Determining key features of X Chromosome Architecture in the context of the Inactive and Dampened States

Present within the nuclei of female human cells are two X chromosomes of differing gene expression states, one active chromosome with a structurally open configuration and one inactive chromosome, harboring a distinct, spatially condensed structure, in which a majority of genes are transcriptionally silenced. A third gene expression state, the dampened X chromosome, is found early in human development in the pre-implantation blastocyst and displays neither the levels of transcription found in the active nor the inactive X chromosome but instead exhibits a reduction of transcriptional activity. Through a combination of current technologies like chromosome conformation capture techniques and microscopy, gene expression has been shown to correlate to chromosome structure. Although the dampened X chromosome is known to feature a unique gene expression profile, the structure of this X chromosome state remains unknown. To understand the impact of structural rearrangements of the human X chromosome throughout the transition period of X chromosome inactivation development we have sought to define the physical features of the active, inactive and dampened states of the X chromosome.To determine the structure of the X chromosome, we chose the sequential fluorescent in situ hybridization (FISH) technique and established the protocol in the lab. To this end, we optimized every step along the way including plating conditions for cells, fixation conditions, microscope relocation and alignment, and image analysis pipelines. We demonstrate that our sequential FISH approach allows for, at minimum, four rounds of hybridization, each containing multiple probes targeting various genomic locations for the purpose of defining chromosome structure in 3D, with imaging acquired on a conventional confocal microscope Applying the optimized sequential FISH, we determined the variance of the size and higher-order organization of the X chromosome not only in different chromosome states of the same cell type but in the active X state across different cell types. We chose two different cell types to analyze: human fibroblast and na�ve embryonic stem cells. The former representing a somatic state that carries an inactive (Xi) and active X chromosome (Xa) while the later illustrates a pluripotent, early in development cell type harboring an Xa and dampened X chromosome (Xd). In both cases, the lncRNA XIST, essential for X chromosome inactivation (XCI) which leads to a change in X chromosome gene transcription, is expressed from the non-active X chromosomes, Xi and Xd. This combination of cell types allows for the investigation of all three gene expression states but also provides an internal comparison between two different gene expression states in each cell. We found that Xa in fibroblast cells, while clearly larger in size and containing more intrachromosomal distance between genes, is absolutely smaller than the na�ve Xa. In comparison, the Xd ranged from slightly smaller to practically the same size as the na�ve Xa. Additionally, some Xd gene locations, such as XACT, were found to be incredibly distant to the other genes unlike in the na�ve Xa. We interrogated the structure of the Xd by comparing it to the structures of the Xa and Xi. We analyzed whole chromosome structure using genomic locations that span the length of the X chromosome as well as specific combinations of genes with relevance towards gene expression. We investigated the genes that become silenced upon XCI and found that the fibroblast Xi predictably contains small intrachromosomal distances between all probe combinations. Conversely, both na�ve and fibroblast Xa tended to be contain large intrachromosomal distances. The na�ve Xd interestingly showed noticeably smaller intrachromosomal distances concerning measurements between genes in the latter half of the X chromosome. Genes that escape X chromosome dampening (XCD) showed similar arrangement between the na�ve X chromosomes but not the fibroblast. Finally, we found that Xd shows intrachromosomal distances the same size or larger than na�ve Xa when measuring between probes on either side of DXZ4. This study ultimately characterizes distinct physical features of the active, inactive and dampened X chromosome states due to the analysis of the spatial locations of key genes regulated by these expression states. The chromosome organization data acquired allowed us to decipher specific features of the X chromosome with different overall activity states and, in the future, will aid in revealing the mechanisms underlying the transitions in chromosome organization during human development