Epigenetic modifications modulate gene expression in normal development and in diseases, such as cancer. X chromosome regulation is an example of coordinate epigenetic regulation of gene expression at the level of an entire chromosome. Our goal is to investigate the epigenetic regulation of X-linked genes in mammals. Genes on the X chromosome are dosage compensated by two processes: upregulation of the active X chromosome in both sexes, and X inactivation in females. A subset of genes escapes X inactivation and thus has higher expression in females. The importance of these genes in normal development is illustrated by the phenotypic anomalies including embryonic lethality in Turner syndrome, which is associated with a single X chromosome. We will investigate the molecular mechanisms that allow genes that escape X inactivation to be expressed within the context of silenced chromatin.
In Aim 1 we will establish a comprehensive list of all genes that escape X inactivation in mouse and determine their chromatin structure. We will use RNAsequencing to distinguish expression from each allele of X-linked genes in cells with two X chromosomes, each from a different mouse species, so that alleles can be distinguished based on polymorphisms between the species. We will map the distribution of chromatin modifications uniquely present on either the active or inactive X chromosomes using chromatin immunoprecipitation (ChIP) together with array and sequencing analyses. Based on these data we will investigate the role of specific enzymes that establish or remove specific histone modifications in relation to X inactivation or escape during female ES cell differentiation when X inactivation takes place. Our previous studies show that escape genes are flanked by binding sites for the chromatin insulator element CTCF that may protect them from adjacent heterochromatin.
In Aim 2 we will determine the functional role of this element in regulating the chromatin structure of the X chromosome by constructing a mouse with mutations at CTCF binding sites that flank an escape gene (Jarid1c). The effects of these mutations will be examined in female ES cells and in mice to determine whether the expression and epigenetic features of Jarid1c are altered in the absence of CTCF binding. This study will advance understanding of the role of chromatin structure in the control of gene expression in normal biology and diseases.
Our study will provide a better understanding of the role of specific proteins in maintaining the structure of expressed and silenced regions of the genome. Our study is relevant to the study of sex differences and sex chromosome disorders, which are poorly understood at the molecular level. Results of our work may also provide new insights into prevalent diseases such as cancer, in which epigenetic modifications can be profoundly altered and lead to uncontrolled cell growth.
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