## 4 Discussion  
According to RNA-Seq data, AP-1 members _Jun_, _Junb_, _Jund_ and _Fosil2_ are the most expressed in GC-1 cells, whereas _Jun_, _Jund_, _Fos_ and _Fosil2_ are highly expressed in spermat  
Figure 7: Correlations between the expressions of _GJA1_, AP-1 members (**A**–**C**,**F**,**G**), _CAMKI_ (**D**), _PRKAR1B_ (**E**), _SOX4_ (**H**) and _SP1_ (**I**) in TGCT. Normalized mRNA expression data was obtained from the TCGA—PanCancer Atlas database ([[https://portal.gdc.cancer.gov/](https://portal.gdc.cancer.gov/)]([https://portal.gdc.cancer.gov/](https://portal.gdc.cancer.gov/)), accessed on 19 December 2021) and analyzed for correlations between gene expressions using the cBioPortal platform [34,35]. gonal cells. Hence, the GC-1 cell line is an excellent model to evaluate the abilities of AP-1 members Jun, Junb and Fosl2, commonly abundant in spermatogonia and GC-1 cells, to regulate _Gja1_ expression. For Jun and Junb, their expression levels are low in intact testes and much higher in isolated cells [36], suggesting that disruption of cell-to-cell contact increases their abundance in isolated spermatogonia. Here, we show that Jun and Junb cooperate with Fosl2 to activate the _Gja1_ promoter in GC-1 cells. While AP-1 family members are important regulators of _Gja1_ expression in different cell types [13; 37], the combinations of these transcription factors vary in a cell-dependent manner. Indeed, we show that Jun and Fosl2 are important regulators of _Gja1_ in GC-1 spermatogonial cells, while Jun and Fos are more involved in the regulation of _Gja1_ in somatic cells of the testis [13]. Precisely, we and others have demonstrated that _Gja1_, being highly expressed in Leydig and Sertoli cells, is regulated by Jun and Fos [13]. According to our ChIP results, the proximal region of the _Gja1_ promoter is required for AP-1-dependent activation in spermatogonia. Although Jun is not expressed in germ cells, we showed that it is being recruited to the proximal region of the _Gja1_ promoter by ChIP assay on GC-1 cells. Interestingly, _Jund_ knockout mice have an impaired spermatogenesis linked to an important reduction in _Gja1_ expression in semimiferous tubules [2]. Hence, Jun-dependent regulation of _Gja1_ may be relevant for other cell populations having high levels of this transcription factor such as Sertoli and Leydig cells (our unpublished data). In our ChIP assays, the recruitments of Junb and Fosl2 to the proximal region of the _Gja1_ promoter could not be observed, as opposed to Jun. This may be attributed to an inability of immunoprecipitation of Junb or Fosl2 by their respective antibodies due to the inaccessibility of the epitopes. Indeed, Junb and/or Fosl2 may be hidden in a major complex involving AP-1 members, cofactors and other transcription factors such as Sp1. It is noteworthy that both 32- and 35-kDa isoforms of Fosl2 are equivalently expressed in GC-1 cells. The 32 kDa isoform has a substitution of amino acids 1-33 of the N-terminus with MSFSLF amino acids. The impact of this substitution on the transcriptional regulatory function of Fosl2 remains to be elucidated. Although _Sp1_ is highly expressed in GC-1 cells, this transcription factor is rather weakly expressed in germ cells. As previously reported in human mymetrial cells [30], the Sp1 factor can regulate _Gja1_ promoter activity in GC-1 cells. Putative Sp1 regulatory elements have been characterized between \(-77\) to \(-69\) and \(-59\) to \(-48\) bp of the human _GJA1_ promoter [37]. Thus, this transcription factor may be an important regulator of _GJA1_ expression in certain types of TGCT. However, the expression of _SP1_ is not correlated with _GJA1_ in TGCT according to TCGA PanCancer Atlas data [38]. _Sox4_ and _Sox12_ are highly expressed in spermatogonia and GC-1 cells. Unlike what has been reported in Sertoli cells [14], Sox4 does not cooperate with AP-1 members to activate the _Gja1_ promoter in GC-1 cells. In addition, _SOX4_ and _SOX12_ are not correlated with _GJA1_ in TGCT according to TCGA PanCancer Atlas data [38].