were exposed to chemical agents that interfere with RHOA, ROCK, or the actin cytoskeleton for up to 8 h, and effects on the blastocyst cavity, HIPPO-YAP signaling, and cell lineage-specific gene expression profiles were examined. Mouse embryos at the embryonic stage E3.5 (expanding blastocysts) and E4.5 (fully expanded blastocysts) were treated with RHOA inhibitor (C3 exoenzyme), ROCK inhibitor (Y27632), or actin filament disruptors (cytochalasin B and latrunculin A). The integrity of the blastocyst cavity was evaluated based on the gross morphology. Effects on HIPPO-YAP signaling were assessed based on the presence of nuclearized YAP protein by immunofluorescence staining and the expression of YAP/TEA domain family member (TEAD) target genes by quantitative RT-PCR (qRT-PCR). The impact of these disruptors on cell lineages was evaluated based on expression of the TE-specific and inner cell mass-specific marker genes by qRT-PCR. The integrity of the apicobasal cell polarity was assessed by localization of protein kinase C zeta (PRKCZ; apical) and scribbled planar cell polarity (SCRIB; basal) proteins by immunofluorescence staining. For comparisons, cultured cell lines, NIH/3T3 (mouse fibroblast) and P19C5 (mouse embryonal carcinoma), were also treated with RHOA inhibitor, ROCK inhibitor, and actin filament disruptors for up to 8 h, and effects on HIPPO-YAP signaling were assessed based on expression of YAP/TEAD target genes by qRT-PCR. Each experiment was repeated using three independent batches of embryos (n = 40-80 per batch) or cell collections. Statistical analyses of data were performed, using one-way ANOVA and two-sample t-test. Inhibition of RHOA deflated the cavity, diminished nuclear YAP (P < 0.01), and down-regulated the YAP/TEAD target and TE-specific marker genes in both E3.5 and E4.5 blastocysts (P < 0.05), indicating that the maintenance of the key TE characteristics is dependent on RHOA activity. However, inhibition of ROCK or disruption of actin filament only deflated the blastocyst cavity, but did not alter HIPPO-YAP signaling or lineage-specific gene expressions, suggesting that the action of RHOA to sustain the TE-specific gene expression program is not mediated by ROCK or the actomyosin cytoskeleton. By contrast, ROCK inhibitor and actin filament disruptors diminished YAP/TEAD target gene expressions in cultured cells to a greater extent than RHOA inhibitor, implicating that the regulation of HIPPO-YAP signaling in expanding blastocysts is distinctly different from that in the cell lines. Furthermore, the apicobasal cell polarity proteins in the expanding blastocyst were mislocalized by ROCK inhibition but not by RHOA inhibition, indicating that cell polarity is not linked to regulation of HIPPO-YAP signaling. Taken together, our study suggests that RHOA activity is essential to maintain the TE lineage in the expanding blastocyst and it regulates HIPPO-YAP signaling and the lineage-specific gene expression program through mechanisms that are independent of ROCK or actomyosin cytoskeleton. Not applicable. This study was conducted using one species, the mouse. Direct translation of the experiments and findings to human fertility preservation and ART requires further investigations. The elucidation of the mechanisms of TE formation is highly pertinent to fertility preservation in women. Our findings may raise awareness among providers of ART that the TE is sensitive to disturbance even in the late stage of blastocyst expansion and that rational approaches should be devised to avoid conditions that may impair the TE and its function. This study was funded by grants from the Ingeborg v.F. McKee Fund of the Hawaii Community Foundation (16ADVC-78882 to V.B.A.), and the National Institutes of Health (P20 GM103457 and R03 HD088839 to V.B.A.). The authors have no conflict of interest to declare.

Chromatin-mediated translational control is essential for neural cell fate specification.

Neural cell fate specification is a multistep process in which stem cells undergo sequential changes in states, giving rise to particular lineages such as neurons and astrocytes. This process is accompanied by dynamic changes of chromatin and in transcription, thereby orchestrating lineage-specific gene expression programs. A pressing question is how these events are interconnected to sculpt cell fate. We show that altered chromatin due to loss of the chromatin remodeler Chd5 causes neural stem cell activation to occur ahead of time. This premature activation is accompanied by transcriptional derepression of ribosomal subunits, enhanced ribosome biogenesis, and increased translation. These untimely events deregulate cell fate decisions, culminating in the generation of excessive numbers of astrocytes at the expense of neurons. By monitoring the proneural factor Mash1, we further show that translational control is crucial for appropriate execution of cell fate specification, thereby providing new insight into the interplay between transcription and translation at the initial stages of neurogenesis.

The histone demethylase UTX regulates the lineage-specific epigenetic program of invariant natural killer T cells.

Invariant natural killer T (iNKT) cells are innate-like lymphocytes that protect against infection, autoimmune disease and cancer. However, little is known about epigenetic regulation of iNKT cell development. Here, we show that the H3K27me3 histone demethylase UTX is an essential cell-intrinsic factor that controls an iNKT lineage-specific gene expression program and epigenetic landscape in a demethylase activity dependent manner. UTX-deficient iNKT cells exhibited impaired expression of iNKT signature genes due to a decrease in activation-associated H3K4me3 and an increase in repressive H3K27me3 marks within the promoters that UTX occupies. We identified JunB as a novel regulator of iNKT development and show that target gene expression of both JunB and iNKT master transcription factor PLZF was UTX-dependent. We determined iNKT super-enhancers and demonstrated that UTX-mediated regulation of super-enhancer accessibility was a key mechanism for iNKT lineage commitment. These findings reveal how UTX regulates iNKT cell development through multiple epigenetic mechanisms.

Organization and Regulation of the Human Genome

The 3-dimensional (3D) chromatin organization plays a critical role in gene regulation. Great strides have been made recently to characterize and identify cis regulatory elements from epigenome profiles in different cell types and tissues, but efforts have just begun to functionally characterize these long-range control elements. Mapping interactions between enhancers and promoters, and understanding how the 3D landscape of the genome constrains such interactions is fundamental to our understanding of genome function. I will present recent findings related to 3D genome organization in mammalian cells, with a particular focus on how chromatin organization contributes to transcriptional regulation. I will describe higher-order organizational features that are observed at the level of both the whole chromosome and individual loci. I will highlight changes in genome organization that occur during the course of differentiation, and discuss the functional relationship between chromatin architecture and gene regulation. Taken together, mounting evidence now shows that the genome organization plays an essential role in orchestrating the lineage-specific gene expression programs through modulating long- range interactions between enhancers and target genes. DisclosuresRen:Arima Genomics, Inc.: Equity Ownership, Patents & Royalties; Eli Lilly: Employment.

Enhancers and their dynamics during hematopoietic differentiation and emerging strategies for therapeutic action.

Cellular differentiation requires precisely regulated tissue-specific and developmental stage-specific gene expression patterns. Numerous studies have highlighted the predictive power of enhancers on lineage-specific gene expression programs and have started to unravel their mechanisms of action. We review here the dynamics of the enhancer landscape during hematopoietic differentiation and how enhancers function in the context of the 3D organization of the genome. We further discuss the involvement of aberrant enhancer activity in human diseases and emerging strategies aiming at controlling enhancer activity and chromosome topology for therapeutic purposes.

Involvement of Polycomb Repressive Complex 2 in Maturation of Induced Pluripotent Stem Cells during Reprogramming of Mouse and Human Fibroblasts.

Induced pluripotent stem cells (iPSCs) provide a reliable source for the study of regenerative medicine, drug discovery, and developmental biology. Despite extensive studies on the reprogramming of mouse and human fibroblasts into iPSCs, the efficiency of reprogramming is still low. Here, we used a bioinformatics and systems biology approach to study the two gene regulatory waves governing the reprogramming of mouse and human fibroblasts into iPSCs. Our results revealed that the maturation phase of reprogramming was regulated by a more complex regulatory network of transcription factors compared to the initiation phase. Interestingly, in addition to pluripotency factors, the polycomb repressive complex 2 (PRC2) members Ezh2, Eed, Jarid2, Mtf2, and Suz12 are crucially recruited during the maturation phase of reprogramming. Moreover, we found that during the maturation phase of reprogramming, pluripotency factors, via the expression and induction of PRC2 complex members, could silence the lineage-specific gene expression program and maintain a ground state of pluripotency in human and mouse naïve iPSCs. The findings obtained here provide us a better understanding of the gene regulatory network (GRN) that governs reprogramming, and the maintenance of the naïve state of iPSCs.

Comprehensive profiling reveals mechanisms of SOX2-mediated cell fate specification in human ESCs and NPCs.

SOX2 is a key regulator of multiple types of stem cells, especially embryonic stem cells (ESCs) and neural progenitor cells (NPCs). Understanding the mechanism underlying the function of SOX2 is of great importance for realizing the full potential of ESCs and NPCs. Here, through genome-wide comparative studies, we show that SOX2 executes its distinct functions in human ESCs (hESCs) and hESC-derived NPCs (hNPCs) through cell type- and stage-dependent transcription programs. Importantly, SOX2 suppresses non-neural lineages in hESCs and regulates neurogenesis from hNPCs by inhibiting canonical Wnt signaling. In hESCs, SOX2 achieves such inhibition by direct transcriptional regulation of important Wnt signaling modulators, WLS and SFRP2. Moreover, SOX2 ensures pluripotent epigenetic landscapes via interacting with histone variant H2A.Z and recruiting polycomb repressor complex 2 to poise developmental genes in hESCs. Together, our results advance our understanding of the mechanism by which cell type-specific transcription factors control lineage-specific gene expression programs and specify cell fate.

Gro/TLE enables embryonic stem cell differentiation by repressing pluripotent gene expression

Gro/TLE proteins (TLE1-4) are a family of transcriptional corepressors acting downstream of multiple signalling pathways. Several TLEs are expressed in a dynamic manner throughout embryonic development and at high levels in embryonic stem cells (ESCs). Here we find that Gro/TLE is not required in ESC for sustaining pluripotency and suppressing differentiation genes, but rather is important for the shutting down of the pluripotency network in differentiation. Consistent with this view, we found that one of the Gro/TLE family, TLE4 is expressed heterogeneously in ESCs in a population that corresponds to a Nanog low subset of ESC culture. TLE4 expression is also increased in response to LIF withdrawal and Fgf/Mek/Erk stimulation. To explore the role of Gro/TLE in more detail we generated an allelic series of knockout ESCs of two TLE genes expressed most dynamically in early differentiation, TLE3 and TLE4. Genetic reduction in TLE dose resulted in an increase in the expression of pluripotency markers and inhibition of ESC differentiation towards both epiblast and endoderm lineages. Overexpression of a drug inducible TLE4 could both rescue TLE3/TLE4 compound phenotypes and induce early expression of endoderm (Hhex-Venus) and neural (Sox1-GFP) reporter genes. Taken together, our results suggest that TLE activity is essential for early differentiation where it acts to suppress the pluripotency network, allowing for the initiation of lineage specific gene expression programs.