SATB1 promotion of trophoblast stem cell renewal through regulation of threonine dehydrogenase
Introduction
The placenta is a transient but essential organ for nourishing the fetus through the production of hormones targeting maternal tissues, the exchange of gas and nutrients, and immunoprotection between mother and fetus [[1], [2], [3]]. These tasks are accomplished by specialized trophoblast cells differentiating from trophoblast stem (TS) cells [4,5]. TS cells arise from polar trophectoderm of the blastocyst or extraembryonic ectoderm, can be propagated in vitro, and maintained in a stem state or induced to differentiate [[6], [7], [8], [9]]. The TS cell stem state is known to be achieved by orchestrated regulatory networks involving key transcription factors, including caudal type homeobox 2 (CDX2), eomesodermin (EOMES), GATA binding protein 3 (GATA3), estrogen related receptor beta (ESRRB), TEA domain transcription factor 4 (TEAD4), and transcription factor AP-2 gamma (TFAP2C) [[6], [7], [8], [9]]. Dysregulation of these key factors leads to pregnancy loss due to extraembryonic hypoplasia [[10], [11], [12], [13], [14], [15], [16], [17]]. Therefore, understanding molecular mechanisms underlying TS cell renewal is important for proper extraembryonic and embryonic development.
Development is precisely regulated through the actions of transcription factors and also through higher order organization of chromatin [[18], [19], [20]]. Special AT-rich binding protein 1 (SATB1) was first identified as a chromatin organizer in thymocytes [[21], [22], [23], [24]]. Subsequently, it was found that SATB1 functions in other tissues and cell types as a regulator of stem cells and cell transformation [[25], [26], [27]]. In embryonic stem (ES) cells, SATB1 regulates the balance between self-renewal and differentiation by repressing Nanog homeobox (NANOG) expression [26]. SATB1 depletion directs ES cell differentiation toward epiblast cells and hinders primitive endoderm lineage development [28]. In the trophoblast cell lineage, SATB1 is highly expressed in trophoblast stem/progenitor cells of the ectoplacental cone and maintains TS cell renewal in mouse and rat TS cells and in the rat Rcho-1 TS cell model [27]. Mechanisms underlying SATB1 action in TS cells are poorly understood.
In this study, we provide insights into SATB1 actions on trophoblast cell development. Rat Rcho-1 TS cells and ex vivo rat blastocysts were used as model systems to elucidate downstream SATB1 signaling. Rcho-1 TS cells can be maintained in a stem/undifferentiated state or induced to differentiate [29] and are exquisitely sensitive to SATB1 manipulation [27]. L-threonine 3-dehydrogenase (TDH) was identified as a mediator of SATB1 actions on trophoblast cell development. TDH is the rate limiting enzyme responsible for the conversion of threonine into glycine and acetyl-coenzyme A [30] and is essential for maintenance of mouse embryonic stem cell self-renewal [30]. The experimental findings described in this report demonstrate that TDH also contributes to SATB1 maintenance of the TS cell stem state.
Section snippets
Animals
Holtzman Sprague-Dawley rats were obtained from Envigo (Indianapolis, IN). Animals were housed in an environmentally controlled facility with lights on from 0600 to 2000 h and were allowed free access to food and water. The presence of a seminal plug or sperm in the vaginal lavage was designated day 0.5 of pregnancy and blastocysts were collected by uterine flushing on day 4.5 of pregnancy. The University of Kansas Animal Care and Use Committee approved protocols for the care and use of animals.
Identification of SATB1 targets
Specific SATB1 knockdown was achieved by lentiviral transduction of two different shRNAs targeting the Satb1 gene in Rcho-1 TS cells [27], Fig. 1A and B). Global expression analysis using RNA-sequencing (RNA-seq) was performed in SATB1 knockdown TS cells at four days (experimentally defined as “Acute”) and ten days (experimentally defined as “Chronic”) after transduction (Fig. 1C and D). Differentially regulated genes were identified in SATB1 knockdown TS cells (Fig. 1C). Transcripts identified
Discussion
Specific transcriptional networks essential for TS cell renewal have been identified [6,7]. SATB1 contributes to the regulation of the TS cell stem state through organizing transcriptional networks [27]. We performed global transcriptome analysis in control and SATB1 knockdown Rcho-1 trophoblast cells to identify potential pathways controlling trophoblast cell development. SATB1 regulated transcripts essential for stem state maintenance consistent with the known actions of SATB1 in promoting
Data availability
The RNA-seq dataset is available in the Gene Expression Omnibus website (https://www.ncbi.nlm.nih.gov/geo/; accession no. GSE151568). The remainder of the data is contained within the manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We thank Dr. Steven McKnight (University of Texas, Southwestern Medical Center, Dallas, TX) for generously providing antibodies to threonine dehydrogenase. We also thank Stacy Oxley and Brandi Miller for administrative assistance. This work was supported by the National Institutes of Health (MJS: HD020676, HD079363, HD099638) the Japan Society for the Promotion of Science (KK: 19K09835), and the International Joint Usage/Research Center, the Institute of Medical Science, the University of Tokyo
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Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, Nasushiobara, Tochigi 329-2793, Japan.