Systemic lupus erythematosus (SLE) involves the inappropriate activation of the adaptive immune system against self-antigens. The adaptive immune system includes B and T lymphocytes. B cells contribute to SLE by the improper production of self-reactive antibodies and induction of effector T cells. A subset of T cells, regulatory T cells (Tregs), contribute to SLE by loss of their suppressive function of effector T cells. An intriguing feature of SLE is that it is far more common in females than males, with a ratio of 9:1, strongly suggesting a genetic link with X chromosome diploidy. My preliminary studies show a significant frequency of chromosome X co-localization or coalescence in human female Tregs as compared to a non-lymphocyte control. In the control the active X (Xa) and inactive X (Xi) chromosome are kept separate from one another at a higher frequency suggesting a protective mechanism not seen in Tregs. I hypothesize that the coalescence of the Xa and Xi chromosome leads to trans-chromosomal gene regulation resulting in inappropriate repression of lymphocyte-specific genes located on the Xa leading to cellular dysfunction and autoimmunity in women.
The first aim of my proposal will identify the human female lymphocyte populations most susceptible to chromosome X coalescence as well as the regulatory effects of the Xa and Xi being in close proximity. The nuclear organization traits, epigenetic modifications, and expression levels of genes important for lymphocyte function that are located on chromosome X will be measured during chromosome coalescence and separation. Measurements and analysis will be carried out by various three-dimensional fluorescence in situ hybridization (FISH) techniques including; RNA/DNA FISH, immuno-DNA FISH, and intron chromosomal expression (ice)/DNA FISH.
The second aim will focus on identifying direct links between chromosome X coalescence and altered expression levels of lymphocyte specific genes in SLE patient lymphocytes. The primary focus will be on chromosome X coalescence in Tregs and its effect on FOXP3 expression. FOXP3 is required for Treg differentiation and Treg suppressive function. Lowered levels of FOXP3+ Tregs is an established hallmark of SLE. RNA/DNA FISH, immuno-DNA FISH, and intron chromosomal expression (ice)/DNA FISH will be utilized for this aim in order to analyze nuclear organization traits, epigenetic modifications, and gene expression levels on a per cell basis in SLE lymphocytes. In summary, this proposal aims to address a long-standing question in the study of SLE with a novel nuclear organization oriented approach. The successful completion of the aims outlined above would identify for the first time the genetic cause and mechanism underlying the increased incidence of SLE in women. Also, the successful completion of this analysis would yield a novel marker for SLE diagnosis. Such an advance is very much needed as current criteria for diagnosis are non-specific and often leads to misdiagnosis and inappropriate treatment. Additionally, this would be the first documented instance of chromosome X trans-chromosomal regulation in differentiated cell types.
Current methodologies for diagnosing the autoimmune disease systemic lupus erythematosus (SLE) in females, lacks definitive genetic markers. This shortcoming results in misdiagnosis and inappropriate treatment plans. The work proposed in this application will determine the genetic causes and mechanisms underlying increased incidence of SLE in women, leading to generation of more effective diagnostic applications.
