Musings of a BIOL463 Student

This assignment was my first exposure to imprinting. I found this paper a little difficult to tackle but after my group discussion and finishing this assignment, I was able to understand what was going on.

Monday March 2^nd in-class “assignment”: the mystery of the lacZ transgene

Assigned paper: Lonfat et al. (2013). Transgene- and locus-dependent imprinting reveals

allele-specific chromosome conformations. PNAS 110(29): 11947-51

Specific questions about the article

1. Recall the general rule, “Figure 1 is often the most important figure in the paper”. Referring to Figure 1:
2. What transgenic lines did the author use (do these lines look somewhat familiar)?
   1. HoxD^rel5
   2. HoxD^{Inv(rel5-Itga6)}

These lines were created by the insertion of the transgene Hoxd9lacZ upstream of the Evx2 HoxD locus.

1. What do the data show (Panel C)?

• Used a stain-and-look approach to observe lacZ
• The data shows that the 2.7Mb inverted genomic fragment containing the reporter resulted in the absence of lacZ expression in the offspring possessing the maternally inherited allele while lacZ expression in the offspring possessing the paternally inherited allele was increased relative to the uninverted HoxD^rel5 line.

1. What is striking/unexpected about the results, and why?

• Upon inversion, there is a substantial change in lacZ expression between the offspring possessing the maternally inherited allele and the offspring possessing the paternally inherited allele while this substantial change in the HoxD^rel5 line is not observed.

1. What conclusions do you make from the data?

• Positioning Hoxd9lacZ closer to Itga6 ex1-24 results in parental-specific expression of the reporter lacZ where expression from the paternal allele is observed, but not from the maternal allele.

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?

• lacZ-specific
  • They relocated the native Hoxd11 gene not including the lacZ transgene into Itga6 Upon staining for Hoxd11 expression, they found no significant difference between the alleles inherited from either parent.  This result implies that the imprinting to repress the expression of the maternal allele requires the presence of the lacZ sequence.
• Position/site-specific?
  • Inserted the Hoxd9lacZ into various positions within the HoxD centromeric landscape, none of which resulted in parental-specific expression. This result showed that this is position specific.
  • When the Hoxd9lacZ reporter was placed before the inversion, the transgene did not show parental-specific expression, illustrating that the imprinting was site-specific.

3. Refer to Figure 2.

1. 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).

• White circles: unmethylated C’s
• Black circles: methylated C’s
• Each row indicates an individual biological replicate.

1. What do the data in Figure 2 show?

• In the inverted line, it is only the maternal alleles that show methylation of the cytosine’s and this result is also seen at the level of oocytes and sperm.
• Inconsistent intermediate methylation was observed in escaper genes from both the maternal and paternal alleles.

1. Why aren’t there a “paternal/+” and a “maternal/+” groups for sperm and oocytes?

• Mothers don’t have sperms.
• Fathers don’t have oocytes.

1. What are “escaper” embryos, and how were they identified prior to bisulphite sequencing?

• Despite inversion of the genomic sequence, escaper embryos did not have maternal-inherited imprinting.
• This was shown by looking at lacZ activity and the researchers determined that the escapers had clonal patches of cells and streaks with 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 ChIP experiments were done to look at the accessibility of the chromatin in these specific locations by analyzing H3K27me3, H3K9me3, and H4K20me3.
• The repressive marks (H3K9me3 and H4K20me3) were more prominent in the maternal allele.

5. Figure 3 depicts the results of a series of 3D chromatin conformation capture (aka “4C”) experiments. Try to “read” the figure and see if you can identify the information described in the text. You don’t need to answer this question in writing.

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

• The figure shows how the change in the local chromatin conformation affects the expression of neighbouring genes, such as Dlx1/2.
• Proposed a hypothesis:
  • In the maternal case for the inversion, because it can no longer associate with the Itga6 locus, the digit enhancers are now closer to the Dlx1/2 promoter, thus increasing expression of these genes. There was a slight up-regulation in the paternal case, but it was much more significant in the maternal case.  The result was observed because the digit enhancer in the paternal case is still able to interact with the transgene, thus reducing the interaction with the Dlx1/2 locus.

7. List any findings that you and your group found surprising.

• Why is this maternal imprinting specific to the lacZ sequence?
• There was a high incidence (33%) of escapers of the maternal imprinting.

In the inversion line, there was an observed increase in lacZ expression in the paternally inherited allele