Exploring Cis Elements and Trans-Acting Factors Involved in the Human Inactive X Chromosome Organization and Compaction

In this dissertation, I have explored cis and trans factors involved in the organization and compaction of the human inactive X chromosome (Xi). I describe here three trans factors (SMCHD1, LRIF1 and SETDB1) that were found to have important roles in Xi chromatin compaction, as demonstrated by a doubling of the Xi volume in their absence. I also report a novel enhancer element on the Xi that is reactivated in SETDB1 mutants and is in part responsible for the Xi decompaction phenotype, and displays complex cis and trans communication between the active X chromosome and Xi. We have generated SMCHD1 and LRIF1 mutants using both TALENs and CRISPR-Cas9 genome engineering platforms. Loss of either protein results in Xi decompaction and reactivation of some Xi genes. Using the X-linked choroideremia locus (CHM) as an example of a reactivated gene, we show that reactivation is coupled with a reduction in the repressive heterochromatin markers histone H3 trimethylated at lysine 9 (H3K9me3) and 27 (H3K27me3) and an increase in the euchromatin marker histone H3 trimethylated at lysine 4 (H3K4me3) in the promoter region. Alongside these chromatin changes, we observed movement of the CHM locus away from the H3K9me3 territory towards the H3K27me3 territory. Previous data from our lab showed that loss of the macrosatellite repeats DXZ4 from the Xi resulted in large-scale changes in cis to the three-dimensional organization of the Xi, including fragmentation of the chromosome territory as observed by light microscopy. Intriguingly, deletion of SMCHD1 in DXZ4 Xi mutants results in a more pronounced Xi decompaction phenotype than that of SMCHD1 loss alone, suggesting that both perform complementary roles to compact the Xi. In the effort to determine which histone lysine methyl-transferase is responsible for H3K9me3 at the Xi, we isolated SETDB1 TALEN mutant clones and discovered that like SMCHD1 and LRIF1, loss of SETDB1 leads to decompaction of the Xi territory. Furthermore, in the SETDB1 mutants, we observed drastic chromatin changes within the 3’ third of the 1.4 megabase Interleukin 1 Receptor Accessory Protein-Like 1 (IL1RAPL1) gene. In this genomic interval, there is localized loss of repressive chromatin defined by H3K9me3 coupled with a gain of the active makers defined by histone H3 di-methylated at lysine-4 (H3K4me2) and acetylated at lysine 27 (H3K27Ac). The DNA underlying the major peak of H3K27Ac possesses very powerful enhancer activity in vitro and is located immediately adjacent to the long terminal repeat of an endogenous retrovirus element ERVL-MaLR that is reactivated from the Xi in the SETDB1 mutants. Reactivation of the ERVL-MaLR results in a significant increase in the transcription of novel bi-directional transcripts originating from the 3’ region, coupled with a significant reduction in full-length IL1RAPL1 transcripts originating from the endogenous 5’ promoter. To determine if this enhancer element contributes to decompaction of the Xi, clones were isolated in which it had been deleted from either the Xa or Xi using CRISPR-Cas9 system. We found that deletion of the enhancer from the Xi increased detection of full-length IL1RAPL1 transcript in trans, but did not result in Xi decompaction. In contrast, deletion of the enhancer from the Xa decompacted the Xi territory and resulted in a total loss of transcript originating from the 5’ promoter on the Xa. These data revealed complex cis and trans effects that affects IL1RAPL1 gene expression and Xi chromatin compaction. Importantly, this same interval is centrally located in a known fragile site on the X chromosome that is frequently lost in patients with intellectual disability. Portions of this dissertation have been published or are being prepared for publication. Parts of Chapter 1 has been published as a book chapter in Epigenetics: Current Research and Emerging Trends, Caister Academic Press (Chadwick, 2015). The data presented in Chapter 3 has been published in Epigenetics & Chromatin (Sun and Chadwick, 2018). Data presented in Chapter 2 are in the process of being prepared as manuscripts for publication.