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Well-TEMP-seq characterizes the temporal dynamics of single-cell gene expression

ABSTRACT: Single-cell RNA sequencing reveals the transcriptional heterogeneity of single cells, but offers static snapshots of gene expression and fails to reveal the temporal dynamics of transcription. Herein, we develop Well-TEMP-seq, a high-throughput, cost-effective, accurate, and efficient method for massively parallel profiling the time-resolved dynamics of single-cell gene expression. Well-TEMP-seq combines metabolic RNA labeling with our recently developed microwell-based single-cell RNA sequencing method (Well-Paired-seq) to distinguish newly transcribed RNAs marked by T-to-C substitutions from pre-existing RNAs in each of thousands of single cells. We further apply Well-TEMP-seq to profiling the dynamics of gene expression of colorectal cancer cells exposed to low-dose of 5-AZA-CdR, a clinically used DNA-demethylating anti-tumor drug. Well-TEMP-seq also for the first time reveals the activation of interferon responsive genes by 5-AZA-CdR induced viral mimicry in the first three days after treatment. Well-TEMP-seq will be broadly applicable to unveil the dynamics of single-cell gene expression in diverse biological processes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE194357 | GEO | 2022/02/01

REPOSITORIES: GEO

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Project description:Background:Aberrant DNA methylation that silences tumor suppressor genes occurs frequently in patients with acute myeloid leukemia (AML). Treatment of AML patients with the inhibitor of DNA methylation, 5-aza-2'-deoxycytidine (5-AZA-CdR) can induce complete remissions, but most patients will relapse. The clinical efficacy of 5-AZA-CdR may be influenced by its limited capacity to activate tumor suppressor genes silenced by methylation of lysine 27 histone H3 (H3K27) by EZH2. In order to overcome this limitation, we investigated previously the antileukemic action of 5-AZA-CdR in combination with the EZH2 inhibitor, 3-deazaneplanocin A (DZNep) on HL-60 AML cells. We observed a remarkable synergistic interaction against these AML cells for this combination. In this study, we investigated in more depth the action of 5-AZA-CdR plus DZNep on gene expression in AML cells using RNA sequence analysis Result:In a colony assay, 5-AZA-CdR in combination with DZNep exhibited also a potent synergy against another human AML cell line: AML-3. The induction of apoptosis in HL-60 and AML3 leukemic cells by 5-AZA-CdR plus DZNep was also synergistic. RNA sequence analysis on HL-60 leukemic cells showed that the combination of 5-AZA-CdR plus DZNep increased the expression of thousands of genes. The genes upregulated by this combination included genes related to differentiation, development, senescence, apoptosis, and tumor suppressor function. Many of the genes activated by 5-AZA-CdR plus DZNep have the potential to suppress leukemogenesis. Conclusion: The activation of many genes by the combination of 5-AZA-CdR plus DZNep correlates with its synergistic antileukemic action. The block in differentiation is one of the hallmarks of AML.The activation of many genes that program differentiation and development by this combination of epigenetic agents has the potential to reverse this block. The reversal of these two epigenetic genesilencing mechanisms by 5-AZA-CdR plus DZNep merits clinical investigation in patients with AML

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Well-TEMP-seq characterizes the temporal dynamics of single-cell gene expression

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