Question

For a given gene mutation reserchers have noticed that histone deacetylase (HDAC) is beneficial in vitro. 1. What method would you utilize to determine gene mutation is loss of function or toxic gain of function? 2. Based on HDAC eveidence would you expect loss of function or toxic gain of function? 3. What approach(es) would be a viable molecular therapy? How would the approach mediate the effect? 4. How would you determine whether the effect is cell type specific?

Answer 1

1. If a gene mutation results in loss of function, then complementation with the wild-type gene (or transfection and ectopic expression of wild-type gene) should restore wild-type phenotype. If the gene mutation results in toxic gain of function, which usually enhances the activity of the protein, then knockdown should restore wild-type phenotype.

2. Histones are basic/positively-charged proteins. The positive charges help histones interact and bind tightly to the negative phosphate groups in the backbone of DNA. Acetylation neutralizes the positive charges on the histone by changing amines into amides. This decreases the strength of histone–DNA interaction, which permits accessibility to transcription factors and allows transcription to occur. HDACs remove these acetyl groups, restoring the positive charge of histones and restoring high-affinity binding between histones and DNA. This condenses the histone–DNA complex, preventing transcription. If HDACs help restore wild-type phenotype, then the mutation is probably gain-of-function.

3. Theoretically, since HDAC inducers are not known, it is possible to inhibit histone acetyltransferases (HATs), which perform the opposite role of HDACs, to get the general effect of increasing HDAC activity, through dis-inhibition. Small-molecule inhibitors of HATs are available, such as anacardic acid and curcumin. However, generally dis-inhibiting/inducing HDACs is a bad idea, because, HDACs perform a very broad range of function. So, broad HDAC induction could have a whole host of non-specific, possibly unwanted effects.

Targeted therapies focussing on inhibiting the particular mutated gene is better. Drugs that selectively and specifically inhibit the gene product is a good idea (for instance, to downregulate the toxic gain of function in HER2/neu and EGFR, trastuzumab and/or lapatinib can be used). If no such drugs can be designed, it is possible to elevate the level of the natural repressor of that gene (for instance, to downregulate oncogenic p53 mutant, HDM2 can be elevated). Otherwise, it is possible to transduce the cells with adenoviral vectors containing siRNA designed specifically against the mutated gene to cause a knockdown (by RNA interference pathway).

4. For determining the cell-type/tissue-specific effect, the tissue-specific expression pattern of the mutated gene should be studied. Usually, the phenotype is due to its activity in the tisse where it is highly expressed. After experimental confirmation, the treatment should be provided, and some cells from the tissue with highest activity should be harvested. The activity of the mutant gene with and without treatment should be measured. Activity of the gene in another cell type with somewhat high activity, but unrelated to the phenotype, can provide a good indicator of cell-type-specific activity. The activity and inhibition in the specific cell type vs. global activity is also a good indicator of whether the effect is cell type specific.