RESUMO
Introduction: We have previously observed phenotypic and developmental changes upon the ectopic expression of the RUNX3 or the ZBTB46 transcription factors in mouse embryonic stem cell (ESC) derived progenitors. In this study, we evaluated the gene expression profiles of the RUNX3- and the ZBTB46-instructed murine ESCs with RNA-seq testing two next-generation sequencing technologies. Methods: We compared the DNA nanoball-based DNBSEQ G400 sequencer (MGI) with the bridge-PCR-based NextSeq 500 instrument (Illumina) for RNA sequencing. Moreover, we also compared two types of MGI sequencing reagents (Standard versus Hot-massive parallel sequencing (MPS)) with the DNBSEQ G400. Results: We observed that both sequencing platforms showed comparable levels of quality, sequencing uniformity, and gene expression profiles. For example, highly overlapping RUNX3- and ZBTB46-regulated gene lists were obtained from both sequencing datasets. Moreover, we observed that the Standard and the Hot-MPS-derived RUNX3- and ZBTB46-regulated gene lists were also considerably overlapped. This transcriptome analysis also helped us to identify differently expressed genes in the presence of the transgenic RUNX3 or ZBTB46. For example, we found that Gzmb, Gzmd, Gzme, Gdf6, and Ccr7 genes were robustly upregulated upon the forced expression of Runx3; on the other hand, Gpx2, Tdpoz4, and Arg2 were induced alongside the ectopic expression of Zbtb46. Discussion: Similar gene expression profile and greatly overlapping RUNX3- and ZBTB46-regulated gene sets were detected with both DNA sequencing platforms. Our analyses demonstrate that both sequencing technologies are suitable for transcriptome profiling and target gene selection. These findings suggest that DNBSEQ G400 represents a cost-effective alternative sequencing platform for gene expression monitoring. Moreover, this analysis provides a resource for exploration of the RUNX3- and ZBTB46-dependent gene regulatory networks.
RESUMO
Zbtb46 is a recently identified dendritic cell (DC)-specific transcription factor with poorly defined biology. Although Zbtb46 is highly expressed in conventional DCs, evidence also points to its presence in erythroid progenitors and endothelial cells suggesting that this factor might influence the early hematopoietic development. Here, we probe the effect of this transcription factor in embryonic stem cell (ESC)-derived blood cell progenitors using chemically inducible mouse cell lines. Unexpectedly, forced expression of this protein elicited a broad repressive effect at the early stage of ESC differentiation. Ectopic expression of Zbtb46 interfered with the mesoderm formation and cell proliferation was also negatively impacted. More importantly, reduced number of CD11b+ myeloid blood cells were generated from ESC-derived Flk1+ mesoderm cells in the presence of Zbtb46. Consistent with this finding, our gene expression profiling revealed that numerous myeloid and immune response related genes, including Irf8, exhibited lower expression in the Zbtb46-primed cells. Despite these repressive effects, however, Zbtb46 overexpression was associated with enhanced formation of erythroid blood cell colonies and increased adult hemoglobin (Hbb-b1) expression at the early phase of ESC differentiation. Moreover, elevated percent of CD105 (Endoglin) positive cells were detected in the Zbtb46-primed samples. In summary, our results support that Zbtb46 suppresses the ESC-derived myeloid development and diverts mesoderm cells toward erythroid developmental pathway. Moreover, our transcriptomic data provide a resource for exploration of the Zbtb46 regulatory network in ESC-derived progenitors.
