Application of CRISPR/Cas9 Pooled Screening for a Non-immediate Readout Model.

Linking phenotypes to genetic components has been an essential part of novel drug discovery, and screening methods have been widely employed to achieve such a goal. Screens can be conducted in either pooled or arrayed formats. Although arrayed screenings provide a better and cheaper alternative in small scale, the larger-scale screenings are conducted in pooled manner. With its adaptability to various models and conditions, CRISPR/Cas9 technology provides an invaluable alternative to classical and RNAi-based screening methods. Combined with high-throughput sequencing and bioinformatics, CRISPR-/Cas9-based pooled screening methods provide unbiased and robust data. In this protocol, we employed CRISPR-/Cas9-based pooled screening for a non-binary and non-immediate readout.

SETD3 regulates endoderm differentiation of mouse embryonic stem cells through canonical Wnt signaling pathway.

With self-renewal and pluripotency features, embryonic stem cells (ESCs) provide an invaluable tool to investigate early cell fate decisions. Pluripotency exit and lineage commitment depend on precise regulation of gene expression that requires coordination between transcription (TF) and chromatin factors in response to various signaling pathways. SET domain-containing 3 (SETD3) is a methyltransferase that can modify histones in the nucleus and actin in the cytoplasm. Through an shRNA screen, we previously identified SETD3 as an important factor in the meso/endodermal lineage commitment of mouse ESCs (mESC). In this study, we identified SETD3-dependent transcriptomic changes during endoderm differentiation of mESCs using time-course RNA-seq analysis. We found that SETD3 is involved in the timely activation of the endoderm-related gene network. The canonical Wnt signaling pathway was one of the markedly altered signaling pathways in the absence of SETD3. The assessment of Wnt transcriptional activity revealed a significant reduction in Setd3-deleted (setd3∆) mESCs coincident with a decrease in the nuclear pool of the key TF ß-catenin level, though no change was observed in its mRNA or total protein level. Furthermore, a proximity ligation assay (PLA) found an interaction between SETD3 and ß-catenin. We were able to rescue the differentiation defect by stably re-expressing SETD3 or activating the canonical Wnt signaling pathway by changing mESC culture conditions. Our results suggest that alterations in the canonical Wnt pathway activity and subcellular localization of ß-catenin might contribute to the endoderm differentiation defect of setd3∆ mESCs.

Roles and Regulation of H3K4 Methylation During Mammalian Early Embryogenesis and Embryonic Stem Cell Differentiation.

From generation of germ cells, fertilization, and throughout early mammalian embryonic development, the chromatin undergoes significant alterations to enable precise regulation of gene expression and genome use. Methylation of histone 3 lysine 4 (H3K4) correlates with active regions of the genome, and it has emerged as a dynamic mark throughout this timeline. The pattern and the level of H3K4 methylation are regulated by methyltransferases and demethylases. These enzymes, as well as their protein partners, play important roles in early embryonic development and show phenotypes in embryonic stem cell self-renewal and differentiation. The various roles of H3K4 methylation are interpreted by dedicated chromatin reader proteins, linking this modification to broader molecular and cellular phenotypes. In this review, we discuss the regulation of different levels of H3K4 methylation, their distinct accumulation pattern, and downstream molecular roles with an early embryogenesis perspective.

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

Objective: 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. Material and Methods: 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. Results: 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. Conclusion: 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.

Directed Differentiation of Mouse Embryonic Stem Cells to Mesoderm, Endoderm, and Neuroectoderm Lineages.

The self-renewal and pluripotency features of mouse embryonic stem cells (mESCs) make them a great tool to study early mammalian development. Various signaling pathways that shape early mammalian development can be mimicked for in vitro mESC differentiation toward primitive lineages first and more specialized cell types later. Since the precise nature of the molecular mechanisms and the crosstalk between these signaling pathways is yet to be fully understood, there is a high level of variability in the efficiency and synchronicity among available differentiation protocols. Commitment of mESCs toward mesoderm, endoderm, or neuroectoderm lineages happens over only a few days and is highly efficient. Here, we provide protocols for the directed differentiation of mESCs toward these lineages in vitro.

Mouse Embryonic Stem Cell Culture in Serum-Containing or 2i Conditions.

With their unique capabilities of self-renewal and differentiation into three germ layers, mouse embryonic stem cells (mESCs) are widely used as an in vitro cellular model for early mammalian developmental studies. mESCs are traditionally cultured in high-serum and LIF-containing medium on a growth-deficient mouse embryonic fibroblast layer. A more recent culturing system with two inhibitors (for GSK3ß (CHIR99021) and MEK1/2 (PD0325901)) and LIF enables the derivation of mESC lines from various mouse strains. Here we describe methods for the mESC growth and maintenance in each medium composition as well as their adaptation to either condition.

Arid4b physically interacts with Tfap2c in mouse embryonic stem cells.

Precise regulation of gene expression is required for embryonic stem cell (ESC) differentiation. Transcription factor (TF) networks coordinate the balance of pluripotency and differentiation in response to extracellular and intracellular signals. Chromatin factors work alongside TFs to achieve timely regulation of gene expression for differentiation process. Our previous studies showed that a member of the Sin3a corepressor complex, Arid4b, is critical for proper mouse ESC differentiation into mesoderm and endoderm. We found elevated histone 3 lysine 27 acetylation (H3K27Ac) in a subset of genomic loci in meso/endoderm directed arid4bΔ cells, coincident with their derepression. We reasoned that Sin3a complex may be required for the suppression of these genes during differentiation. To identify TFs that might cooperate with Arid4b for this function, we found consensus TF binding sequences enriched in H3K27Ac elevated regions in arid4bΔ cells. Of these candidate TFs, we validated expression of Bach1, Ddit3, Prrx2, Znf354c and Tfap2c in mESCs. We then demonstrated a physical interaction between Arid4b and Tfap2c in mESCs using endogenous coimmunoprecipitation and proximity ligation assay experiments. Our results point to a role of Arid4b in the Sin3a complex in repression of a subset of Tfap2c-regulated genes during meso/endoderm differentiation.

Arid4b alters cell cycle and cell death dynamics during mouse embryonic stem cell differentiation.

Cell division and death play an important role in embryonic development. Cell specialization is accompanied with slow proliferation and quiescence. Cell death is important for morphogenesis. Gene expression changes during differentiation is coordinated by lineage-specific transcription factors and chromatin factors. It is not yet fully understood how alterations in gene expression and cell cycle/death mechanisms are connected. We previously identified a chromatin protein Arid4b as a critical factor for meso/endoderm differentiation of mouse embryonic stem cells (mESCs). The differentiation defect of Arid4b-deficient mESCs might be due to misregulation of cell proliferation or death. Here, we identified a role for Arid4b in cell cycle rewiring at the onset of differentiation. Arid4b-deficient differentiating cells have less proliferative capacity and their cell cycle profile is more similar to mESC stage than the differentiating wild-type cells. We found no evidence of increased DNA damage or checkpoint activation. Our investigation of cell death mechanisms found no contribution from autophagy but revealed a slight increase in Caspase-3 activation implying early apoptosis in Arid4b-deficient differentiating cells. Taken together, our data suggest Arid4b regulates cell cycle alterations during exit from pluripotency. Future studies will be instrumental in understanding whether these changes directly contribute to Arid4b-dependent differentiation control.

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.