Dynamic regulation of chromatin loops by cohesins and CTCF in real time: physiology and pathology

The human genome is spatially organised at multiple levels. In the range of tens to hundreds kilobases of DNA, two distant chromosome sites are brought together to form a chromatin loop. Chromatin loops are found throughout the genome and they represent basic units of genome organisation. They are also important for regulation of gene expression. The genome-wide organisation of chromatin loops has recently been studied in detail, but it is still poorly understood how individual chromatin loops change their shape over time.

In this project, we will visualise selected chromosome sites, using advanced microscopy techniques. Using these we can analyse the formation and dissolution of individual chromatin loops in human cells and, in addition, can study the molecular mechanisms regulating the dynamics of chromatin loops by depleting candidate regulators and studying the outcomes in cells. Moreover, to explain the dynamics of chromatin loops, we will develop mathematical models, which will integrate several steps involved in chromatin-loop formation and dissolution.

Our recent data suggest that common regulators facilitate both formation of chromatin loops and cohesion between duplicated chromosomes at the same chromosome sites. However, it is unknown how these two functions are interlinked. We will address whether the two functions compete with each other or whether they are co-regulated, i.e. up-regulated or down-regulated together.

Regulators of chromosome loops are often mutated in inborn diseases and some types of cancers e.g. bladder cancer and leukaemia. To address how these diseases develop, we will investigate how such mutations change the dynamic behaviour of chromosome loops. Overall, our study will provide crucial information on how a basic unit of genome organisation is dynamically regulated in human cells and how this process could go wrong in human diseases.

Chromatin loops represent basic units of genome organisation. The formation of a chromatin loop relies on cohesins and the CCCTC-binding factor (CTCF). The 4C and Hi-C analyses revealed formation of chromatin loops genome-wide. However, it is still unclear how the conformations of individual chromatin loops change over time and what molecular mechanisms drive such changes. We will visualize conformation of chromatin loops and observe their dynamic changes in real-time imaging of human cells. For this, we will select repetitive DNA sequences adjacent to two CTCF binding sites involved in loop formation and visualise them using nuclease-deficient Cas9 and single guide RNAs. Our pilot experiments show that this method is feasible and we will further develop and optimise it.

Using the above method, we will investigate the formation rate and lifetime of chromatin loops. We will also study molecular mechanisms regulating their formation and dissolution. Based on these analyses, we will develop mathematical models for loop formation and dissolution. These analyses will establish an important framework for the real-time dynamics of chromatin loops. Meanwhile, cohesins facilitate both chromatin-loop formation and sister chromatid cohesion at CTCF-binding sites. However it is unknown how cohesins interlink two roles at the same chromosome sites - we will address this question.

Mutations of cohesins and their regulators cause various congenital disorders and promote specific types of cancers. It is suspected that these mutations impair chromosome organisation, leading to such diseases. However, the specific mechanisms by which defects of cohesins and their regulators lead to these diseases are unknown. To obtain insight into such mechanisms, we will study how these mutations affect the dynamics of chromatin loops. In particular, we will focus on NIPBL haplo-insufficiency, which causes a congenital disorder, and STAG2 mutations, which are frequently found in cancers.

The proposed research will have the following academic and economic/societal impacts:

Academic impact:

1) In the proposed project, we will investigate the dynamic regulation of chromosome organisation, focusing on its basic units, chromatin loops. Our research will reveal novel molecular regulation of chromatin loops, which will contribute to research in chromosome organisation and gene regulation. In addition, we will study how chromatin loops and sister chromatid cohesion are interlinked by common regulators. Our research will give important insight into dynamic chromosome organisation, which will contribute not only to research in chromosome biology but also to cell and developmental biology in general.

2) Our research will produce useful reagents for the research community. Such reagents include DNA constructs and human cell lines. In the proposed work, we will also develop computer models for the dynamics of chromatin loops. We will swiftly share our reagents and computer codes with the research community, following publication.

3) We will address fundamental mechanisms involved in the dynamic regulation of chromosome organisation. Dynamic chromosome organisation plays important roles in regulation of gene expression. So, our project should give important information to university students and early-career researchers, who are interested in genetics, cell and developmental biology. We hope our research will give them inspiration and encouragement to become life scientists.


Economic and societal impact:

1) We aim to deliver high quality and internationally competitive science. This should help the UK to maintain its reputation as a leader country in biomedical science research.

2) Several human diseases are caused by mutations of cohesins and their regulators. In fact, their germline mutations cause congenital disorders, which are collectively called cohesinopathies. For example, mutations of the chromosome cohesin loader, NIPBL cause Cornelia de Lange Syndrome. Moreover, somatic mutations of cohesins lead to specific types of cancers such as bladder cancer, Ewing sarcoma and myeloid leukaemia. In particular, mutations of cohesin STAG2 are frequently found in these cancers. We will address whether (and, if so, how) dynamics of chromatin loops are altered by these mutations associated with the diseases. Our research will give important insights into the mechanism of these diseases. In the long term, our research should have clinical relevance to their diagnosis and treatment.

3) Our research will provide excellent materials for public engagement regarding cell biology and life sciences. For example, our microscopy images in the proposed work will showcase dynamic cellular events. Moreover, we will be able to demonstrate how defects in dynamic chromosome behaviours could lead to various diseases. We will organise inspiring public engagement events using materials from our research.