Functional analysis of a conserved nuage/piRNA pathway gene, tapas.

Piwi-interacting RNAs (piRNAs) have been implicated in defending the germline genome against the deleterious retroelements. Most of the piRNA pathway components have been shown to localize to the nuage, a unique perinuclear structure found in the animal germlines. Recently, a tudor domain protein Tejas that localizes to the nuage, has been reported to be involved in the germline piRNA pathway in Drosophila. Later, a homolog of Tejas, Tapas, was also found to be functioning in the piRNA pathway and localize to the nuage in Drosophila. Here, I report the characterization of Tapas for its role in the piRNA pathway. I show that tapas is involved in the hierarchical assembly of nuage: the proper localization of Vasa, Spindle-E, Tejas, Aubergine, Krimper, Argonaute 3 and Maelstrom to the nuage depends on tapas, and localization of Tapas is independent of any of the known nuage components. These results suggest that tapas probably functions upstream in the hierarchical assembly. Further, I found that Tapas physically interacts with some of the piRNA pathway components such as Aubergine, Argonaute3, Vasa, Tejas and Spindle-E. These results imply that Tapas probably aids in the formation of macromolecular complex at the perinuclear region, supporting the idea that piRNA components form a macrocomplex or sub-complexes at the perinuclear nuage by being engaged in piRNAs production.Bachelor of Science in Biological Science

High levels of progesterone receptor B in MCF-7 cells enable radical anti-tumoral and anti-estrogenic effect of progestin

The widely reported conflicting effects of progestin on breast cancer suggest that the progesterone receptor (PR) has dual functions depending on the cellular context. Cell models that enable PR to fully express anti-tumoral properties are valuable for the understanding of molecular determinant(s) of the anti-tumoral property. This study evaluated whether the expression of high levels of PR in MCF-7 cells enabled a strong anti-tumoral response to progestin. MCF-7 cells were engineered to overexpress PRB by stable transfection. A single dose of Promegestone (R5020) induced an irreversible cell growth arrest and senescence-associated secretory phenotype in MCF-7 cells with PRB overexpression (MCF-7PRB cells) but had no effect on MCF-7 cells with PRA overexpression. The growth-arresting effect was associated with downregulations of cyclin A2 and B1, CDK2, and CDK4 despite an initial upregulation of cyclin A2 and B1. R5020 also induced an evident activation of Nuclear Factor κB (NF-κB) and upregulation of interleukins IL-1α, IL-1β, and IL-8. Although R5020 caused a significant increase of CD24+CD44+ cell population, R5020-treated MCF-7PRB cells were unable to form tumorspheres and underwent massive apoptosis, which is paradoxically associated with marked downregulations of the pro-apoptotic proteins BID, BAX, PARP, and Caspases 7 and 8, as well as diminution of anti-apoptotic protein BCL-2. Importantly, R5020-activated PRB abolished the effect of estrogen. This intense anti-estrogenic effect was mediated by marked downregulation of ERα and pioneer factor FOXA1, leading to diminished chromatin-associated ERα and FOXA1 and estrogen-induced target gene expression. In conclusion, high levels of agonist-activated PRB in breast cancer cells can be strongly anti-tumoral and anti-estrogenic despite the initial unproductive cell cycle acceleration. Repression of ERα and FOXA1 expression is a major mechanism for the strong anti-estrogenic effect.Ministry of Education (MOE)Published versionThis work was supported by the Singapore Ministry of Education Academic Research Fund Tier II, MOE2014-T2-2-125 and MOE-T2EP30121-0018

Tetratricopeptide repeat domain 9A knockout induces social anxiety and impairs offense behaviors in female mice

The involvement of tetratricopeptide repeat domain 9A (TTC9A) in anxiety-like behaviors through estrogen action has been reported in female mice, this study further investigated its effects on social anxiety and aggressive behaviors.Agency for Science, Technology and Research (A*STAR)Published versionThis work was supported by the Ministry of Education, Singapore; the A*STAR Biomedical Research Council, Singapore (06/1/221/19/455); and the Hong Kong Research Grants Council

Estrogen exacerbates mammary involution through neutrophil-dependent and -independent mechanism

There is strong evidence that the pro-inflammatory microenvironment during post-partum mammary involution promotes parity-associated breast cancer. Estrogen exposure during mammary involution drives tumor growth through neutrophils' activity. However, how estrogen and neutrophils influence mammary involution are unknown. Combined analysis of transcriptomic, protein, and immunohistochemical data in BALB/c mice showed that estrogen promotes involution by exacerbating inflammation, cell death and adipocytes repopulation. Remarkably, 88% of estrogen-regulated genes in mammary tissue were mediated through neutrophils, which were recruited through estrogen-induced CXCR2 signalling in an autocrine fashion. While neutrophils mediate estrogen-induced inflammation and adipocytes repopulation, estrogen-induced mammary cell death was via lysosome-mediated programmed cell death through upregulation of cathepsin B, Tnf and Bid in a neutrophil-independent manner. Notably, these multifaceted effects of estrogen are mostly mediated by ERα and unique to the phase of mammary involution. These findings are important for the development of intervention strategies for parity-associated breast cancer.Ministry of Education (MOE)Published versionThis research is funded the Ministry of Education of Singapore. Academic Research Fund Tier I, MOE2017-T1-002-081. We thank Drs. Natasa Bajalovic, Amanda Woo and Mr. Lee Shi Hao for their technical assistance