Abstract
BackgroundMesendodermal formation during early gastrulation requires the expression of lineage-specific genes, while the regulatory mechanisms during this process have not yet been fully illustrated. TATA box-binding protein (TBP) and TBP-like factors are general transcription factors responsible for the transcription initiation by recruiting the preinitiation complex to promoter regions. However, the role of TBP family members in the regulation of mesendodermal specification remains largely unknown.MethodsWe used an in vitro mesendodermal differentiation system of human embryonic stem cells (hESCs), combining with the microarray and quantitative polymerase chain reaction (qRT-PCR) analysis, loss of function and gain of function to determine the function of the TBP family member TBP-related factor 3 (TRF3) during mesendodermal differentiation of hESCs. The chromatin immunoprecipitation (ChIP) and biochemistry analysis were used to determine the binding of TRF3 to the promoter region of key mesendodermal genes.ResultsThe mesendodermal differentiation of hESCs was confirmed by the microarray gene expression profile, qRT-PCR, and immunocytochemical staining. The expression of TRF3 mRNA was enhanced during mesendodermal differentiation of hESCs. The TRF3 deficiency did not affect the pluripotent marker expression, alkaline phosphatase activity, and cell cycle distribution of undifferentiated hESCs or the expression of early neuroectodermal genes during neuroectodermal differentiation. During the mesendodermal differentiation, the expression of pluripotency markers decreased in both wild-type and TRF3 knockout (TRF3−/−) cells, while the TRF3 deficiency crippled the expression of the mesendodermal markers. The reintroduction of TRF3 into the TRF3−/− hESCs rescued inhibited mesendodermal differentiation. Mechanistically, the TRF3 binding profile was significantly shifted to the mesendodermal specification during mesendodermal differentiation of hESCs based on the ChIP-seq data. Moreover, ChIP and ChIP-qPCR analysis showed that TRF3 was enriched at core promoter regions of mesendodermal developmental genes, EOMESODERMIN, BRACHYURY, mix paired-like homeobox, and GOOSECOID homeobox, during mesendodermal differentiation of hESCs.ConclusionsThese results reveal that the TBP family member TRF3 is dispensable in the undifferentiated hESCs and the early neuroectodermal differentiation. However, it directs mesendodermal lineage commitment of hESCs via specifically promoting the transcription of key mesendodermal transcription factors. These findings provide new insights into the function and mechanisms of the TBP family member in hESC early lineage specification.
Highlights
Mesendodermal formation during early gastrulation requires the expression of lineage-specific genes, while the regulatory mechanisms during this process have not yet been fully illustrated
chromatin immunoprecipitation (ChIP) and ChIP-qPCR analysis showed that TBP-related factor 3 (TRF3) was enriched at core promoter regions of mesendodermal developmental genes, EOMESODERMIN, BRACHYURY, mix paired-like homeobox, and GOOSECOID homeobox, during mesendodermal differentiation of human embryonic stem cells (hESCs). These results reveal that the TATA box-binding protein (TBP) family member TRF3 is dispensable in the undifferentiated hESCs and the early neuroectodermal differentiation
Microarray analysis revealed the upregulation of ME signature genes EOMES, T, Mix paired-like homeobox (MIXL1), and GOOSECOID homeobox (GSC), while the expression of early neuroectodermal marker genes paired box 6 (PAX6), neuronal differentiation 1 (NEUROD1), neurogenin 1 (NEUROG1), and achaete-scute family BHLH transcription factor 1 (ASCL1) remained unchanged (Fig. 1b)
Summary
Mesendodermal formation during early gastrulation requires the expression of lineage-specific genes, while the regulatory mechanisms during this process have not yet been fully illustrated. The differentiation of hESCs into mesoderm and endoderm undergoes an intermediate state called mesendoderm (ME), which is equivalent to the primitive streak during gastrulation [55] During this process, numerous genes, encoding the transcription factors critical for the ME specification, such as EOMESODERMIN (EOMES), BRACHYURY (T), mix paired-like homeobox (MIXL1), and GOOSECOID homeobox (GSC), are transcribed [8, 14, 18, 34]. The fine-tuned spatiotemporal transcription requires the coordination of various signaling pathways, epigenetic modifications, specific transcriptional factors, and general transcription factors (GTFs) Epigenetic modifiers, such as SETD7 and EZH2, are orchestrated to turn on the transcription of ME genes by the induction of wingless-type MMTV integration site family (WNT) and NODAL signals [38, 61]. Deciphering the function of GTFs during early differentiation would help us to understand more about the lineage fate commitment, and facilitate the application of hPSC derivatives in cell therapy and drug development [16, 47, 68]
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