In-class assignment: Fukuda et al. (2016) paper

While the main claim of the paper is related to the maintenance of Xist imprinting, we will focus more on three aspects of the mechanism of this imprinting: chromatin ‘relaxation’, the effects of Kdm4b and TSA, and the real of Oct 4.

You are welcome to work on these questions individually or as part of a group. If you work in a group, your group will submit one set of answers for the whole group. Please ensure that all group members participate in the development of the answers.

The answers are to be submitted by email (as an attachment) by the end of class.

PLEASE FOCUS ON ANSWERING THE QUESTIONS IN PURPLE (2, 4, 6, 8, 9).

Chromatin ‘relaxation’ and Xist

2. What can be said about the chromatin of and around the Xist locus in bimaternal, very early embryos that express Kdm4b and are treated with TSA? How do Kdm4b  and TSA affect the levels of chromatin ‘relaxation’? How does this relate to Xist expression and XCI?

It was found that loss of H3K9me3 via Kdm4b (a H3K9me3 demethylase), or gain of histone acetylation by TSA induced Xm-Xist expression. In an investigation of whether chromatin decondensation is associated with Xist expression through Kdm4b and TSA, Fukuda et al. found that Kdm4b/TSA-XmXm embryos showed significantly relaxed chromatin states in both stages compared to Egfp/DMSO (control) – XmXm embryos. This shows that Kdm4b and TSA affect chromatin by promoting the relaxed state, and therefore increasing expression of Xist. This increased expression of Xist induces X chromosome inactivation.

Lethality of embryos without paternal Xist.

4. How do you think Kdm4b and TSA can rescue the lethality of a missing paternal Xist in Xm Xp embryos?

Since Kdm4b and TSA promote chromatin relaxation at the Xist region of the chromosome, they increase the expression of Xist. Once the chromatin is decondensed in early preimplantation phase, Xm-Xist can be derepressed and result in the rescue of lethality by Xist paternal deletion (XmXpΔ). This indicates that genetic lethality can be overcome without direct gene manipulation. Since Xp lacks Xist, it will continue to be active, but decondensation of the Xist region on Xm induces XCI on Xm. This prevents two X chromosomes from being active, and thus is able to rescue lethality.

Roles of the pluripotency factor Oct 4

6. What can you directly conclude from Figure 5a?

From Figure 5, we see that in Oct4 knockdown Xist is expressed at the same levels as in the Scramble-treated cells, but Tsix is expressed at significantly lower levels. From this, we can conclude that Oct 4 is required for the expression of Tsix (at normal levels).

Mega-model

9. Now, try to fit everything you have learned about XCI into a sensible model that explains how XCI occurs in mouse at the pre-implantation and post-implantation stages. Your model should include at least Xist, Tsix, Jpx, CTCF, Oct 4, Nanog, Cdx2, H3K27me3 and H3K(various)ac. If you feel adventurous, make a note of which part of the model corresponds to which hypothetical mechanism seen at the start of the unit (i.e. blocking factor, symmetry-breaking factor, alternate states of the two Xs, etc).

Start with Xist expressed on paternal and Tsix expressed on maternal. Cdx2 comes with the paternal X – it is what silences Tsix and keeps Xist expressed on the paternal X. Oct 4 is turned on and activates the transcription of Xm Tsix in cis. Nanog comes in and dimerizes with Oct 4 having no effect on Xm as its function is repressing Xist. Cdx2 expression is turned off at Xp. This allows Nanog Oct 4 dimers to bind and cause Xp silencing by repressing Xist and promoting Tsix expression in the embryo. One of the chromosomes (paternal or maternal) is then inactivated randomly by dissociation of dimers. On the other hand, Cdx2 remains bound  to Xp in the extraembryonic tissues, thus Xp is kept inactive. Expression of Nanog and Oct 4 is turned off – thus there is no expression of Xist or Tsix on the Xm.

In cases where no Nanog Oct 4 dimers are bound. Acetylation and methylation of H3 play a role in determining whether Tsix/Xist are/aren´t expressed.

Furthermore, expression and binding of CTCF close to Xist promoter prevents Xist transcription. However Jpx can bind CTCF and drag it away – allowing expression of Xist.