Single-stranded DNA binding protein Ssbp3 induces differentiation of mouse embryonic stem cells into trophoblast-like cells.
Single-stranded DNA binding protein Ssbp3 induces differentiation of mouse embryonic stem cells into trophoblast-like cells.
复制标题
单链DNA结合蛋白Ssbp3诱导小鼠胚胎干细胞分化为滋养层样细胞
DOI：
10.1186/s13287-016-0340-1
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发表时间：
2016-05-28
影响因子：
7.5
通讯作者：
Li H
中科院分区：
文献类型：
--
作者：
Liu J;Luo X;Xu Y;Gu J;Tang F;Jin Y;Li H
控制胚胎干细胞（ESCs）无限自我更新和发育多能性的内在因素和外在信号已经被广泛研究。然而，涉及胚胎外滋养细胞从ESCs分化的因素数量要少得多。在这项研究中，我们研究了单链DNA结合蛋白Ssbp3在诱导小鼠ESCs向滋养细胞样分化中的作用。方法在自我更新培养条件下，通过过表达或敲低Ssbp3对小鼠ESCs进行功能获得和功能丧失实验。通过实时定量反转录聚合酶链反应（qRT-PCR）检测多能性和谱系标记的表达水平。通过affymetrix微阵列检测ssbp3过表达细胞的基因表达谱。通过qRT-PCR和Western blotting对基因本体和通路项进行分析和进一步验证。通过亚硫酸盐测序检测ssbp3过表达细胞中Elf5启动子的甲基化状态。Ssbp3诱导的滋养细胞样表型也通过畸胎瘤形成和早期胚胎注射试验进行了评估。结果Ssbp3在小鼠ESCs中的强制表达上调了谱系相关基因的表达水平，其中滋养细胞标志物的表达水平最高。相反，在ESCs中，Ssbp3的缺失减弱了Oct4下调或BMP4和bFGF处理诱导的滋养细胞谱系标记基因的表达。有趣的是，全球基因表达谱分析表明，Ssbp3过表达并没有显著改变多能性相关转录因子的转录水平。相反，Ssbp3促进了早期滋养外胚层转录因子Cdx2的表达，激活了MAPK/Erk1/2和TGF-β通路。此外，Ssbp3的过表达降低了Elf5启动子的甲基化水平，促进了伴有内出血的畸胎瘤的产生，表明滋养细胞的存在。结论单链DNA结合蛋白Ssbp3是小鼠ESCs向滋养细胞样细胞分化的调节因子。这一发现有助于理解ESC分化为胚胎外谱系的调控网络。
BackgroundIntrinsic factors and extrinsic signals which control unlimited self-renewal and developmental pluripotency in embryonic stem cells (ESCs) have been extensively investigated. However, a much smaller number of factors involved in extra-embryonic trophoblast differentiation from ESCs have been studied. In this study, we investigated the role of the single-stranded DNA binding protein, Ssbp3, for the induction of trophoblast-like differentiation from mouse ESCs.MethodsGain- and loss-of-function experiments were carried out through overexpression or knockdown of Ssbp3 in mouse ESCs under self-renewal culture conditions. Expression levels of pluripotency and lineage markers were detected by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analyses. The global gene expression profile in Ssbp3-overexpressing cells was determined by affymetrix microarray. Gene ontology and pathway terms were analyzed and further validated by qRT-PCR and Western blotting. The methylation status of the Elf5 promoter in Ssbp3-overexpressing cells was detected by bisulfite sequencing. The trophoblast-like phenotype induced by Ssbp3 was also evaluated by teratoma formation and early embryo injection assays.ResultsForced expression of Ssbp3 in mouse ESCs upregulated expression levels of lineage-associated genes, with trophoblast cell markers being the highest. In contrast, depletion of Ssbp3 attenuated the expression of trophoblast lineage marker genes induced by downregulation of Oct4 or treatment with BMP4 and bFGF in ESCs. Interestingly, global gene expression profiling analysis indicated that Ssbp3 overexpression did not significantly alter the transcript levels of pluripotency-associated transcription factors. Instead, Ssbp3 promoted the expression of early trophectoderm transcription factors such as Cdx2 and activated MAPK/Erk1/2 and TGF-β pathways. Furthermore, overexpression of Ssbp3 reduced the methylation level of the Elf5 promoter and promoted the generation of teratomas with internal hemorrhage, indicative of the presence of trophoblast cells.ConclusionsThis study identifies Ssbp3, a single-stranded DNA binding protein, as a regulator for mouse ESCs to differentiate into trophoblast-like cells. This finding is helpful to understand the regulatory networks for ESC differentiation into extra-embryonic lineages.