Browsing by Subject "RNA-Seq"

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Open Access
RNA based biomarkers for prediction of the endometrial window of implantation
(2020-12) Dedeoğlu, Ege
Early reproductive failure is the most common issue related to successful pregnancies, as around 30% of all conceptions reach live birth. The path to a successful pregnancy is reliant on the successful implantation of the embryo to the endometrium. This event requires three major components; a viable embryo ready for implantation, a receptive endometrium in which the implantation will occur, and healthy crosstalk between the embryo and receptive endometrium. It is estimated that two out of all three implantation failures are related to endometrial origin. This has led many researchers to attempt to elucidate the mechanism behind endometrial receptivity and generate a prediction of successful implantation of endometrial origin. Although there have been plenty of articles on this subject, there is still no consensus regarding standard endometrial receptivity biomarkers. Additionally, most of these articles’ findings cannot find their way into clinics. This is highlighted by the fact that the success rate of embryo implantation in ART applied in clinics is only around 10%. This study aimed to identify novel methods and biomarkers to predict the endometrium's receptivity, which could be applied in clinics easier and faster than the current kits in the market. We took several different approaches to achieve this aim. The first was to identify and validate particular miRNAs that showed a change in expression levels of the different days of the endometrial cycle in a healthy women’s serum. Our bioinformatical analysis has yielded ten miRNAs that show statistical differences in the human endometrium and being expressed in the serum. Downstream RNA-Seq and qPCR experiments have validated specific miRNAs previously predicted and identified novel miRNAs used for this purpose. The second was using in silico methods, identifying novel genes present in the endometrium that can predict the optimal point of receptivity. If considered and validated in vitro, this novel gene-list will be a cheaper but still as powerful alternative to the current endometrial test kit used in clinics today. Further validation RNA-Seq experiments on healthy and infertile females will elucidate our novel biomarkers' strength, designed to be used in ART clinics worldwide. Furthermore, upon building on these findings, it is possible to uncover previously overlooked mechanisms leading to women's implantation success.
Open Access
Role of histone variant H3.3 in transcription and mitotic progression
(2017-04) Örs, Ayşegul
Chromatin structure needs to be dynamic and flexible in order for the eukaryotic cellular processes to function correctly. Incorporation of histone variants into chromatin serves to increase epigenetic plasticity by conferring new structural and functional properties to chromatin. Histone variants are implicated in many cellular processes such as transcription or cell division and their deregulation is involved in tumorigenesis. H3.3 is an evolutionarily well conserved histone variant that differs by only a few amino-acids from its replicationdependent counterparts. With the aim of determining H3.3 function, novel knockin/ conditional knock-out mouse models were established and characterized. In these models, one of the two genes coding for H3.3, H3f3a or H3f3b has been modified to code for an N-terminal FLAG-FLAG-HA tagged H3.3A or H3.3B which can be depleted upon Cre expression. Nucleosome resolution genome-wide mapping FH-H3.3A and FH-H3.3B determined that H3.3A and H3.3B were similarly enriched at promoter regions and their enrichment levels positively correlated with high expression and gene body enrichment. They were also found enriched in telomeres and some repetitive DNA sequences. In a subset of these repetitive regions H3.3A and H3.3B showed differential enrichment properties. As double H3.3-KO mouse generation resulted lethal, mouse embryonic fibroblasts (MEFs) were isolated from FH-H3.3 mice and transformed. Using a combination of Cre recombinase mediated knock-out and RNA interference technology, a new cellular model was established where H3.3 expression was essentially depleted. Although H3.3 enrichment profiles were indicative of a role in active transcription, whole transcriptome analysis upon single H3.3 depletion in livers and an almost complete H3.3 depletion in MEFs yielded very few differentially regulated genes. Interestingly, H3.3 depleted MEFs showed a high increase in mitotic defects and abnormal nuclear structures. Thus, an important yet often understudied role for H3.3 in genomic maintenance during mitotic progression was highlighted.