A Chemical Biology Study of Human Pluripotent Stem Cells Unveils HSPA8 as a Key Regulator of Pluripotency

摘要

Highlights ? A human ECC-based screen identified novel chemical regulators of pluripotency ? Displurigen disrupts human embryonic stem cell pluripotency by targeting HSPA8 ? HSPA8 maintains pluripotency by facilitating the DNA-binding activity of OCT4 Summary Chemical biology methods such as high-throughput screening (HTS) and affinity-based target identification can be used to probe biological systems on a biomacromolecule level, providing valuable insights into the molecular mechanisms of those systems. Here, by establishing a human embryonal carcinoma cell-based HTS platform, we screened 171,077 small molecules for regulators of pluripotency and identified a small molecule, Displurigen, that potently disrupts hESC pluripotency by targeting heat shock 70-kDa protein 8 (HSPA8), the constitutively expressed member of the 70-kDa heat shock protein family, as elucidated using affinity-based target identification techniques and confirmed by loss-of-function and gain-of-function assays. We demonstrated that HSPA8 maintains pluripotency by binding to the master pluripotency regulator OCT4 and facilitating its DNA-binding activity. prs.rt("abs_end"); Introduction The pluripotency regulators OCT4, NANOG, and SOX2 form a core transcription regulatory network through auto- and reciprocal activations at the transcription level, which is believed to be responsible for the maintenance of human embryonic stem cell (hESC) pluripotency ( Boyer et?al., 2005 ). At the same time, multiple protein factors belonging to a diversity of functional categories, such as transcription factors, epigenetic factors, and signaling components, work cooperatively to form an expanded pluripotency factor network that supports the core pluripotency network ( Boyer et?al., 2005 ). In contrast to the well-defined core network, our knowledge of this expanded pluripotency network, including its components, the interactions between these components, and the mechanism of interaction between the expanded network and the core network, remains insufficient. Bioactive small molecules have been applied to the field of hESC research with success. Many such studies have applied small molecules as modulators of lineage-specific differentiations ( Borowiak et?al., 2009 , Chen et?al., 2009 , Chen et?al., 2012 , Gonzalez et?al., 2011a , Lian et?al., 2012 and Mahmood et?al., 2010 ). Other studies have exploited small molecules as chemical probes to uncover novel molecular mechanisms underlying hESC pluripotency or differentiation ( Chen et?al., 2006 , Xu et?al., 2010 and Zhu et?al., 2009 ). High-throughput screenings (HTS) were usually conducted for the search of such molecules. If the mechanism of action was unknown for a given molecule, affinity-based target identification methods can be used to identify its?biological target(s). These studies have been used to identify novel protein factors and to unveil previously unknown molecular mechanisms that regulate hESC fate determination ( Xu et?al., 2008 ). In recent years, hESCs and human induced pluripotent stem cells (hiPSCs) have been used successfully for HTS in several studies ( Barbaric et?al., 2010 , Ben-David et?al., 2013 , Desbordes et?al., 2008 , Gonzalez et?al., 2011b , Kameoka et?al., 2014 , Kumagai et?al., 2013 , Manganelli et?al., 2014 and Xu et?al., 2010 ). However, the high cost associated with the maintenance and scale-up of human pluripotent stem cells (hPSCs) inevitably limits the scale of their application in HTS studies. We chose to explore an alternative source of pluripotent stem cells, human embryonal carcinoma cells (hECCs), as a robust platform for HTS with low cost. hECCs are pluripotent stem cells derived from human teratocarcinomas and are considered to be the malignant counterparts of hESCs. The molecular regulatory mechanism of hECC pluripotency has been shown to mimic that of hESCs ( Josephson et?al., 2007 ). Because of their cancerous nature, hECCs are not prone to spontaneous differentiation and require a less demanding culture condition compared with hPSCs. Experimental results acquired from studies using hECCs have been proven to be highly stable and readily reproducible ( Josephson et?al., 2007 ), making hECCs ideal candidate platforms for HTS purposes. Based on the concept of hECC-based HTS, we established a pluripotency reporter system using the hECC line NTERA-2. Using this system, we conducted a large-scale chemical screening and found 122 small molecules that disrupt hESC pluripotency. One of these molecules, which we named Displurigen, potently disrupts hESC pluripotency by targeting heat shock 70-kDa protein 8 (HSPA8, the constitutively expressed member of the 70-kDa heat shock protein family), as discovered using affinity-based target identification methods and functional validations. We demonstrated that HSPA8 helps maintain pluripotency by direct binding to the OCT4 protein and facilitating OCT4 binding to DNA. Results Establishment of an NTERA-2 Cell-Based Pluripo