Methyltransferase DOT1L as a regulator of splicing during neuronal differentiation

VILLARREAL A; CHUNG WY; FRANZ H; VOGEL T

Freiburg

Congreso; 3rd German-Catalan Workshop on Epigenetic and Disease; 2016

CRC992: Medical Epigenetics-From basic mechanisms to clinical applications

DOT1L is a methyltransferase responsible for the mono-, di- and tri-methylation of histone 3 (H3) at lysine 79 (K79) located in the globular domain of the histone. Published data from our group indicate that DOT1L is involved in brain cortex development in part by disrupting upper layer neurons fate. We created then a DOT1L conditional knock out mouse (DOT1L cKO) driven by FOXG1-Cre expression which resulted in a reduced cortical plate. Recent RNAseq analysis from these animals showed that the expression levels of many mRNAs were altered in the DOT1LcKO and interestingly several of this transcripts were present as altered splicing variants. We aim now to verify if DOT1L is indeed capable of directly regulating splicing events in genes that are crucial for brain development. Correlative analyses of RNA- and ChIP-seq showed that H3K79me2 is present in only a subset of the genes with altered splicing variants indicating that this function of DOT1L could also be independent of its histone methyltransferase activity. In an in vitro model of neuronal differentiation of mouse embryonic stem cells we observed that some of these altered splicing variants are expressed only at later time points of neuronal differentiation when DOT1L transcription is downregulated. In a different set of experiments, we overexpressed DOT1L fused to the tags HA and FLAG in Neuro2A cells. Co-IP and mass spectroscopy analysis showed that DOT1L interacts with several members of the U2 splicing complex such as SF3B1 and SF3B3. Cell fractionation assay followed by Co-IP indicated that DOT1L and SF3B1 interact at the chromatin level. Together our data suggest, for the first time, that DOT1L might be involved in regulation of splicing events probably by direct interaction with components of the spliceosome.