Activity Analysis Of Chicken ERα Promoter And Study On Spatio-temporal Expression Of Alternative Splicing Variants

Avian gonadal development and sex differentiation has been the hotspot of researchs on avian developmental biology, but regulatory mechanism of gonadal development and sex differentiation remains to be further studied. It was reported that ERa gene which is closely related to the reproductive system development and fuctions maintain palys an important role in early stage of chick gonadal development and sex differentiation. It is known that ERa gene has multiple promoters whose products are different transcripts (namely different alternative splicing variants).In this research,5’upstream region of ERa was analyzed firstly and different deletion fragments were cloned and recombined into vectors to find activity differences of multiple promoters. On this basis, expression differences of alternative splicing variants regulated by different promoters in different tissues during embryonic development periods and sexual maturity stage were identified, and then, results were compared with ERa expression under the same condition to explore the relationship between the two. The dual luciferase reporter gene system was used to detect promoter activities of different deletion fragments; semi-quantitative RT-PCR method was used to obtain quantitative analysis on expression of alternative splicing variants and ERa. The main results are as follows.1. Deletion fragments which recombined into vectors of pGL3-basic plasmids were transfected into three kinds of cells and activities were detected. Promoter activities were unfound in transfection experiment of DF1cells, but conversely, promoter activitie differences were detected in PK15cells and Hela cells.The activitie differences are as follows:ERaPF1(+52～+216)>ERaPF3(-238～+216)>ERaPF4(-403～+216)> ERaPF2(-65～+216)>ERaPF5(-2125～+216).It is convinced that Recombinants of pGL3-basic plasmids are cell-specified.Additionally, promoter activitie differences of ERa may be influenced by different transcription factors in gene regulatory pathways or interaction among promoters. 2. Alternative splicing variant C which is regulated by promoter C was detected in different tissues during different stage (E10.5, E12.5, E14.5, E16.5, E18.5) of chick embryo and6-month-old chicken by semi-quantitative RT-PCR. It is found that alternative splicing variant C is tissue-specified, its expression was only detected in liver. It is also found that expression of alternative splicing variant C is developmental period specified and sexual differentiated. In addition, some elements are present to down-regulate the expression of alternative splicing variant C in E14.5male embryo which maybe related to epigenetic modification or other factors acting on promoter.3. ERa which is regulated by multiple promoters in liver was detected by semi-quantitative RT-PCR. No significant difference of ERa expression between male and female chick liver tissue was found during the embryonic development stages. However a very significant difference of the ERa expression was found between6-month-old male and female liver.It is speculated that the expression difference of ERa may be associated with egg yolk and egg protein synthese in liver of layers.4. By comparing with ERa expression, we found that when spliceosome C expression of liver tissue in female is higher than male, ERa expression of liver tissue in female will be lower than male,and vice versa. It is suggest initially that expressions of spliceosome C and ERa between liver tissues of female and male are negative correlation.In other words promoter C and multiple promoters of ERa are negative correlation in liver tissues.5. ERa and alternative splicing variant D were detected in different tissues of6-month-old chicken by semi-quantitative RT-PCR. It is confirmed that expression of ERa and spliceosome D are found in all detected tissues, ERa expression in oviduct is greater than seminiferous duct,but spliceosome D expression in these two tissues are opposite.It is inferred initially that promoter D is negatively regulated by some regulation mechanism to make spliceosome D expression decreased in oviduct. ER gene is initiated from multiple promoters in the5’flanking regions, thus expression of ERa is unaffected.6. Expression levels of ERa and spliceosome D in male and female chick tissues from five periods detected by semi-quantitative RT-PCR.It is identified that the two expression are most active in gonads. Compared with other tissues, ERa expression in liver is relatively higher, but spliceosome D expression relatively lower. It is initially considered that promoter D is positive correlated with ERa expression in gonads, but it is negative correlated with ERa expression in liver of chick embryonic development periods.