Suppressor mutations in Mecp2-null mice implicate the DNA damage response in Rett syndrome pathology.

Mutations in X-linked methyl-CpG-binding protein 2 (MECP2) cause Rett syndrome (RTT). To identify functional pathways that could inform therapeutic entry points, we carried out a genetic screen for secondary mutations that improved phenotypes in Mecp2/Y mice after mutagenesis with N-ethyl-N-nitrosourea (ENU). Here, we report the isolation of 106 founder animals that show suppression of Mecp2-null traits from screening 3177 Mecp2/Y genomes. Whole-exome sequencing, genetic crosses, and association analysis identified 22 candidate genes. Additional lesions in these candidate genes or pathway components associate variant alleles with phenotypic improvement in 30 lines. A network analysis shows that 63% of the genes cluster into the functional categories of transcriptional repression, chromatin modification, or DNA repair, delineating a pathway relationship with MECP2. Many mutations lie in genes that modulate synaptic signaling or lipid homeostasis. Mutations in genes that function in the DNA damage response (DDR) also improve phenotypes in Mecp2/Y mice. Association analysis was successful in resolving combinatorial effects of multiple loci. One line, which carries a suppressor mutation in a gene required for cholesterol synthesis, Sqle, carries a second mutation in retinoblastoma binding protein 8, endonuclease (Rbbp8, also known as CtIP), which regulates a DDR choice in double-stranded break (DSB) repair. Cells from Mecp2/Y mice have increased DSBs, so this finding suggests that the balance between homology-directed repair and nonhomologous end joining is important for neuronal cells. In this and other lines, two suppressor mutations confer greater improvement than one alone, suggesting that combination therapies could be effective in RTT.

A twist code determines the onset of osteoblast differentiation.

Runx2 is necessary and sufficient for osteoblast differentiation, yet its expression precedes the appearance of osteoblasts by 4 days. Here we show that Twist proteins transiently inhibit Runx2 function during skeletogenesis. Twist-1 and -2 are expressed in Runx2-expressing cells throughout the skeleton early during development, and osteoblast-specific gene expression occurs only after their expression decreases. Double heterozygotes for Twist-1 and Runx2 deletion have none of the skull abnormalities observed in Runx2(+/-) mice, a Twist-2 null background rescues the clavicle phenotype of Runx2(+/-) mice, and Twist-1 or -2 deficiency leads to premature osteoblast differentiation. Furthermore, Twist-1 overexpression inhibits osteoblast differentiation without affecting Runx2 expression. Twist proteins' antiosteogenic function is mediated by a novel domain, the Twist box, which interacts with the Runx2 DNA binding domain to inhibit its function. In vivo mutagenesis confirms the antiosteogenic function of the Twist box. Thus, relief of inhibition by Twist proteins is a mandatory event precluding osteoblast differentiation.