Genetic dissection of the Down syndrome critical region.

Down syndrome (DS), caused by trisomy 21, is the most common chromosomal disorder associated with developmental cognitive deficits. Despite intensive efforts, the genetic mechanisms underlying developmental cognitive deficits remain poorly understood, and no treatment has been proven effective. The previous mouse-based experiments suggest that the so-called Down syndrome critical region of human chromosome 21 is an important region for this phenotype, which is demarcated by Setd4/Cbr1 and Fam3b/Mx2. We first confirmed the importance of the Cbr1-Fam3b region using compound mutant mice, which carry a duplication spanning the entire human chromosome 21 orthologous region on mouse chromosome 16 [Dp(16)1Yey] and Ms1Rhr. By dividing the Setd4-Mx2 region into complementary Setd4-Kcnj6 and Kcnj15-Mx2 intervals, we started an unbiased dissection through generating and analyzing Dp(16)1Yey/Df(16Setd4-Kcnj6)Yey and Dp(16)1Yey/Df(16Kcnj15-Mx2)Yey mice. Surprisingly, the Dp(16)1Yey-associated cognitive phenotypes were not rescued by either deletion in the compound mutants, suggesting the possible presence of at least one causative gene in each of the two regions. The partial rescue by a Dyrk1a mutation in a compound mutant carrying Dp(16)1Yey and the Dyrk1a mutation confirmed the causative role of Dyrk1a, whereas the absence of a similar rescue by Df(16Dyrk1a-Kcnj6)Yey in Dp(16)1Yey/Df(16Dyrk1a-Kcnj6)Yey mice demonstrated the importance of Kcnj6. Our results revealed the high levels of complexities of gene actions and interactions associated with the Setd4/Cbr1-Fam3b/Mx2 region as well as their relationship with developmental cognitive deficits in DS.

Systematic profiling of conditional degron tag technologies for target validation studies.

Conditional degron tags (CDTs) are a powerful tool for target validation that combines the kinetics and reversible action of pharmacological agents with the generalizability of genetic manipulation. However, successful design of a CDT fusion protein often requires a prolonged, ad hoc cycle of construct design, failure, and re-design. To address this limitation, we report here a system to rapidly compare the activity of five unique CDTs: AID/AID2, IKZF3d, dTAG, HaloTag, and SMASh. We demonstrate the utility of this system against 16 unique protein targets. We find that expression and degradation are highly dependent on the specific CDT, the construct design, and the target. None of the CDTs leads to efficient expression and/or degradation across all targets; however, our systematic approach enables the identification of at least one optimal CDT fusion for each target. To enable the adoption of CDT strategies more broadly, we have made these reagents, and a detailed protocol, available as a community resource.