SPEN protein expression and interactions with chromatin in mouse testicular cells.

SPEN (spen family transcription repressor) is a nucleic acid-binding protein putatively involved in repression of gene expression. We hypothesized that SPEN could be involved in general downregulation of the transcription during the heat shock response in mouse spermatogenic cells through its interactions with chromatin. We documented predominant nuclear localization of the SPEN protein in spermatocytes and round spermatids, which was retained after heat shock. Moreover, the protein was excluded from the highly condensed chromatin. Chromatin immunoprecipitation experiments clearly indicated interactions of SPEN with chromatin in vivo However, ChIP-Seq analyses did not reveal any strong specific peaks both in untreated and heat shocked cells, which might suggest dispersed localization of SPEN and/or its indirect binding to DNA. Using in situ proximity ligation assay we found close in vivo associations of SPEN with MTA1 (metastasis-associated 1), a member of the nucleosome remodeling complex with histone deacetylase activity, which might contribute to interactions of SPEN with chromatin.

The periphilin 1-like BFAR isoform 3 is highly expressed in transcriptionally silent oocytes and involved in RNA metabolism.

The mouse 3110001I22Rik gene located in the first intron of Bfar is considered as a Bfar variant coding for the BFARv3 protein. However, it differs from other BFAR isoforms and resembles periphilin 1 (PPHLN1) due to its two (Lge1 and serine-rich) conserved domains. We identified the BFARv3/EGFP-interacting proteins by co-immunoprecipitation coupled to mass spectrometry, which revealed 40S ribosomal proteins (RPS3, RPS14, RPS19, RPS25, RPS27), histones (H1.2, H1.4, H3.3C), proteins involved in RNA processing and splicing (SFPQ, SNRPA1, HNRNPA3, NONO, KHDRBS3), calcium signaling (HPCAL1, PTK2B), as well as HSD17B4, GRB14, POSTN, and MYO10. Co-immunoprecipitation revealed that both Lge1 and Ser-rich domains of BFARv3 were necessary for binding to RNA-interacting factors NONO and SFPQ, known to be components of paraspeckles. Reciprocal co-immunoprecipitation and the proximity ligation assay confirmed that both BFARv3 and PPHLN1 could interact with NONO and SFPQ, suggesting a new function for PPHLN1 as well. BFARv3 and its Lge1 or Ser-rich-deficient mutants preferentially localize in the nucleus. We found an accumulation of BFARv3/EGFP (but not its mutated forms) in the nuclear granules, which was enhanced in response to arsenite treatment and ionizing radiation. Although Bfar v3 is expressed ubiquitously in mouse tissues, its expression is the highest in metaphase II oocytes. The BFARv3 interactome suggests its role in RNA metabolism, which is critical for the transcriptionally silent MII oocyte. Mouse BFARv3 has no ortholog in the human genome, thus it may contribute to the differences between these two species observed in oocyte maturation and early embryonic development.

Pro-death signaling of cytoprotective heat shock factor 1: upregulation of NOXA leading to apoptosis in heat-sensitive cells.

Heat shock can induce either cytoprotective mechanisms or cell death. We found that in certain human and mouse cells, including spermatocytes, activated heat shock factor 1 (HSF1) binds to sequences located in the intron(s) of the PMAIP1 (NOXA) gene and upregulates its expression which induces apoptosis. Such a mode of PMAIP1 activation is not dependent on p53. Therefore, HSF1 not only can activate the expression of genes encoding cytoprotective heat shock proteins, which prevents apoptosis, but it can also positively regulate the proapoptotic PMAIP1 gene, which facilitates cell death. This could be the primary cause of hyperthermia-induced elimination of heat-sensitive cells, yet other pro-death mechanisms might also be involved.

Galactosylceramide affects tumorigenic and metastatic properties of breast cancer cells as an anti-apoptotic molecule.

It was recently proposed that UDP-galactose:ceramide galactosyltransferase (UGT8), enzyme responsible for synthesis of galactosylceramide (GalCer), is a significant index of tumor aggressiveness and a potential marker for the prognostic evaluation of lung metastases in breast cancer. To further reveal the role of UGT8 and GalCer in breast cancer progression, tumorigenicity and metastatic potential of control MDA-MB-231 cells (MDA/LUC) and MDA-MB-231 cells (MDA/LUC-shUGT8) with highly decreased expression of UGT8 and GalCer after stable expression of shRNA directed against UGT8 mRNA was studied in vivo in athymic nu/nu mice. Control MDA/LUC cells formed tumors and metastatic colonies much more efficiently in comparison to MDA/LUC-shUGT8 cells with suppressed synthesis of GalCer after their, respectively, orthotopic and intracardiac transplantation. These findings indicate that UGT8 and GalCer have a profound effect on tumorigenic and metastatic properties of breast cancer cells. In accordance with this finding, immunohistochemical staining of tumor specimens revealed that high expression of UGT8 accompanied by accumulation of GalCer in MDA-MB-231 cells is associated with a much higher proliferative index and a lower number of apoptotic cells in comparison to the MDA/LUC-shUGT8 cells. In addition, it was found that expression of UGT8 in MDA-MB-231 cells increased their resistance to apoptosis induced by doxorubicin in vitro. Therefore, these data suggest that accumulation of GalCer in tumor cells inhibits apoptosis, which would facilitates metastatic cells to survive in the hostile microenvironment of tumor in target organ.