1. Recall the general rule, “Figure 1 is often the most important figure in the paper”. Referring to Figure 1:

1a)  What transgenic lines did the author use (do these lines look somewhat familiar)? Hoxd9/lacz (HoxD^rel5) and HoxD^{Inv(rel5-Itga6)}. These lines look similar to the line we discussed on Friday where there was also a large inversion.

b) What do the data show (Panel C)? There is normal expression of both parental copies without the inversion. Inversion causes loss of expression when inherited from the mother.

c) What is striking/unexpected about the results, and why? F1 progeny either had strong or almost no expression of lacZ. This indicates that the inversion places the lacZ sequence into a region where it is then maternally imprinted. This is striking because this is very site-specific.

d) What conclusions do you make from the data? Maternal imprinting is position dependent and occurred near the Itga6 locus.

2. How did the authors show that the observed imprinting is lacZ-specific and position/site-specific? Do you agree with their data interpretation and with their conclusion? The researchers used a different mouse line that has HoxD11 replacing the HoxD/lacZ transgene in the inverted sequence. Then they looked at the expression of HoxD11. There was no change when the inverted allele was maternally or paternally passed down, so when lacZ is not present there is no imprinting of the allele. The position specific aspect is shown in Figure 1. Their data interpretation and conclusion sound plausible.

3. Refer to Figure 2.

a) Briefly explain how to “read” the diagrams shown (i.e. what do the rows of circles represent, what do the white vs. black circles represent). Circles represent potential methylation sites at C residues. White circles are un-methylated and black circles are methylated.

b) What do the data in Figure 2 show? Methylation in escapers is much more variable. Maternal inverted alleles are heavily methylated and paternal inverted alleles are mostly un-methylated. This pattern is also seen in eggs and sperm. D shows the normal amount of methylation in the non-inverted line.

c) Why aren’t there a “paternal/+” and a “maternal/+” groups for sperm and oocytes? Sperm and eggs are haploid, so only have one allele. This only shows the methylation that is sex specific.

d) What are “escaper” embryos, and how were they identified prior to bisulphite sequencing? These embryos had some lacZ expression when the inverted reporter as maternally transmitted. Other maternal/- embryos had little to no expression of lacZ.

4. What was the purpose of the authors’ ChIP experiments, and why did they choose to look for specific histone modifications? What did they find? (Expected answer: max two sentences)

The purpose of ChIP was to look at which regions were associated with H3K27me3. They found that the histones associated with the HoxD9 promoter and lacZ were heavily methylated compared to wild type.

6. What does Figure 4 show? (Please describe/summarize its content including specific information).

A shows a strong interaction between Dlx1/Dlx2 and the transgenes in paternal/+. B shows that Dlx1/Dlx2 expression is much higher in Maternal/+, less in Paternal/+, and even less in WT when the transgene was inverted. C and D show chromosome compaction models for how the digit enhancers can interact with Dlx1/Dlx2.