Linking Chromosomal Silencing With Xist Expression From Autosomal Integrated Transgenes

Xist is the master regulator of X-Chromosome Inactivation (XCI), the mammalian dosage compensation mechanism that silences one of the two X chromosomes in a female cell. XCI is established during early embryonic development. Xist transgene (Tg) integrated into an autosome can induce transcriptional silencing of flanking genes; however, the effect and mechanism of Xist RNA on autosomal sequence silencing remain elusive. In this study, we investigate an autosomal integration of Xist Tg that is compatible with mouse viability but causes male sterility in homozygous transgenic mice. We observed ectopic Xist expression in the transgenic male cells along with a transcriptional reduction of genes clustered in four segments on the mouse chromosome 1 (Chr 1). RNA/DNA Fluorescent in situ Hybridization (FISH) and chromosome painting confirmed that Xist Tg is associated with chromosome 1. To determine the spreading mechanism of autosomal silencing induced by Xist Tg on Chr 1, we analyzed the positions of the transcriptionally repressed chromosomal sequences relative to the Xist Tg location inside the cell nucleus. Our results show that the transcriptionally repressed chromosomal segments are closely proximal to Xist Tg in the three-dimensional nucleus space. Our findings therefore support a model that Xist directs and maintains long-range transcriptional silencing facilitated by the three-dimensional chromosome organization.

Global chromatin conformation differences in the Drosophila dosage compensated chromosome X

In Drosophila melanogaster the single male chromosome X undergoes an average twofold transcriptional upregulation for balancing the transcriptional output between sexes. Previous literature hypothesised that a global change in chromosome structure may accompany this process. However, recent studies based on Hi-C failed to detect these differences. Here we show that global conformational differences are specifically present in the male chromosome X and detectable using Hi-C data on sex-sorted embryos, as well as male and female cell lines, by leveraging custom data analysis solutions. We find the male chromosome X has more mid-/long-range interactions. We also identify differences at structural domain boundaries containing BEAF-32 in conjunction with CP190 or Chromator. Weakening of these domain boundaries in male chromosome X co-localizes with the binding of the dosage compensation complex and its co-factor CLAMP, reported to enhance chromatin accessibility. Together, our data strongly indicate that chromosome X dosage compensation affects global chromosome structure.

The detection of female cell activity in male sex chromosome chimeric Rideau Arcott sheep, using the Xist gene product as a marker

The detection of the SRY (Sex-determining region on the Y chromosome) gene is a popular method used for the identification of freemartins (XX/XY female chimeras). This method relies on the fact that the SRY gene is a Y chromosome specific gene and is thus normally only present in males therefore detecting its presence in a female indicates the presence of male cells (XY cells) within the female. This concept can be extrapolated to the male counterparts of freemartins with regards to the Xist gene. This gene is normally only widely expressed in females and can be used as a marker for identifying females. Therefore, detecting Xist gene expression in males (in tissues other than the testes, as the Xist gene is expressed exclusively in the testes of males) may indicate that these males contain transcriptionally competent female cells and thus necessarily labels them as sex-chromosome chimeras. In the present study four previously identified male sex chromosome chimeras were screened for the expression of the Xist gene using reverse transcription Polymerase Chain Reaction (PCR), and it was detected in three of the four chimeras. Xist expression was not detected in one of the chimeras because the proportion of female cells in its blood is significantly low and thus it is likely that the blood sample used in the study did not possess female cells. None-the-less it was concluded that the detection of Xist expression in male sex chromosome chimeras can be used as an indication of the presence and transcriptional competence of female cells within them.

Kinetochore size variation in mammalian chromosomes: an image analysis study with evolutionary implications

The kinetochore, a proteinaceous plate that is the site for attachment of spindle microtubules to the metaphase chromosome, can be visualized using anti-kinetochore indirect immunofluorescence. We have used computer-assisted image analysis to measure the variation of kinetochore surface areas, as reflected by immunofluorescence areas, in cell lines derived from rat kangaroo, Chinese hamster and common rat, to determine if our size estimates correlate well with those obtained using measurements from electron micrographs. In addition, we used male and female human fibroblast cell lines, as well as a transformed human female cell line as well as a transformed human female cell line (HeLa), to examine kinetochore size variation among cells, between sexes, and between cell lines. We found that our system gave reproducible estimates of kinetochore size, and that these sizes correlated very well (r = 0.95) with the electron micrograph measurements. In examining variation within humans, we observed measurable differences between cell lines. Despite this difference, all the human lines had size distributions that were leptokurtotic and positively skewed. The fact that very few chromosomes exhibited areas smaller than the mode gives support to the idea that mammalian chromosomes may require a specific, minimum amount of kinetochore material in order to maintain stable attachment to the mitotic spindle. On the other hand, the positive skewness seems to indicate that larger kinetochores, possibly the result of events such as Robertsonian fusions, are fully functional. The retention of this plasticity may allow the chromosomes to maintain an evolutionary adaptability that might otherwise be lost.