Some scientists have proposed the “X Chromosome Nucleolus Nexus” hypothesis to explain XCI escape. This hypothesis states that when the cells are exposed to stress such as viral infection, it leads to nucleolus expansion. Since the nucleolus is very active, if the Xi is located near the nucleolus it can be disrupted. The nucleolus contains high concentrations of polyamines and nucleolin. Polyamines such as spermidine and spermine function as counter ions to the anionic RNA, allowing RNA stabilization and intra-strand hybridization. They are also involved in expansion of the nucleolus. If the Xi is exposed to polyamines, the polyamines can disrupt the Xi chromatin state by binding to DNA and competing with histones. Polyamines can also stabilize alternate DNA conformations such as those at fragile sites on the X chromosome. The nucleolin can also alter the Xi chromatin and disrupt gene silencing.
Other features of the X chromosome may contribute to escape from XCI. The X chromosome contains 6 fragile sites. Fragile sites are segments of DNA vulnerable to altered rates of replication, gaps, breaks, and viral insertions. Problems at fragile sites can result in gene loss, gaps in the chromosome, and interference of epigenetic regulation. Some fragile sites are enriched with AT repeats and thus the DNA strands are more susceptible to separation and intra-strand hybridization which can interfere with polymerase activities. 29% of PAR1 on the X chromosome consists of Alu elements, and the composition of the adjacent S5 region is 19% Alu elements. The abundance of Alu elements on the X chromosome may also influence XCI escape because Alu elements are CpG rich and require pervasive methylation to prevent improper transcription. These elements contain an intragenic RNA pol III promoter which is silenced by the placement of a nucleosome over it.
Potential issues can arise during cell replication because the Xi is the last chromosome to be replicated. If the supply of suppressor proteins and methyl donors like SAM is low, the Alu elements will be erroneously transcribed and the resulting Alu RNA transcripts can disrupt methylation, translation, and translocation, potentially interfering with initiation and maintenance of XCI. Additionally, according to the X chromosome nucleolus nexus hypothesis, proximity of Xi to the nucleolus exposes it to nucleolins and polyamines which can displace the nucleosomes and enable unwanted RNA polymerase activity. Previous studies have observed significantly elevated serum levels of Alu DNA in SLE patients compared to healthy controls, giving credence to the role of Alu elements in XCI escape. Finally, disrupting the nucleosomes blocking Alu transcription would liberate the stored negative supercoiling stress that could then ripple through the region and temporarily unsettle other complexes like those involved in Xist RNA anchoring, further advancing XCI escape.
Despite ample research into XCI, XCI escape and its relationship to the sex bias in autoimmunity and allergy, there remains many uncertainties. Observations from some studies may contradict findings from others and frustrate attempts to pinpoint an exact answer. For example, the exact role of FOXP3 in SLE is still contradictory. While overexpression of FOXP3 in mice offers protection from kidney dysfunction, high numbers of CD4+ FOXP3+ T cells are associated with more severe SLE in human patients. Another example is the role of toll like receptors in SLE. While TLR7 and TLR9 are both part of the TLR family, they seem to cause very disparate effects. Mice that lack TLR9 exhibit severe cases of SLE whereas mice that lack TLR7 exhibit a mild form of SLE. A further point of concern is the accuracy of using mice models to draw conclusions on human autoimmune diseases and allergies. There are several differences between the murine X chromosome and the human version that should be highlighted. The murine version is telocentric, with the centromere located at the end of the chromosome arm, whereas the human version is submetacentric, with the centromere located relatively closer to the middle. Therefore, in mice, the centromere would not impede the spread of XCI down the chromosome arm, as it was hypothesized to do in the human X chromosome.
Another important difference is that the murine X chromosome lacks Alu elements, which were thought to have a role in promoting XCI escape, thus the murine Xi is predicted to be more resistant to XCI escape. Overall, mice models might not be suitable when it comes to the study of X-linked events preceding the adaptive immune response. Unfortunately, that is an issue associated with the use of every model organism, and studies involving human participants come with their own set of restrictions and limitations.To this day there is no clear-cut answer thoroughly explaining the mechanisms behind the increased risk of females for developing autoimmune and allergic diseases. Many scientists believe that escape of X-linked features such as immune-related genes results in the sex bias, but questions remain. For example, as mentioned in the previous section, there are some contradictory findings such as the role of FOXP3 in SLE.
There should be studies into the exact involvement of FOXP3 and why overexpression in mice confers protective effects while simultaneously being associated with serious SLE in humans. Another area to further study is the potential role of miRNAs in the sex bias. Since miRNAs are present on the X chromosome while absent on the Y, and are known to have immune response related functions, there is a great likelihood of them contributing to the sex disparity in development of autoimmune and allergic diseases. In addition, some studies have found that naïve female lymphocytes are deficient in epigenetic modifications to the Xi such as Xist RNA and heterochromatin marks and that these modifications only return upon antigen stimulation. Research should be conducted into the reason behind this phenomenon, as well as the mechanisms and cell extrinsic factors involved. Furthermore, researchers should investigate the effects of this phenomenon on autoimmune disease risk and severity. Studies on this subject should gradually shift to focus more on the epigenomics and away from trying to find specific genes and mutations. Advances in technology have opened new realms of exploration. For instance, there are now tools that can alter epigenetics in a cell -specific manner, enabling research into the effects of specific epigenetic changes on specific DNA segments.
However, researchers should not just concentrate on methylation and histone patterns. Epigenetics also involves less obvious changes, such as the fleeting changes caused by release of supercoiled stress and transient alternative DNA conformations at fragile sites. Human females display higher incidences of autoimmune disease, with some diseases such as SLE having female: male ratios as skewed as 9:1. Adult females also display an increased risk of developing allergies compared to male counterparts. Many researchers believe this sex bias in autoimmune and allergic diseases stems from escape of X-linked genes from X chromosome inactivation. In this paper the process of XCI and its hypothesized mechanisms were explained. We also examined several X-linked genes with functions involving the immune response that probably contribute to the sex bias in autoimmunity and allergies. Other features of the X chromosome such as fragile sites, Alu elements, and miRNAs may further reinforce the sex bias. Future studies on this subject will continue to expand our understanding of why females are more susceptible to autoimmune and allergic diseases.
