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Focus on Annoroad' Hi-C Sequencing: Analyzed the Structural Organization of the Inactive X Chromosome in the Mouse Published on Nature

Annoroad 2016-07-26

Annoroad cooperated with Institut Curie, etc., analyzed the structural organization of the inactive X chromosome in the mouse through Hi-C technology, and the relative research published on Natureon July 18th, 2016.



X-chromosome inactivation (XCI) involves major reorganization of the X chromosome as it becomes silent and heterochromatic. During female mammalian development, XCI is triggered by up-regulation of the non-coding Xist RNA from one of the two X chromosomes. 


The researcher investigated the structure, chromatin accessibility and expression status of the mouse Xi chromosome in highly polymorphic clonal neural progenitors (NPCs) and embryonic stem cells. Xist and DXZ4-containing boundary play roles in organizing the inactive X (Xi) chromosome. The Xi chromosome lacks active/inactive compartments (compartment A/B)and topologically associating domains (TADs), except around genes that escape XCI.


Materials and Methods


Materials: mouse highly polymorphic clonal neural progenitors (NPCs) and embryonic stem cells


Methods: Hi-C sequencing, ATAC-seq and RNA-seq


Bioinformation analysis: Compartment A/B analysis, TAD analysis, Integrative analysis of Hi-C and RNA-seq


Results


1. Structural organization of the inactive X chromosome


To investigate the structure of the Xi chromosome, the researcher performed allele-specific Hi-C in a clonal neural progenitor cell (NPC) line that was derived from highly polymorphic F1 mouse embryonic stem (ES) cells. They performed Hi-C in ES cells, in which XCI has not yet occurred, and found that autosomes and both active X chromosomes displayed prominent active/inactive (A/B) compartmentalization and TAD structures(Figure 1).

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Fig 1: Chromatin three-dimensional structure characteristics of ES cells


However, the Xi chromosome displayed no A/B compartments (Figure 1), but was instead partitioned into two massive interaction domains separated by a hinge region of ~200 kb including the DXZ4 macrosatellite(Figure 2).


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Fig 2: Structure characteristics of inactive X chromosome


2. The structure of the inactive X chromosome confirmed by FISH at the single cell level


Through DNA fluorescence in situ hybridization (FISH), the researcher investigated the Chromatin spatial structure within one mega-domain (probes a–b) or spanning the mega-domain boundary (probes b–c) at the single cell level. Despite extensive cell-to-cell variation, regions within the same mega-domain showed greater overlap on the Xi than on the Xa chromosome, whereas regions spanning the boundary showed lower overlap on the Xi chromosome(Figure 3).


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Fig 3: Structure characteristics of inactive X chromosome verified with FISH


3. The role of Xist in the reconstruction of Xi chromosome


Through induced Xist expression in undifferentiated male (XY) ES cells, Hi-C revealed that 48 h of Xist induction resulted in notable structural changes along the X chromosome (Fig. 4a, b). While these changes were not observed after induction of a Xist mutant lacking the A-repeat region, which were not silence genes. Wild-type Xist induction did not lead to detectable changes in TAD structure, but resulted in increased interaction frequencies along the chromosome(Figure 4d),and the mega-domain segmentation boundary is confirmed to be correct by RNA/DNA FISH(Figure 4c).


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Fig 4: Xist-mediated silencing is sufficient to generate a boundary at DXZ4 in ES cells



Hi-C, as a characteristic technology of Annoroad, is constantly exploring innovative. Hi-C could reveal the three dimensional structure of chromosome, and can be used for assisting genome assembly. Our technology experience has ranged from Group Hi-C, 1Kb Hi-C, Target Hi-C, Single Cell Hi-C to assisted assembly Hi-C, and the species experience spanning animals, plants and microorganisms. Additionally, the cooperation units have involved throughout the domestic and international research institutes. Annoroad' senior Hi-C is sincerely inviting you to join us.





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