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Single-cell lineage analysis reveals extensive multimodal transcriptional control during directed beta-cell differentiation.

ABSTRACT: The in vitro differentiation of insulin-producing beta-like cells can model aspects of human pancreatic development. Here, we generate 95,308 single-cell transcriptomes and reconstruct a lineage tree of the entire differentiation process from human embryonic stem cells to beta-like cells to study temporally regulated genes during differentiation. We identify so-called 'switch genes' at the branch point of endocrine/non-endocrine cell fate choice, revealing insights into the mechanisms of differentiation-promoting reagents, such as NOTCH and ROCKII inhibitors, and providing improved differentiation protocols. Over 20% of all detectable genes are activated multiple times during differentiation, even though their enhancer activation is usually unimodal, indicating extensive gene reuse driven by different enhancers. We also identify a stage-specific enhancer at the TCF7L2 locus for diabetes, uncovered by genome-wide association studies, that drives a transient wave of gene expression in pancreatic progenitors. Finally, we develop a web app to visualize gene expression on the lineage tree, providing a comprehensive single-cell data resource for researchers studying islet biology and diabetes.

SUBMITTER: Weng C

PROVIDER: S-EPMC7744443 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Commitment of pluripotent stem cells into differentiated cells and associated gene expression necessitate specific epigenetic mechanisms that modify the DNA and corresponding histone proteins to render the chromatin in an open or closed state. This in turn dictates the associated genetic machinery, including transcription factors, acknowledging the cellular signals provided. Activating histone methyltransferases represent crucial enzymes in the epigenetic machinery that cause transcription initiation by delivering the methyl mark on histone proteins. A number of studies have evidenced the vital role of one such histone modifier, DOT1L, in transcriptional regulation. Involvement of DOT1L in differentiating pluripotent human embryonic stem (hES) cells into the cardiac lineage has not yet been investigated. METHODS:The study was conducted on in-house derived (KIND1) and commercially available (HES3) human embryonic stem cell lines. Chromatin immunoprecipitation (ChIP) was performed followed by sequencing to uncover the cardiac genes harboring the DOT1L specific mark H3K79me2. Following this, dual immunofluorescence was employed to show the DOT1L co-occupancy along with the cardiac progenitor specific marker. DOT1L was knocked down by siRNA to further confirm its role during cardiac differentiation. RESULTS:ChIP sequencing revealed a significant number of peaks characterizing H3K79me2 occupancy in the proximity of the transcription start site. This included genes like MYOF, NR2F2, NKX2.5, and HAND1 in cardiac progenitors and cardiomyocytes, and POU5F1 and NANOG in pluripotent hES cells. Consistent with this observation, we also show that DOT1L co-localizes with the master cardiac transcription factor NKX2.5, suggesting its direct involvement during gene activation. Knockdown of DOT1L did not alter the pluripotency of hES cells, but it led to the disruption of cardiac differentiation observed morphologically as well as at transcript and protein levels. CONCLUSIONS:Collectively, our data suggests the crucial role of H3K79me2 methyltransferase DOT1L for activation of NKX2.5 during the cardiac differentiation of hES cells.

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Single-cell lineage analysis reveals extensive multimodal transcriptional control during directed beta-cell differentiation.

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