Stem cell-based approach to identify regulatory TFs during mammalian cell differentiation
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PROVIDER: PRJNA1201124 | ENA |
REPOSITORIES: ENA
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Project description:Cell differentiation is controlled by transcription factors (TFs). However, we still do not fully understand which TFs are needed for even one mammalian differentiation pathway. To address this, we developed a detailed and integrated method. This method combines pluripotent stem cell differentiation in the lab, single-cell transcriptomics, and a CRISPR-based TF loss-of-function screen. Using this approach, we identified all the TFs required for spinal motor neuron (MN) differentiation in mice and their requirement in human MN differentiation. Our transcriptomic analysis revealed that while over 1,300 TFs are transcribed during ventral spinal cord differentiation, only 55 have annotated functions in the literature. The screen identified 232 TFs and other transcription-regulating genes essential for the progression of ventral spinal cord differentiation, including 116 TFs specifically required for MN differentiation. This approach led to three critical observations. First, the motor neuron progenitor (pMN) stage is a regulatory hub that correlates with a reduction in cell potential. Second, a secondary screen in human ventral spinal cord differentiation revealed a greater degree of conservation j between mouse pMNs and the human-specific ventral MN progenitors (vpMNs). This finding was unexpected, as the conservation was anticipated to be with the canonical human pMN stage. Third, the screen underscored the importance of the numerous and yet understudied ZF TFs controlling mammalian cell differentiation. In summary, we present a universal, unbiased strategy for studying TFs in mammalian differentiation, comparable to methodologies used in simpler organisms. This approach enhances our understanding of MN differentiation and provides a practical framework to identify all TFs required for any differentiation trajectory that can be recapitulated in vitro, offering many applications in various fields of developmental biology.
2025-09-01 | GSE285057 | GEO
Project description:Enhancers play a central role in cell-type-specific gene expression and are marked by H3K4me1/2. Active enhancers are further marked by H3K27ac. However, the methyltransferases responsible for the deposition of H3K4me1/2 on enhancers remain elusive. Furthermore, the functions of these methyltransferases on enhancers and associated cell-type-specific gene expression are poorly understood. Here, we identify MLL4 (KMT2D) as a major H3K4 mono- and di-methyltransferase in mammalian cells. Using adipogenesis and myogenesis as model systems, we show that MLL4 exhibits cell-type- and differentiation-stage-specific genomic binding and is predominantly localized on enhancers. MLL4 co-localizes with lineage-determining transcription factors (TFs) on active enhancers during differentiation. Deletion of MLL4 dramatically decreases H3K4me1/2 and H3K27ac on enhancers and leads to severe defects in cell-type-specific gene expression and cell differentiation. Finally, we provide evidence that lineage-determining TFs recruit and require MLL4 to establish enhancers critical for cell-type-specific gene expression. Together, these results identify MLL4 as an H3K4 mono-/di-methyltransferase required for enhancer activation during cell differentiation. ChIP-Seq analyses of adipogenic TF (C/EBPalpha, C/EBPbeta, and PPARgamma) and Pol II profiles at D0 (day 0) and D2 (day 2) of adipogenesis in WT (MLL3-/-) and MLL4 KO (MLL3-/-;MLL4-/-) brown preadipocytes.
2013-12-17 | E-GEOD-50416 | biostudies-arrayexpress
Project description:Evolutionarily recent TFs partake in human cell cycle regulation
| PRJNA1179459 | ENA
Project description:Enhancers play a central role in cell-type-specific gene expression and are marked by H3K4me1/2. Active enhancers are further marked by H3K27ac. However, the methyltransferases responsible for the deposition of H3K4me1/2 on enhancers remain elusive. Furthermore, the functions of these methyltransferases on enhancers and associated cell-type-specific gene expression are poorly understood. Here, we identify MLL4 (KMT2D) as a major H3K4 mono- and di-methyltransferase in mammalian cells. Using adipogenesis and myogenesis as model systems, we show that MLL4 exhibits cell-type- and differentiation-stage-specific genomic binding and is predominantly localized on enhancers. MLL4 co-localizes with lineage-determining transcription factors (TFs) on active enhancers during differentiation. Deletion of MLL4 dramatically decreases H3K4me1/2 and H3K27ac on enhancers and leads to severe defects in cell-type-specific gene expression and cell differentiation. Finally, we provide evidence that lineage-determining TFs recruit and require MLL4 to establish enhancers critical for cell-type-specific gene expression. Together, these results identify MLL4 as an H3K4 mono-/di-methyltransferase required for enhancer activation during cell differentiation.
2013-12-17 | GSE50416 | GEO
Project description:mammalian cell-lines Transcriptome or Gene expression
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Project description:Evolutionarily recent TFs partake in human cell cycle regulation [BRDU]
| PRJNA1179464 | ENA
Project description:A multiplexed in vivo gene knockout approach to identify cancer drivers in small cell lung cancer
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Project description:Integrated Genomics Approach to Identify Biologically Relevant Alterations in Fewer Samples.
| PRJEB11800 | ENA