Methylation of histone 3 lysine 4 (H3K4) is a major epigenetic system associated with gene expression. In mammals there are six H3K4 methyltransferases related to yeast Set1 and fly Trithorax, including two orthologs of fly Trithorax-related: MLL3 and MLL4. Exome sequencing has documented high frequencies of MLL3 and MLL4 mutations in many types of human cancer. Despite this emerging importance, the requirements of these paralogs in mammalian development have only been incompletely reported. Here, we examined the null phenotypes to establish that MLL3 is first required for lung maturation, whereas MLL4 is first required for migration of the anterior visceral endoderm that initiates gastrulation in the mouse. This collective cell migration is preceded by a columnar-to-squamous transition in visceral endoderm cells that depends on MLL4. Furthermore, Mll4 mutants display incompletely penetrant, sexdistorted, embryonic haploinsufficiency and adult heterozygous mutants show aspects of Kabuki syndrome, indicating that MLL4 action, unlike MLL3, is dosage dependent. The highly specific and discordant functions of these paralogs in mouse development argues against their action as general enhancer factors.

statement 26 The H3K4 methyltransferases MLL3 and MLL4 have strikingly different null 27 phenotypes during mouse development; MLL3 is required for lung maturation 28 whereas MLL4 is required for anterior visceral endoderm migration. 29 30 Abstract 31 Methylation of histone 3 lysine 4 (H3K4) is a major epigenetic system 32 associated with gene expression. In mammals there are six H3K4 33 methyltransferases related to yeast Set1 and fly Trithorax, including two 34 orthologs of fly Trithorax-related: MLL3 and MLL4. Exome sequencing has 35 documented high frequencies of Mll3 and Mll4 mutations in many types of 36 human cancer. Despite this emerging importance, the requirements of these 37 sister genes in mammalian development have only been incompletely reported. 38Here we examined the null phenotypes to establish that MLL3 is first required 39 for lung maturation whereas MLL4 is first required for migration of the anterior 40 visceral endoderm (AVE) that initiates gastrulation and is the first collective cell 41 migration in development. This migration is preceded by a columnar to 42 squamous transition in visceral endoderm cells that depends on MLL4. 43 Furthermore, Mll4 mutants display incompletely penetrant, sex distorted, 44 embryonic haploinsufficiency and adult heterozygous mutants show aspects of 45 Kabuki syndrome, indicating that MLL4 action, unlike MLL3, is dosage 46 dependent. The highly specific and discordant functions of these sister genes 47 argues against their action as general enhancer factors. 48 49 50 histone 3 tail on key nucleosomes (Schmitges et al., 2011). This opposition is 85 central to epigenetic regulation in development, differentiation, homeostasis 86and more recently, oncogenesis (Chi et al., 2010;Rao and Dou, 2015; Soshnev 87 et al., 2016) with several Trx-G and Pc-G factors, including the H3K27 88 methyltransferase, EZH2, implicated as oncogenes or tumor suppressors in a 89 variety of malignancies. Mll1 was discovered as the major leukemia gene at the 90 11q23.1 translocation involved in early onset childhood leukemia (Li and Ernst, 91 2014). The N-terminal half of MLL1 fused to many (now more than 70) different 92 C-terminal partners, including AF4 and AF9 (Slany, 2009; Meyer et al., 2018) 93 is leukemiogenic without the need for secondary mutations (Dobson et al., 94 2000). These MLL1 fusion proteins promote both acute lymphocytic (ALL) and 95 acute myeloid (AML) leukemias, collectively termed mixed lineage leukemias. 96Massively parallel sequencing of cancer exomes by the international 97 cancer genome projects revealed somatic mutations in Mll3 and Mll4 in almost 98 all cancers analyzed (Rao and Dou, 2015). Inactivating heterozygous mutations 99 have been identified in patients with medulloblastoma,