X inactivation is an important mechanism of dosage compensation in mammals, which silences at random one of the two X chromosomes in female cells and thus equalizes gene expression between males (XY) and females (XX). X chromosome silencing is associated with the accumulation of inactive epigenetic marks. The inactive X chromosome is highly condensed and preferentially located adjacent to specific nuclear compartments, either the nuclear membrane or the surface of the nucleolus. Despite efficient silencing some genes escape X inactivation and thus remain bi-allelically expressed in females, resulting in sexually dimorphic gene expression levels. A number of these genes escape X inactivation in multiple species, indicating that escape from silencing is an essential feature of these genes. To track allele-specific gene expression changes during cell differentiation at single-cell resolution and determine pathways that govern X-linked gene silencing and escape during development we will employ mouse models that we previously developed to measure allele-specific gene expression and chromatin structure. Our previous findings demonstrate tissue-specific differences in genes that escape X inactivation. We will now address cell-to-cell and time-related variability in X-linked gene expression. This research will help develop analytical methods to follow dynamic changes in gene expression applicable to the whole genome. To determine the 3D architecture of the inactive X chromosome and of domains of inactivation and escape we will use a novel DNase Hi-C method combined with allele-specific analyses. By this approach we have obtained the first 3D view of the whole inactive X chromosome, which reveals a surprising bipartite structure with an apparent hinge that binds CTCF on the inactive allele. These preliminary results will serve as the basis for further experiments to obtain a higher resolution view using X chromosome capture libraries. We will examine the 3D data in relation to lncRNA loci and chromatin elements such as CTCF binding sites that may govern the architecture of the silenced X chromosome and of domains of escape and inactivation. In particular, we will examine the role of specific elements in positioning the inactive X chromosome within the nucleus. This research is relevant to the understanding of sex differences and of congenital and acquired diseases associated with sex chromosome and other genomic abnormalities.
Our goal is to use the X chromosome as a model system for the study of the control of gene expression at single-cell resolution during differentiation, and o epigenetic mechanisms related to the architecture and positioning of genomic regions within the nucleus. This research will help understand disease states such as cancer in which epigenetic regulation and nuclear conformation are clearly disrupted.
Showing the most recent 10 out of 40 publications
