Abstract MP142: Genome-wide Studies Reveal the Essential and Opposite Roles of Arid1a in Controlling Human Cardiogenesis and Neurogenesis From Pluripotent Stem Cells

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Juli Liu ◽  
Sheng Liu ◽  
Hongyu Gao ◽  
Lei Han ◽  
Xiaona Chu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Binding ◽  
Brain Development ◽  
Chromatin Remodeling ◽  
Neural Development ◽  
Human Heart ◽  
Chromatin Accessibility ◽  
Neurogenic Genes ◽  
Genome Wide ◽  
Early Human

Background: Early human heart and brain development simultaneously occur during embryogenesis. Notably, in human newborns, congenital heart defects strongly associate with neurodevelopmental abnormalities, suggesting a common gene/complex underlying both cardiogenesis and neurogenesis. However, due to lack of in vivo studies, the molecular mechanisms that govern both early human heart and brain development remain elusive. The evolutionarily conserved ATP-dependent SWI/SNF complex is one of the largest chromatin remodeling complexes, consisting of ~15 subunits, including SMARCA2 (also known as BRM) or SMARCA4 (also known as BRG1) as the ATPase catalytic subunit. Several BRG1-associated factors (BAFs), such as ARID1A (Baf250a), have DNA binding capacity and assemble with either BRM or BRG1 to form a functional chromatin-remodeling complex. A single amino acid mutation (Arid1aV 1068G/V1068G ), impaired Arid1a-DNA interactions and resulted in both cardiac neural defects. Mutations in 4 different SWI/SNF subunits including ARID1A/B were identified in human congenital syndromes that include both neural and cardiac defects. Results: Here, we report ARID1A, which is a DNA-binding-subunit of the SWI/SNF epigenetic complex, controls both neurogenesis and cardiogenesis from human embryonic stem cells (hESCs) via employing distinct mechanisms. CRISPR/Cas-9 knockout of ARID1A (ARID1A -/- ) led to spontaneous differentiation of neural cells together with globally enhanced expression of neurogenic genes in undifferentiated hESCs. Additionally, when compared with WT hESCs, cardiac differentiation from ARID1A -/- hESCs was prominently suppressed, whereas neural differentiation was significantly promoted. Whole genome-wide ChIP-seq and ATAC-seq analyses revealed that ARID1A was required to open chromatin accessibility on promoters of essential cardiogenic genes, and temporally associated with key cardiogenic transcriptional factors T and MEF2C during early cardiac development. However, during neural development, transcription of most essential neurogenic genes was dependent on ARID1A and ARID1A could interact with REST, which is a known transcriptional repressor. Conclusions: We uncovered the key and opposite roles by ARID1A to govern both early human cardiac and neural development and characterized the mechanisms. We found global chromatin accessibility on cardiogenic genes was dependent on ARID1A, whereas transcriptional activity of neurogenic genes was regulated by ARID1A, possibly through ARID1A-REST interaction.

scholarly journals Genome-wide Studies Reveal the Essential and Opposite Roles of ARID1A in Controlling Human Cardiogenesis and Neurogenesis from Pluripotent Stem Cells

2020 ◽  
Author(s):  
Juli Liu ◽  
Sheng Liu ◽  
Hongyu Gao ◽  
Lei Han ◽  
Xiaona Chu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Brain Development ◽  
Neural Development ◽  
Pluripotent Stem Cells ◽  
Human Heart ◽  
Chromatin Accessibility ◽  
Open Chromatin ◽  
Neurogenic Genes ◽  
Genome Wide ◽  
Early Human

AbstractBackgroundEarly human heart and brain development simultaneously occur during embryogenesis. Notably, in human newborns, congenital heart defects strongly associate with neurodevelopmental abnormalities, suggesting a common gene/complex underlying both cardiogenesis and neurogenesis. However, due to lack of in vivo studies, the molecular mechanisms that govern both early human heart and brain development remain elusive.ResultsHere, we report ARID1A, which is a DNA-binding-subunit of the SWI/SNF epigenetic complex, controls both neurogenesis and cardiogenesis from human embryonic stem cells (hESCs) via employing distinct mechanisms. Knockout of ARID1A (ARID1A-/-) led to spontaneous differentiation of neural cells together with globally enhanced expression of neurogenic genes in undifferentiated hESCs. Additionally, when compared with WT hESCs, cardiac differentiation from ARID1A-/- hESCs was prominently suppressed, whereas neural differentiation was significantly promoted. Whole genome-wide scRNA-seq, ATAC-seq, and ChIP-seq analyses revealed that ARID1A was required to open chromatin accessibility on promoters of essential cardiogenic genes, and temporally associated with key cardiogenic transcriptional factors T and MEF2C during early cardiac development. However, during early neural development, transcription of most essential neurogenic genes was dependent on ARID1A, which could interact with a known neural restrictive silencer factor REST/NRSF.ConclusionsWe uncovered the opposite roles by ARID1A to govern both early cardiac and neural development from pluripotent stem cells. Global chromatin accessibility on cardiogenic genes is dependent on ARID1A, whereas transcriptional activity of neurogenic genes is under control by ARID1A, possibly through ARID1A-REST/NRSF interaction.

scholarly journals Satb2 acts as a gatekeeper for major developmental transitions during early vertebrate embryogenesis

2020 ◽  
Author(s):  
Saurabh J. Pradhan ◽  
Puli Chandramouli Reddy ◽  
Michael Smutny ◽  
Ankita Sharma ◽  
Keisuke Sako ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Dna Binding ◽  
Chromatin Accessibility ◽  
Dna Binding Proteins ◽  
Developmental Transitions ◽  
Pluripotency Factors ◽  
Neural Crest Development ◽  
Genome Wide ◽  
Genome Activation ◽  
Zygotic Genome

AbstractZygotic genome activation (ZGA) initiates regionalized transcription responsible for the acquisition of distinct cellular identities. ZGA is dependent upon dynamic chromatin architecture sculpted by conserved DNA-binding proteins. However, whether the tissue-specific transcription is mechanistically linked with the onset of ZGA is unknown. Here, we have addressed the involvement of chromatin organizer SATB2 in orchestrating these processes during vertebrate embryogenesis. Integrative analysis of transcriptome, genome-wide occupancy and chromatin accessibility revealed contrasting molecular functions of maternal and zygotic pools of Satb2. Maternal Satb2 represses zygotic genes by influencing the interplay between the pluripotency factors. By contrast, zygotic Satb2 activates transcription of the same group of genes during neural crest development and organogenesis. Comparative analysis of maternal versus zygotic function of Satb2 underscores how these antithetical activities are temporally coordinated and functionally implemented. We discuss the evolutionary implications of the biphasic and bimodal regulation of landmark developmental transitions by a single determinant.

scholarly journals CHD7 promotes neural progenitor differentiation in embryonic stem cells via altered chromatin accessibility and nascent gene expression

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hui Yao ◽  
Douglas F. Hannum ◽  
Yiwen Zhai ◽  
Sophie F. Hill ◽  
Ricardo D.’Oliveira Albanus ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Chromatin Remodeling ◽  
Embryonic Stem ◽  
Neural Progenitor ◽  
Chromatin Accessibility ◽  
Nervous System Development ◽  
Conditional Knockout ◽  
Glial Differentiation ◽  
Cell Stage

Abstract CHARGE syndrome, a rare multiple congenital anomaly condition, is caused by haploinsufficiency of the chromatin remodeling protein gene CHD7 (Chromodomain helicase DNA binding protein 7). Brain abnormalities and intellectual disability are commonly observed in individuals with CHARGE, and neuronal differentiation is reduced in CHARGE patient-derived iPSCs and conditional knockout mouse brains. However, the mechanisms of CHD7 function in nervous system development are not well understood. In this study, we asked whether CHD7 promotes gene transcription in neural progenitor cells via changes in chromatin accessibility. We used Chd7 null embryonic stem cells (ESCs) derived from Chd7 mutant mouse blastocysts as a tool to investigate roles of CHD7 in neuronal and glial differentiation. Loss of Chd7 significantly reduced neuronal and glial differentiation. Sholl analysis showed that loss of Chd7 impaired neuronal complexity and neurite length in differentiated neurons. Genome-wide studies demonstrated that loss of Chd7 leads to modified chromatin accessibility (ATAC-seq) and differential nascent expression (Bru-Seq) of neural-specific genes. These results suggest that CHD7 acts preferentially to alter chromatin accessibility of key genes during the transition of NPCs to neurons to promote differentiation. Our results form a basis for understanding the cell stage-specific roles for CHD7-mediated chromatin remodeling during cell lineage acquisition.

Chromatin State Dynamics Underlying CD8 T Cell Differentiation and Dysfunction in Cancer

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 861-861
Author(s):  
Mary Philip ◽  
Lauren Fairchild ◽  
Liping Sun ◽  
Agnes Viale ◽  
Taha Merghoub ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Chromatin Remodeling ◽  
Cd8 T Cell ◽  
Chromatin Accessibility ◽  
T Cell Differentiation ◽  
Therapeutic Interventions ◽  
Transcriptional Networks ◽  
Genome Wide

Abstract T cells recognizing tumor-specific antigens are detected in cancer patients but are dysfunctional. Upon antigen encounter, T cells differentiate into discrete phenotypic and functional states. Cellular differentiation is driven by epigenetic remodeling, however, it is not known whether and how epigenetic programming establishes and regulates tumor-specific T cell (TST) dysfunction and determines a T cell's ability to respond to therapeutic interventions such as immune checkpoint blockade (PD-1 and CTLA-4). Here for the first time, we (1) identify chromatin dynamics underlying T cell differentiation to the dysfunctional state in mouse and human tumors and (2) provide insights into the epigenetic and transcriptional regulatory mechanisms determining T cell susceptibility to therapeutic reprogramming. Using a genetic cancer mouse model, we previously showed that CD8 TST become unresponsive early during carcinogenesis at the pre-malignant stage, even before the emergence of a pathologically-defined malignant tumor. While T cell dysfunction was initially reversible, it ultimately became a fixed state that could not be rescued by therapeutic interventions such as PD1 checkpoint blockade. To identify the hierarchical changes in chromatin states resulting in "dysfunction imprinting," we used the Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq) to map the genome-wide changes in chromatin accessibility in TST cells over the course of cancer development. In parallel, we carried out RNA-Seq to determine the interplay between chromatin remodeling and transcriptional networks. Substantial chromatin remodeling occurred during early T cell activation in the pre-malignant lesion (days 5-7) followed by a second wave of chromatin accessibility changes between days 7 and 14. Strikingly, after the second wave, no further CD8 T cell chromatin remodeling occurred during carcinogenesis, even after progression to an advanced late-stage tumor with an immunosuppressive microenvironment. Interestingly, these 2 distinct chromatin accessibility patterns in TST correlated temporally with the plastic and fixed dysfunctional states and susceptibility to therapeutic reprogramming in vivo. To understand the transition from plastic to fixed dysfunction, we analyzed the differential expression of transcription factors (TF) in conjunction with changes in peak accessibility at TF-binding motifs genome-wide. We identified a network including CD8 T cell regulatory TF such as TCF1, LEF1, BLIMP1, and BACH2 as well as less-well-characterized TF (NR4A2, TOX) potentially controlling differentiation to the dysfunctional state. Moreover, ATAC-Seq analysis of human tumor-infiltrating CD8 T cells revealed similar tumor-associated changes in peak accessibility, and studies are ongoing to assess the associated TF networks. In this study, we have defined discrete chromatin states and associated transcriptional networks underlying plastic and fixed dysfunction in TST, thus providing new insights into the genomic control circuitry of T cell differentiation/dysfunction that may point to new strategies for cellular reprogramming of T cells for cancer immunotherapy. Disclosures No relevant conflicts of interest to declare.

scholarly journals Human iPSC-derived Down syndrome astrocytes display genome-wide perturbations in gene expression, an altered adhesion profile, and increased cellular dynamics

2020 ◽  
Vol 29 (5) ◽  
pp. 785-802 ◽  
Author(s):  
Blandine Ponroy Bally ◽  
W Todd Farmer ◽  
Emma V Jones ◽  
Selin Jessa ◽  
J Benjamin Kacerovsky ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Down Syndrome ◽  
Cell Adhesion ◽  
Brain Development ◽  
Trisomy 21 ◽  
Chromatin Accessibility ◽  
Chromosome 21 ◽  
Genome Wide ◽  
And Function ◽  
The Impact

Abstract Down syndrome (DS), caused by the triplication of human chromosome 21, leads to significant alterations in brain development and is a major genetic cause of intellectual disability. While much is known about changes to neurons in DS, the effects of trisomy 21 on non-neuronal cells such as astrocytes are poorly understood. Astrocytes are critical for brain development and function, and their alteration may contribute to DS pathophysiology. To better understand the impact of trisomy 21 on astrocytes, we performed RNA-sequencing on astrocytes from newly produced DS human induced pluripotent stem cells (hiPSCs). While chromosome 21 genes were upregulated in DS astrocytes, we found consistent up- and down-regulation of genes across the genome with a strong dysregulation of neurodevelopmental, cell adhesion and extracellular matrix molecules. ATAC (assay for transposase-accessible chromatin)-seq also revealed a global alteration in chromatin state in DS astrocytes, showing modified chromatin accessibility at promoters of cell adhesion and extracellular matrix genes. Along with these transcriptomic and epigenomic changes, DS astrocytes displayed perturbations in cell size and cell spreading as well as modifications to cell-cell and cell-substrate recognition/adhesion, and increases in cellular motility and dynamics. Thus, triplication of chromosome 21 is associated with genome-wide transcriptional, epigenomic and functional alterations in astrocytes that may contribute to altered brain development and function in DS.

scholarly journals An interpretable bimodal neural network characterizes the sequence and preexisting chromatin predictors of induced TF binding

2019 ◽  
Author(s):  
Divyanshi Srivastava ◽  
Begüm Aydin ◽  
Esteban O. Mazzoni ◽  
Shaun Mahony
Keyword(s):  
Neural Network ◽  
Dna Binding ◽  
Dna Sequences ◽  
Binding Specificity ◽  
Chromatin Accessibility ◽  
Cell Type ◽  
Genome Wide ◽  
Binding Preference ◽  
Cell Type Specific ◽  
Insight Into

AbstractTranscription factor (TF) binding specificity is determined via a complex interplay between the TF’s DNA binding preference and cell type-specific chromatin environments. The chromatin features that correlate with TF binding in a given cell type have been well characterized. For instance, the binding sites for a majority of TFs display concurrent chromatin accessibility. However, concurrent chromatin features reflect the binding activities of the TF itself, and thus provide limited insight into how genome-wide TF-DNA binding patterns became established in the first place. To understand the determinants of TF binding specificity, we therefore need to examine how newly activated TFs interact with sequence and preexisting chromatin landscapes.Here, we investigate the sequence and preexisting chromatin predictors of TF-DNA binding by examining the genome-wide occupancy of TFs that have been induced in well-characterized chromatin environments. We develop Bichrom, a bimodal neural network that jointly models sequence and preexisting chromatin data to interpret the genome-wide binding patterns of induced TFs. We find that the preexisting chromatin landscape is a differential global predictor of TF-DNA binding; incorporating preexisting chromatin features improves our ability to explain the binding specificity of some TFs substantially, but not others. Furthermore, by analyzing site-level predictors, we show that TF binding in previously inaccessible chromatin tends to correspond to the presence of more favorable cognate DNA sequences. Bichrom thus provides a framework for modeling, interpreting, and visualizing the joint sequence and chromatin landscapes that determine TF-DNA binding dynamics.

scholarly journals Mitotic chromosome binding predicts transcription factor properties in interphase

2018 ◽  
Author(s):  
Mahé Raccaud ◽  
Andrea B. Alber ◽  
Elias T. Friman ◽  
Harsha Agarwal ◽  
Cédric Deluz ◽  
...  
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Mitotic Chromosome ◽  
Specific Binding ◽  
Site Occupancy ◽  
Chromatin Accessibility ◽  
Live Cells ◽  
Specific Binding Sites ◽  
Genome Wide ◽  
Chromosome Binding

SummaryMammalian transcription factors (TFs) differ broadly in their nuclear mobility and sequence-specific/non-specific DNA binding affinity. How these properties affect the ability of TFs to occupy their specific binding sites in the genome and modify the epigenetic landscape is unclear. Here we combined live cell quantitative measurements of mitotic chromosome binding (MCB) of 502 TFs, measurements of TF mobility by fluorescence recovery after photobleaching, single molecule imaging of DNA binding in live cells, and genome-wide mapping of TF binding and chromatin accessibility. MCB scaled with interphase properties such as association with DNA-rich compartments, mobility, as well as large differences in genome-wide specific site occupancy that correlated with TF impact on chromatin accessibility. As MCB is largely mediated by electrostatic, non-specific TF-DNA interactions, our data suggests that non-specific DNA binding of TFs enhances their search for specific sites and thereby their impact on the accessible chromatin landscape.

scholarly journals ARID1A Is Critical for Maintaining Normal Hematopoiesis in Mice

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3833-3833
Author(s):  
Lin Han ◽  
Vikas Madan ◽  
Anand Mayakonda ◽  
Pushkar Dakle ◽  
Weoi Woon Teoh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Chromatin Remodeling ◽  
B Cell Lymphoma ◽  
Chromatin Accessibility ◽  
Brdu Incorporation ◽  
Rna Seq ◽  
Hematopoietic Stem ◽  
Hematopoietic Development

Abstract ARID1A is a key component of ATP-dependent SWI/SNF complex involved in chromatin remodeling. Chromatin remodeling mediated by SWI/SNF complex is crucial for gene expression and affects a broad range of biological processes including hematopoietic development. ARID1A is frequently mutated across several solid tumors as well as hematopoietic malignancies, including Burkitt's lymphoma, diffuse large B-cell lymphoma and acute promyelocytic leukemia. Nevertheless, function of ARID1A in adult hematopoiesis and implications of its deficiency in development and progression of hematopoietic diseases has not been explored. In this study, we used a murine model of ARID1A deficiency to establish its essential function in maintaining normal hematopoietic development. Germline loss of Arid1a is embryonic lethal; therefore, we generated mice with deletion of Arid1a specifically in the hematopoietic compartment using Vav-iCre and Mx1-Cre transgenic mice. Arid1afl/fl;Vav-iCre+ mice occurred at a lower than expected frequency, suggesting some perinatal mortality. For the Mx1-Cre model, Arid1a exon 9 was excised by administrating poly(I:C) to adult mice and hematopoiesis was evaluated using flow cytometry. An increase in both percentage and absolute number of long-term hematopoietic stem cells (LTHSCs) defined as Lin-Sca1+Kit+CD34-FLT3- or Lin-Sca1+Kit+CD48-CD150+ occurred in the bone marrow using both models of Arid1a deficiency. RNA-sequencing of sorted LTHSCs from Arid1a KO bone marrow revealed dysregulated expression of several genes involved in cell cycle, G2/M checkpoint and related pathways. In vivo BrdU incorporation assays showed a substantially lower proportion of quiescent hematopoietic stem cells in Arid1a deficient bone marrow. To assess the reconstitution ability of ARID1A deficient HSCs, sorted KO or WT LTHSCs were transplanted into irradiated congenic recipient mice in competitive repopulation assays. Proportion of donor-derived cells in recipients transplanted with KO cells was strikingly lower compared to wild-type cells, suggesting poor reconstitution ability of Arid1a KO LTHSCs. Also, differentiation of both myeloid and lymphoid lineages was impaired in Arid1a KO mice compared to WT controls. To investigate the mechanism of perturbed differentiation of the myeloid and erythroid lineages, RNA-Seq was performed on sorted CMPs, GMPs and MEPs from WT and Arid1a KO BM. Our analysis showed significant decrease in expression of several transcription factors (Runx1, Gata2, Cebpa), which play a crucial role in lineage differentiation. To determine how Arid1a deficiency alters chromatin accessibility in myeloid precursors, Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-Seq) was performed on sorted Lin-Kit+ BM cells from both Arid1a KO and WT mice. A global reduction in open chromatin in Arid1a KO cells was noted compared to WT cells. A substantial overlap occurred between down regulated genes (RNA-seq) and reduced chromatin accessibility in Arid1a KO myeloid progenitors. Motifs for PU.1, RUNX1, GATA and CEBPA were significantly enriched in loci with reduced ATAC-seq signals in Arid1a KO cells. Our findings demonstrate an indispensable function of Arid1a in hematopoietic development and underline the importance of precise chromatin dynamics maintained by ARID1A-containing SWI/SNF complex in hematopoiesis. Disclosures No relevant conflicts of interest to declare.

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