Regulation of trophoblast differentiation by BAF complex chromatin remodelling factors

The placenta is a critical organ required to maintain a healthy pregnancy. All organs are made up of many distinct cell types. In the placenta the foetus contributes a range of different highly specialized cells called "trophoblasts", which form from a single specialized cell type called Trophoblast Stem Cells (TSCs) that we can culture and study in the laboratory. Pregnancy complications such as preeclampsia, and its more severe form HELLP Syndrome affect approximately 5% of pregnancies, have potentially long-term health implications and are one of the leading causes of maternal and infant death. Defective development and behaviour of trophoblasts is heavily implicated in these pregnancy complications so understanding how trophoblasts form is critical to developing better diagnostic, treatment and management methodologies to tackle such disorders.

The identity and behaviour of cells is largely controlled by the genes that are active within the cell. Whether genes are active, and thus the identity of cells, depends on whether DNA is accessible to the molecular machinery that controls gene activation. The accessibility of DNA is controlled by specialised proteins known as "chromatin remodelling factors", so these factors control trophoblast development and behaviour. In particular, previous studies suggest that the BAF complex of chromatin remodelling factors are important for the correct formation of different trophoblast types, and thus a viable pregnancy.

In this project we will investigate how BAF complexes control the balance of different trophoblast types arising from TSCs, and how they interact with a network of other proteins to do so. Our research will tell us how cell fate decisions are controlled in the placenta. It will reveal proteins and DNA sequences that control trophoblast formation, and which may therefore be mutated leading to pregnancy complications. The ultimate result will therefore be key advances in our fundamental understanding of how the placenta develops. This new knowledge can be exploited in future to develop therapeutic intervention strategies in pregnancy disorders that are associated with problems of placental function.

Trophoblast stem cells (TSCs) are multipotent self-renewing cells giving rise to multiple trophoblast subtypes within the placenta, essential to maintaining a viable, healthy pregnancy. Mouse TSCs are a well-established model system that can be manipulated in vitro to interrogate molecular mechanisms controlling trophoblast differentiation. Pregnancy complications such as preeclampsia and HELLP (haemolysis, elevated liver enzymes, low platelet count) Syndrome are particularly linked to defective formation and behaviour of invasive endovascular trophoblasts that enhance maternal blood supply to the foetus. Our preliminary analyses (see Case for Support) suggest formation of this essential trophoblast subtype is heavily influenced by BAF complex chromatin remodelling factors.

I recently showed the chromatin landscape undergoes widespread changes during TSC differentiation. We will continue to exploit mouse TSCs to understand how BAF chromatin remodelling complex bromodomain(BRD)-containing proteins BRG1, BRM and BAF180 influence the chromatin landscape and thus trophoblast cell fate and behaviour by:
1) Using CRISPR/Cas9 gene knockout and a highly potent and specific small molecule inhibitor specifically targeting BRG1, BRM and BAF180 BRDs, coupled with RNA-seq and qRT-PCR profiling of well characterized markers of individual trophoblast subtypes;
2) Profiling BRG1, BRM and BAF180 chromatin binding locations using ChIP-seq in control and BRD-attenuated trophoblasts;
3) Profiling BAF complex-dependent chromatin accessibility using ATAC-seq on control, BRG1, BRM, BAF180 and BRG1;BRM CRISPR knockout and BRD-inhibited trophoblasts;
4) Determining the composition of BRG1-, BRM- and BAF180-containing protein complexes in wild type trophoblasts using co-IP with mass spectrometry.

This will provide valuable insights into trophoblast subtypes controlled by BAF complexes, how they control use of cis-regulatory sequences and their molecular mechanism of action.

Who will benefit?
The main immediate impact of this fundamental research project will be on the projects of other research scientists, as detailed elsewhere. In the medium- to long-term the principal beneficiaries will be:
1) women suffering from preeclampsia and other pregnancy complications, their unborn children and families;
2) The NHS and other healthcare organisations through better predictive, prognostic and management strategies for pregnancy complications;
3) Commercial organizations willing to exploit our genomic data and findings to provide medical tests to inform medical practice in the management of pregnancy complications, or to produce trophoblast cell types of therapeutic value.

How will they benefit?
Pregnancy complications such as preeclampsia and HELLP (haemolysis, elevated liver enzymes, low platelet count) Syndrome can have health implications to both mother and child beyond the pre- and early post-natal period. Research providing key insights into gene regulation in the placenta will lead to improved prediction and management strategies will therefore have a range of benefits. It will reduce hospital time for sufferers, reduce the incidence of premature births in response to pregnancy complications, allow patients to be appropriately prioritised for antenatal care and facilitate timely implementation of treatment regimens to negate and control long-term complications. This will have the potential effect of enhancing the lifelong health and wellbeing of both mother and child, through reducing incidences of health complications linked to premature birth, or likelihood of cardiovascular disease in later life. It will simultaneously benefit the economy through their enhanced ability to work and a reduced healthcare burden. Benefits to the NHS are a reduced patient burden and fewer negative outcomes, which can potentially lead to costly litigation. Clinically and commercially exploitable strategies arising from the novel knowledge we will provide, such as to identify genetic variants associated with pregnancy complications, or to produce trophoblasts of therapeutic value will also help drive innovation and economic growth.

Further impact
By championing and exploiting in vitro approaches using trophoblast stem cells to generate highly meaningful functional data to complement necessary in vivo approaches we are also acting to replace and reduce numbers of animals used in research, thus providing a Replacement, Refinement and Reduction of Animals in Research (3Rs) impact

The biology and medical management of pregnancy complications is an emotive issue likely to be of broad public interest. We plan to communicate our research outside of academia to raise awareness of developmental biology and stem cell research and its potential applications to understanding disorders of pregnancy by continuing collaboration with the Public Engagement team at University of Warwick. Our outreach and public engagement activities will also contribute to a more informed public debate. Consistent with this, the PI will continue to communicate with the public and promote the visibility of our research through all available forums including our website, public debates, and individual events tailored for this purpose. This will further increase public awareness of cutting-edge technology and approaches, thus generating greater public interest and enthusiasm for science and promoting a more scientifically knowledgeable society.