Our Journey so far
The central interest in the Brockdorff lab is to understand how X inactivation works. We are probably biased but we think this is simply the best model system to delve into the secrets of how genes and genomes are regulated as organisms develop from a single cell, the fertilized egg, to the incredibly complex multicellular system that makes up a living breathing animal. One of the great joys of studying X inactivation has been the journey through so many fascinating dimensions of biology.
Classical genetics and the study of animals/cells with rearranged X chromosomes played a key role in our early work on defining the X inactivation centre, a locus on the X chromosome that is critical for the X inactivation process. Taking this forward we were eventually able to identify the X inactive specific transcript (Xist) gene, the master regulator of X inactivation.
Images from our early studies mapping the location of the X inactivation centre and Xist gene on the X chromosome
Turning to molecular biology we found that the Xist gene produces a very large non-coding RNA that is expressed from the inactive X chromosome when X inactivation begins, and continually thereafter. We went on to show that Xist is necessary for X inactivation by deleting the Xist gene in XX embryonic stem cells, a model for studying X inactivation in vitro.
Molecular assays from our work showing Xist is a large RNA expressed in XX females and not XY males (left), and that Xist RNA is expressed exclusively from the inactive X (centre). Sequencing of Xist RNA revealed absence of significant open reading frames, suggesting it is a non-coding RNA (right).
The application of cell biology methods that were developed to visualise specific RNAs by fluorescence microscopy allowed us to visualise Xist RNA accumulating from the site of transcription to cover the entire length of the X chromosome.
Images from our work using RNA fluorescence in situ hybridisation to show that Xist localizes in a banded pattern over the length of the X chromosome at metaphase (left), and that Xist RNA is progressively upregulated in differentiating XX female ES cells forming a cloud of RNA that corresponds to the inactive X chromosome at interphase (right).
We immersed ourselves in studying X inactivation during mammalian development, understanding how the process is regulated and coordinated so that a single X chromosome is inactivated at the right time and only in cells of female embryos. Equally fascinating have been studies on the reversal of X inactivation that occurs during maturation of female germ cells and also during experimental reprogramming for example in animal cloning.
Image illustrating the cycle of X inactivation during female embryogenesis in mouse (left) and an immunofluorescence staining of a protein that is concentrated on the inactive X chromosome in an early (blastocyst) stage mouse embryo (right).
The inactive X chromosome is a large structure and this has allowed us to apply advanced imaging methods or super-resolution microscopy, to investigate its structure and organisation and to visualise Xist RNA and the key molecules that drive the process.
Images from our work using super-resolution microscopy illustrating a female cell with an Xist RNA cloud made up of around 100 individual Xist RNA molecules (left) and showing the close spatial relationship between Xist RNA and proteins of the Polycomb repressor family (right).
Much of our work in the past 30 years has involved the identification and analysis of the molecules that underpin X inactivation using the techniques of molecular genetics, molecular biology, genomics, epigenomics, proteomics and biochemistry.
Examples from our work illustrating X chromosome wide analysis of gene expression (top) and the increased deposition of specific histone modifications (bottom) during X inactivation.
Through these approaches we have been able to understand the link between Xist RNA and chromatin, notably histone protein modification, DNA methylation, variant histones and molecules that regulate higher order chromosome folding, for example SmcHD1.
A model illustrating the major protein factors that bind to Xist RNA to initiate modification of underlying chromatin and gene silencing
As we have moved into an era where most of the key molecules needed for X inactivation have been identified, we have begun to better understand the dynamics of the process by watching them in action using live-cell imaging.
Movie showing the dynamic behaviour of fluorescently tagged Xist RNA molecules after induction in mouse embryonic stem cells