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March 28, 2013

“Yes, MADM”

IST Austria Professor Simon Hippenmeyer with collaborators succeeds in extending MADM method • Technique enables new insights into so far unclear phenomenon of genomic imprinting • Effects of imprinting on single cells analyzed for the first time • Impact on neural circuits as next step

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According to Gregor Mendel, we can thank our mother and father equally for the way we turn out, as their genes contribute to our genetic make-up in an equal manner. This is, however, not so for a specific set of genes which display “genomic imprinting”. Imprinted genes are active when they are inherited from one parent, but inactive when inherited from the other parent. Thus, some of these imprinted genes are expressed only when they are on a chromosome inherited from the mother (maternal), but not when the chromosome is inherited from the father (paternal), while for other imprinted genes it is the other way around. Scientists have been debating how many genes of the total human genome are imprinted, with the estimates ranging from about a hundred to over a thousand. Also, the precise function of imprinting is still unclear: It is certainly crucial for prenatal development, metabolism and behavior, and its deregulation causes many diseases including cancer and brain disorders. However, the role of most imprinted genes is so far not thoroughly studied.

In their recent publication in Cell Reports (10.1016/j.celrep.2013.02.002), Simon Hippenmeyer – Assistant Professor at IST Austria – and his colleagues Randy Johnson at MD Anderson Cancer Center and Liqun Luo at Stanford University extended the ‘Mosaic Analysis with Double Markers’ (MADM) technique in mice to probe for the effects of imprinting at the level of single cells. Hippenmeyer and his colleagues were, for the first time, able to analyze the effects of imprinting on single cells. Their work using the MADM technique demonstrates that imprinting has specific effects, dependent on cell type and chromosome.

For MADM, two reciprocally chimeric marker genes are placed separately on identical positions on homologous chromosomes. The chimeric marker genes consist of partial coding sequences for green and red fluorescent proteins. If MADM is activated during cell division, the two daughter cells can, under certain conditions, each express one of the two fluorescent proteins, and so glow in different colors. This enables researchers to easily spot and analyze these cells. In their recent work, Hippenmeyer and colleagues used MADM to create so called uniparental disomy (UPD) for sets of whole chromosomes. In maternal UPD, cells carry two copies of a particular chromosome from the mother but lack the father’s chromosome. In paternal UPD, cells inherit two chromosomes from the father but have no chromosome from the mother. With the use of the MADM technique, cells with maternal UPD can be labeled in one color (e.g. red) and cells with paternal UPD with the other color (e.g. green). For imprinted genes, either the mother’s or the father’s copy is consistently active, while the other is inactive. Therefore, cells with UPDs carry either two active or two inactive copies of an imprinted gene. By comparing the phenotype of certain maternal UPD cells to paternal UPD cells, the researchers could then probe whether imprinted genes on a particular chromosome have any effect.

Hippenmeyer and colleagues focused their analysis on UPDs for mouse chromosomes 7 and 12, and assessed the number and shape of cells with maternal UPD or paternal UPD, respectively. They obtained two key results: firstly, the effects of imprinted genes are highly specific, depending on the type of cell. Secondly, UPDs for distinct chromosomes lead to different effects on cells. The scientists observed the most extreme changes with chromosome 7. Cells carrying two paternal copies of chromosome 7 are much more numerous than cells carrying two maternal copies. They are particularly numerous in the liver and lung, while no change is seen in the heart and for certain types of nerve cells in the brain. The changes are therefore specific to the cell type. Conversely, when looking at cells carrying two paternal copies of chromosome 12, the number of cells does not appear to be changed in any tissue. This suggests that an imbalance of imprinted gene expression on chromosome 7 has a different effect from an imbalance on chromosome 12.

With this further development of the MADM technique, Hippenmeyer and his colleagues will lay the groundwork to systematically analyze the effect of imprinting on a whole genome level. The researchers are now in progress to apply the MADM method to rigorously assay the effect of imprinting on the brain and its role in the development of neural circuits.


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