An ENU-induced mutation in Twist1 transactivation domain causes hindlimb polydactyly with complete penetrance and dominant-negatively impairs E2A-dependent transcription.

Twist1 encodes a basic helix-loop-helix transcription factor (TF), which forms homodimer or heterodimer with other TFs, like E2A, to regulate target genes' expression. Mutations in TWIST1 are associated with Saethre-Chotzen syndrome (SCS), a rare congenital disorder characterized with osteogenesis abnormalities. However, how dysfunction of TWIST1 leads to SCS is still largely unknown. Here, using an unbiased ENU-induced mutagenesis screening, we identified a novel Twist1 mutation and the mutant mouse phenocopies some features of SCS in a dominant manner. Physically, our mutation p.F191S lies at the edge of a predicted α-helix in Twist1 transactivation (TA) domain. Adjacent to F191, a consecutive three-residue (AFS) has been hit by 3 human and 2 mouse disease-associated mutations, including ours. Unlike previously reported mouse null and p.S192P alleles that lead to hindlimb polydactyly with incomplete penetrance but a severe craniofacial malformation, our p.F191S causes the polydactyly (84.2% bilateral and 15.8% unilateral) with complete penetrance but a mild craniofacial malformation. Consistent with the higher penetrance, p.F191S has stronger impairment on E2A-dependent transcription than p.S192P. Although human p.A186T and mouse p.S192P disease mutations are adjacent to ours, these three mutations function differently to impair the E2A-dependent transcription. Unlike p.A186T and p.S192S that disturb local protein conformation and unstabilize the mutant proteins, p.F191S keeps the mutant protein stable and its interaction with E2A entire. Therefore, we argue that p.F191S we identified acts in a dominant-negative manner to impair E2A-dependent transcription and to cause the biological consequences. In addition, the mutant mouse we provided here could be an additional and valuable model for better understanding the disease mechanisms underlying SCS caused by TWIST1 dysfunction.

Whole-genome analysis of 5-hydroxymethylcytosine and 5-methylcytosine at base resolution in the human brain.

BACKGROUND: 5-methylcytosine (mC) can be oxidized by the tet methylcytosine dioxygenase (Tet) family of enzymes to 5-hydroxymethylcytosine (hmC), which is an intermediate of mC demethylation and may also be a stable epigenetic modification that influences chromatin structure. hmC is particularly abundant in mammalian brains but its function is currently unknown. A high-resolution hydroxymethylome map is required to fully understand the function of hmC in the human brain. RESULTS: We present genome-wide and single-base resolution maps of hmC and mC in the human brain by combined application of Tet-assisted bisulfite sequencing and bisulfite sequencing. We demonstrate that hmCs increase markedly from the fetal to the adult stage, and in the adult brain, 13% of all CpGs are highly hydroxymethylated with strong enrichment at genic regions and distal regulatory elements. Notably, hmC peaks are identified at the 5'splicing sites at the exon-intron boundary, suggesting a mechanistic link between hmC and splicing. We report a surprising transcription-correlated hmC bias toward the sense strand and an mC bias toward the antisense strand of gene bodies. Furthermore, hmC is negatively correlated with H3K27me3-marked and H3K9me3-marked repressive genomic regions, and is more enriched at poised enhancers than active enhancers. CONCLUSIONS: We provide single-base resolution hmC and mC maps in the human brain and our data imply novel roles of hmC in regulating splicing and gene expression. Hydroxymethylation is the main modification status for a large portion of CpGs situated at poised enhancers and actively transcribed regions, suggesting its roles in epigenetic tuning at these regions.