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Origination of novel gene regulatory networks in the evolution of mammalian pregnancy

Periodic Reporting for period 1 - EVOLPREG (Origination of novel gene regulatory networks in the evolution of mammalian pregnancy)

Reporting period: 2016-04-12 to 2018-04-11

The basic understanding of gene regulatory networks important for mammalian pregnancy has remained enigmatic. The pregnancy in placental mammals (eutherians) includes internal fetal development facilitated by an invasive placenta and a richly vascularized uterine endometrium that can accommodate implantation. These functions are associated with the capacity of endometrial stromal fibroblasts (ESF) to differentiate (decidualize) to decidual stromal cells (DSC). In this project I studied on the gene regulatory networks involved in decidualization.
Recently it was reported that ancestral function of DSCs was to manage the inflammatory reaction upon implantation, whereas maintenance of pregnancy is a derived function. These characteristics may correspond to decidualization phases of DSCs, and my first objective was in vitro characterization of gene regulatory networks typical for decidualization phases. We documented extensive, dynamic changes in the early (3 days) and late (8 days) decidual cell transcriptomes (Rytkönen et al. 2018). Transcription of transcription factors in both phases is characterized by putative or known PGR target genes, indicating that both phases are under progesterone control. Regarding the inflammatory status of the cells, the data suggest the existence of an early STAT pathway dominated state and a later NFKB regulated state, and these definitions may be useful for understanding endometrial and pregnancy states in vivo (Figure 1).

The second objective of this project was to study how internal and external signaling compounds mediate transcriptional changes via a known endometrial regulator, Aryl Hydrocarbon Receptor (AHR). We studied AHR activation upon kynurenine or TCDD induction. In early decidualization phase both activators decreased transcription of interferon (IFNG) induced genes and modulated transcription in acute inflammatory pathways (TNF/IL-8) indicating that AHR regulates inflammation in the early phase. In the late decidualization phase kynurenine activation affected extracellular matrix (ECM) genes via downregulation of TGFB signaling pathway suggesting that in addition to inflammation, AHR also regulates fibrosis in the endometrium.

Thirdly, as oxygen delivery is a main placental function and stress tolerance is important for the decidual phenotype, we investigated the effects of hypoxic stress on endometrial gene regulation. Transcriptomic analysis revealed that both ESF and DSC respond to hypoxia by upregulating classical hypoxia pathways such as glycolytic genes. However, in ESF hypoxia induced repression included several inflammatory genes whereas in DSCs general stress response genes and cell cycle mediators were repressed. These findings further support that in specifically in DSCs general stress response is replaced with decidual specific regulatory circuits.

We also conducted ChIP-seq on chromatin histone markers and observed that the number of actively transcribed H3K4me3 associated promoters did not change upon hypoxia. To explore the peak dynamics we correlated the H3K4me3 promoter mark height and breadth to transcription levels, and found that in both conditions, the H3K4me3 height correlated with the gene being turned on or off, whereas breadth also partly captured the intensity of the transcription. Investigation of the broadest and sharpest H3K4me3 peaks in hypoxic DSCs indicated that only the genes with broad peaks were highly enriched in functional categories relevant for endometrial physiology, such as blood vessel development and ECM.

These results are being disseminated to scientific audience via several scientific publications and conferences such as Keystone Symposia. The knowledge is delivered to students and younger scientist trough courses and supervision, and for general audience via science fair and a newspaper article. Our results on gene regulatory networks will be directly useful for understanding the basic characteristics of endometrium and may provide mechanistic insights for therapeutic applications. On a conceptual level our findings specifically move the research focus more towards on how changes in temporal regulatory states of decidualization may underlie reproductive disorders. We are exploiting the results by initiating a project where clinical data will be studied with special emphasis on the decidualization state specific gene regulation.
Overall the results of this project provide novel molecular and mechanistic insights into decidual gene regulation. This is important from the perspective of basic reproductive biology as well as maternal and fetal health, because majority of reproductive and pregnancy complications involve defects in endometrial decidualization. Recently it has also been discovered that decidualization defects contribute to maternal age-related decline in reproductive success.

References:

Rytkönen KT, Erkenbrack EM, Poutanen M, Elo LL, Pavlicev M and Wagner GP (2018) Decidualization of human endometrial stromal fibroblasts is a multi-phasic process involving distinct transcriptional programs. Reprod. Sci., accepted
Figure 1. The main inflammatory transcription factor pathways in decidualization