Whole chromosome silencing
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CRISPR/Cas9 genome engineering revolutionized the way we approach biological research. In the past we contributed to pioneering work that adapted this technology to C. elegans. We currently develop new ways to utilize this technology to study gene control during germ cell development.
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We recently published our success in using CRISPR to bisect chromosomes. We created worm strains in which both X chromosomes were bisected. In one case one half circularized into a ring chromosome. Surprisingly, this ring chromosome was maintained through multiple mitotic and meiotic cycles.
Top: Illustration of the strategy that resulted in bisection of the X chromosome into a linear and circular segments. The genomic sites of the multiple CRISPR cleavage sites are marked in black. The genomic sites that was used in the images to detect the chromosomes is marked in green. Bottom: IF image of WT linear and mutant ring chromosome. Blue: DAPI. Green FISH foci.
We used this strain to show that the continuity of the sex chromosome is necessary for their meiotic silencing. In many heterogametic (i.e., XY cells) meiocytes, sex chromosomes do not express genes during meiosis. Moreover, any unsynapsed chromatin, from both sex chromosomes and autosomes is also silenced. In the meiocytes of the strain with the bisected X chromosomes, the X segments expressed genes in similar levels to autosomes. We observed lower levels of heterochromatin markers on the X:
IF images of WT and mutant male pacytene nuclei stained with DAPI (blue), H3K9me2 heterochromatin marker (green) and FISH foci marking the X chromosome.
Higher levels of markers associated with transcription:
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IF images of WT and mutant pacytene nuclei stained with DAPI (blue), HIM-8 marking the X chromosome (green), and H3K4me3 (left, red) and active RNAPol-II (right, red) .
As well by comparing specific genes using RNASeq from worm gonads:
RNA-seq results. Plotted is log2 of the fold-change maximum likelihood estimate (lfcMLE) of YBT7 vs. wild type for each gene along the X chromosome. Only highly differentially expressed genes are illustrated. Alignments of the linear (green) and internal (orange) segments of YBT7 are shown schematically below the graph.
Moreover, these segments were not silenced, even when they were not synapsed, contrary to all known cases of unsynapsed chromatin, and to the main model:
IF images of WT and mutant pacytene nuclei stained with DAPI (blue), FISH probe marking the X chromosome (white), SYP-4 marking synapsed chromatin (red) and active RNAPol-II (green) .
All of these results suggest that special features of X chromosome chromatin allow it to escape meiotic silencing when the chromosome is not continuous. Read more in:
Open questions and current work:
How is the continuity of the X chromosome “sensed”?
What differences in the X chromosome chromatin leads to its different gene expression control?
