Targeting the inactive X chromosome in lupus

Lupus affects nine times more women than men. One explanation for this phenomenon lies in the difference between women’s and men’s chromosomes, the long pieces of DNA within each cell that contain our genes lined up one after the other like recipes in a cookbook. Women have two X chromosomes, while men have one X and one Y chromosome. Dr. Laura Carrel is investigating how this difference in the number of X chromosomes might cause women to be more susceptible to lupus than men.

Normally, in a woman’s cells, the genes on one of her X chromosomes are active and those on the other are turned off or inactive. But Dr. Carrel has shown that in all women, about 10% of genes on the “inactive” X chromosome escape from the inactivation process and are actually active. She hypothesizes that the level of gene activity on the inactive chromosome might be even higher in women with lupus. To test this theory, Dr. Carrel will study the immune cells of women with and without lupus to find X chromosome genes that are more active in women with lupus.

What this study means to people with lupus

This research project will help us understand the biology of lupus and point to new targets for drug discovery.

Systemic Lupus Erythematosus (SLE) is heavily biased 9:1 towards females. This gender discrepancy appears to reflect both hormonal and sex chromosome differences between men and women. This application will focus on the X chromosome in SLE, as X-linked genes may underlie gender biased disorders based on how they respond to X-chromosome inactivation (XCI), the epigenetic process that silences one X in females to achieve dosage equivalence with males. Because of XCI, females are mosaics with cells that inactivate the maternally or paternally-inherited X. While XCI represses most genes on one X in females, we established that ~10% “escape” XCI and are expressed from both the active and inactive X chromosomes. Additionally, XCI states for >15% of human X genes are heterogeneous and escape XCI from a subset of Xs, but are silenced or reduced in expression in other females. These genes are responsible for an extraordinary degree of expression variation that is unique to females. Yet, how they contribute to traits with gender differences has not been addressed.

Multiple studies point to an X chromosome role in SLE. In particular, some X-linked genes are overexpressed and/or hypomethylated in women with lupus. Nevertheless, direct evaluation of inactive X expression has not been examined, as it cannot be ascertained from microarray data or even RNAseq data without allele-specific analysis and consideration of XCI mosaicism. Moreover, sex chromosomes are excluded from most genome wide association study (GWAS) efforts. The objective of this proposal is to evaluate inactive X gene expression in lupus and to determine whether small molecule inhibitors of epigenetic modifiers modulate inactive X gene expression in lupus cells.

We hypothesize that females with lupus have higher expression of genes due to XCI escape. Supporting this hypothesis, our preliminary data in normal lymphoblasts indicate that the X-linked gene NAA10 carrying a SLE high-risk allele has higher inactive X expression (higher XCI escape) than the low-risk allele. Further, genes in regions implicated in SLE have XCI escape heterogeneity. In order to directly test our hypothesis it is necessary to evaluate individuals with SLE.

We aim to test inactive X expression in B cells, T cells, and macrophages from SLE and control individuals by high-throughput RNA sequencing (RNAseq) using our custom computational pipeline that assesses allele-specific expression following normalization for XCI mosaicism. We will validate RNAseq results by RNA FISH and directly test candidate genes including the immune relevant genes TLR7, TLR8, IRAK1, and CD40LG. We will further test SLE cells to determine how genes that escape XCI respond to active gene expression inhibitors including histone demethylase inhibitor GSKJ1, DOT1L inhibitor EPZ5676, and BRD4 inhibitor JQ1. These studies will give key insight into SLE targets and mechanisms and will begin to test their response to potential therapeutics.