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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.