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ING5 Case Study

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The ING (INhibitor of Growth) family of genes and proteins (ING1–ING5) was identified in 1996, from PCR-mediated subtractive hybridization between a normal mammary epithelial cell strain and several transformed breast cancer cell-lines, followed by an in vivo functional screen for tumourigenesis1. The ING proteins target histone acetyltransferase (HAT) and histone deacetylase (HDAC) complexes to the H3K4me3 mark, altering histone acetylation and gene expression2. ING4 was identified by computational analysis and is expressed in various tissues; also several splice variants and transcripts were described for this protein3–5. The ING5 protein of the ING family, was described by computational homology search and shares a high amino acid …show more content…

Also, due to the presence of the LID in all INGs, we will study if ING5 and ING4 are capable of interacting with Lamin A in differentiating stem cells and the importance of this interaction to stem cell differentiation.
To achieve these objectives, we will use three different mouse embryonic stem (ES) cell lines (R1, D3, E14), already present in the lab. In ongoing studies, it was noted that these cells presented all five of the ING genes with altered expression when they were induced to differentiate. We will first study if altering ING5 and ING4 expression using siING5 and siING4 to decrease it and pCI-ING5, pCI-ING4 to increase levels, will affect differentiation and the self-renewal rate of these lines. We will use sphere formation assays to assess self-renewal. We will also induce differentiation with these different expression constructs, thus measuring the influence of these proteins in the process. To determine the degree of differentiation with varying expression of the ING genes, we will use flow cytometry to follow changes in stem cells markers, such as OCT4, OLIG2 and Nestin16.
Previous studies from our group already described the LID and the interaction of several ING proteins with Lamin A12. In the conditions used in this study, ING4 was co-precipitated with Lamin A. However, even though ING5 presents the LID, it was not co-precipitated12. In this work we aim to evaluate the effects of the possible interaction between the ING4 and

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