Do H3K4 methyltransferases MLL4 and MLL3 instruct the mammalian body plan?

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The major epigenetic regulator, MLL4, is prominently mutated in many cancers and is one of six mammalian histone 3 lysine 4 (H3K4) methyltransferases. Heterozygous mutations of MLL4 provoke Kabuki syndrome, which involves mental retardation, facial and other morphological changes, and metabolic disorders. Despite its medical importance, the role of MLL4 in mouse development has only been partially examined. We have been systematically documenting the embryonic roles of all six H3K4 methyltransferases and report that Mll4 null embryos undergo defective gastrulation owing to a defect in establishing the anterior visceral endoderm (AVE) and consequently, the anterior-posterior (A-P) axis during early development. To probe later into development, we generated a new loxP conditional Mll4 allele. Using Sox2Cre, we identified a role for MLL4 in the epiblast and using R26CreERT2 with tamoxifen-induction at different times, found evidence for MLL4 contribution until the completion of A-P axis elongation at E13.5. We propose that MLL4 is continuously required for neuromesodermal progenitor (NMP) specification of trunk elongation, segmentation and the tail, potentially exerted through regulation of Hox cluster colinearity. Conditional mutagenesis also revealed that MLL3, which is not required until birth, can substitute for MLL4 during the elongation of the A-P axis. Using conditional mutagenesis in utero and in embryonic stem cell lines (ESCs) established from our conditional knockout mouse lines, we aim to dissect the contributions of MLL4 (and MLL3) to the establishment of the mammalian body plan. We will investigate whether embryonic development can be recapitulated in vitro through NMPs and gastruloids in the absence of MLL4 (+/-MLL3) with a focus on colinear induction of Hox cluster expression. These analyses include a structure/function dissection of MLL4 using a deletion series of Mll4 BAC transposons introduced into the conditional Mll4 ESCs. Using in situ hybridization, imaging techniques, and conditional mutagenesis methods developed in part by our lab, together with expression profiling, deep sequencing and in vitro embryogenesis, we aim to identify the relationship between MLL4 (and MLL3) in specification of the mammalian body plan.