Hypertranscribe free trial5/5/2023 ![]() ![]() Interestingly, both male and female PGCs are larger than somatic cells and similar to ES cells ( Figure S1D-E). CNN qRT-PCR confirmed that pre-rRNA ( Figure 1C), as well primary mRNAs ( Figure 1D), are significantly upregulated in male and female E13.5 PGCs. Next, to rule out increased mRNA stability in PGCs compared to soma, the expression of both primary (unspliced) and mature RNA Polymerase (RNA Pol) I and II transcripts was profiled at E13.5. Moreover, PGC transcription is elevated in comparison to a range of E13.5 tissues, and is comparable to cultured ES cells ( Figure S1C). Elevated gene expression is apparent throughout PGC development from E9.5 to E13.5 ( Figure S1A-B), confirming that this phenomenon occurs over a wide window of PGC development. Relative to soma, PGCs exhibit significantly higher levels of housekeeping genes typically used for qRT-PCR normalization, including Gapdh, Ubb, Rpl7 and H2A ( Figure 1B). ![]() We next performed cell number-normalized (CNN) qRT-PCR for common housekeeping genes. ![]() Quantification of total RNA from equal numbers of sorted PGCs or soma revealed approximately 4-fold higher amounts of RNA in male PGCs than in soma, and 3-fold in female PGCs ( Figure 1A). PGCs and soma were profiled at E13.5, when sexual differentiation is readily apparent and cells are entering cell cycle arrest ( Bowles and Koopman, 2007). To investigate the transcriptome of PGCs and neighboring somatic cells of the gonad (soma), we first took advantage of the Oct4/GFP transgenic mouse line ( Yeom et al., 1996). Increased RNA Polymerase I and II transcripts in PGCs These results demonstrate that global hypertranscription occurs in vivo and reveal that the PGC transcriptome is much more dynamic than previously thought. Moreover, we find that PGC hypertranscription depends on the activity of Myc/Max and pTEFb, highlighting a potentially important role for these proteins in germ cell development. Our results document a dramatic upregulation of the PGC transcriptome in male and female E13.5 gonads, inclusive of many genes associated with increased biosynthetic capacity and growth. In this study we focused on the developing germline, and asked whether PGCs can be distinguished from their neighboring somatic cells by their transcriptional output. Thus, many cases of hypertranscription may have gone unnoticed. Moreover, traditional methods of analyzing large sequencing datasets do not take into account global changes to gene expression. However, the genome-wide extent of hypertranscription has not been investigated during development in vivo ( Percharde et al., 2017). The available data point to a critical role of global transcriptional elevation in embryogenesis. In addition, c-Myc-driven global transcriptional activation has been documented in ES cells, activated lymphocytes, and cancer cells ( Lin et al., 2012 Nie et al., 2012) where it may drive rapid proliferation. Chd1 promotes hypertranscription in ES cells and is essential for growth of the post-implantation epiblast ( Guzman-Ayala et al., 2015) and for emergence of definitive hematopoietic stem/progenitor cells at midgestation ( Koh et al., 2015). This globally elevated transcriptional state has been proposed to occur in several contexts of embryonic development. These studies revealed that PGCs express a pluripotency program with high similarity to that of Embryonic Stem (ES) cells, along with unique modules specific to PGCs related to migration and sexual differentiation.ĮS cells can exist in a state of hypertranscription, which involves a global nascent amplification of most of the transcriptome ( Efroni et al., 2008 Guzman-Ayala et al., 2015 Percharde et al., 2017). In parallel, several groups, including our own, have investigated the transcriptional profile of PGCs. Significant insights have been gained into the unique chromatin reprogramming that PGCs undergo during their development. PGC development is accompanied by a remarkable level of epigenetic reprogramming, such as genome-wide DNA demethylation including the removal of genomic imprints, widespread changes to histone modifications and variants, and X-chromosome reactivation in females. ![]() From E7.75-E10.5, these cells migrate to the developing gonads where they proliferate and, from E12.5, commence sexual differentiation ( Tam and Snow, 1981 Western et al., 2008). In mice, PGCs arise from a small group of epiblast cells that are first identifiable at embryonic day (E)7.25. Primordial germ cells (PGCs), the embryonic precursors to mature oocytes and spermatozoa, are thus essential for ontogeny and species survival. The germline carries on to the next generation both developmental totipotency as well as genetic and epigenetic predispositions to disease. ![]()
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