Download File - Alexis Kezirian

Maggie Hamidi
Alexis Kezirian
Lonfat et al. Assignment
1. The general effect of DNA methylation on gene expression is gene expression
silencing.
2. “Imprinting” refers to the genomic silencing of one of two parental alleles,
depending on the parent of origin in marsupials and mammals.
3. Large centromeric deserts are genomic DNA that flank genes and contain
regulatory sequences such as enhancers.
4. If the LoxP sites are inverted, rather than using a direct repeat of LoxP sites,
then an inversion will occur rather than an excision. When the crossover
occurs with inverted LoxP sites, the genomic region becomes inverted. In
other words, the LoxP sites should be opposite orientation of each other for
an inversion, whereas an excision would have LoxP sites facing in the same
direction.
5. Digit enhancer sequences active on HoxD cluster genes
6. a) One transgenic line was the Hoxd9lacZ transgene, inserted into the rel5
site with a loxP site, upstream of the Lnp gene. The other transgenic line used
the same (inverted) Hoxd9lacZ transgene, inserted into the rel5 site with a
loxP site, downstream of the ltga6 ex1-24 gene.
b) Figure C compares the expression of the LacZ gene product in maternallyand paternally-inherited transgenes for the normal versus inverted
transgenes. The maternal and paternal transgenes show relatively similar
localization and intensity for LacZ expression when the transgene is not
inverted. When the transgene is inverted, LacZ expression for the paternallyinherited transgene is highly upregulated and expressed throughout most of
the embryo interior, whereas almost a complete absence of LacZ expression
is observed for the maternally-inherited transgene.
c) It is surprising that one embryo carrying the same transgene with the
same (inverted) orientation would show different expression patterns for its
reporter gene (LacZ), but that this difference in expression levels is not
observed for embryos carrying either the same maternally- or paternallyinherited transgene with normal orientation.
d) Inversion and positioning of the Hoxd9lacZ transgene near the ltga6 ex124 gene is sufficient for imprinting which silences the maternally-inherited
transgene but upregulates the paternally-inherited transgene.
7. This study first performed a control experiment, with the Hoxd9lacZ
transgene (with the LacZ gene) in its normal location and orientation. No
imprinting was observed between maternally- and paternally-inherited
transgenes. Then the transgene was inverted and placed at a different
position (next to ltga6 ex1-24 rather than rel5) and LacZ expression was
observed. Imprinting was observed for this treatment, with embryos with the
paternally-inherited transgene expressing upregulated levels of LacZ and
those with the maternally-inherited transgene having almost no expression
of LacZ.
8. a) These diagrams show differential allelic methylation. Black circles
represent methylated cytosine sites in the region of interest. White circles
are unmethylated cytosine sites in the region of interest. Each row of circle
represents a different individual.
b) In Figure 2A, the inverted transgene results in most of the paternallyinherited cysteine sites being unmethylated, and nearly all of the maternallyinherited cysteine sites being methylated. In Figure 2B shows escapers,
meaning those individuals whose methylation patterns differed from what
was expected. Maternal escaper embryos showed a variable but lower rate of
methylation. Figure 2C confirms that DNA methylation occurs to relatively
the same extent as in somatic cells, with most of the paternally-inherited
cysteine sites being unmethylated, and nearly all of the maternally-inherited
cysteine sites being methylated. Figure 2D shows no differential methylation
when the transgene is not inverted; a similar pattern is seen for both
maternally- and paternally-inherited transgenes.
9. ChIP experiments are used to identify protein-DNA interactions to look for
histone modifications in the brains of HoxDInv(rel5-Itga6) mutants at locations
such as H3K27me3 and other specific inactive marks.