Oral Presentation 46th Lorne Genome Conference 2025

Chromatin tethering by the Trithorax homolog MLL during the ESC naive to primed transition (113823)

Francis Stewart 1 2 , Maike Steindel 3 , Oliver Davis 3 , Katrin Neumann 1 , Kranz Andrea 1 , Liviu Pirvan 3 , Jörg Morf 3 , Anastassiadis Konstantinos 1 , Srinjan Basu 3 4
  1. Technische Universität Dresden, Dresden, SAXONY, Germany
  2. Key State Laboratory for Microbial Technology, Shandong University, Qingdao, China
  3. Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
  4. Department of Life Scences, Imperial College, London, UK

The histone 3 lysine 4 (H3K4) methyltransferases, MLL1-4, are extremely large nucleoplasmic proteins. None of the four are required in embryonic stem cells (ESCs) or in very early mouse development until Mll4 (Kmt2d) is required for establishment of the embryonic anterior-posterior axis (1). Although Mll2 (Kmt2b) encodes the major H3K4 methyltransferase active in late oogenesis (2) and for bivalency in ESCs (3), knockout embryos are only retarded from E6.5 (4). Mll2 knockout ESCs can be differentiated towards neural stem cells until neuroectodermal differentiation stops before the onset of Nestin positive neural rosettes. Using time courses of conditional mutagenesis, we found that the requirement for Mll2 in neuroectodermal differentiation is exerted during ESC exit from naïve to primed pluripotency at least 6 cell cycles earlier than the onset of the mutant phenotype. Single-cell transcriptomic analysis during the transition from naïve to primed pluripotency revealed that Mll2 is not required for the transition but is required for the transcriptional upregulation of several genes including Otx2, Ntx2 and Sox2. Through transcriptomic, CUT&Tag and Micro-C/Hi-C correlations, we found that local loss of Mll2-dependent 3D chromatin looping accompanies the loss of transcription. Consequently, we propose that MLL2 is a multivalent chromatin tethering factor that secures long-range interactions during priming for the transition to neuroectoderm. Notably MLL2 H3K4 methylation is not required for this tethering. To address our hypothesis, we used live-cell 3D single-molecule localisation microscopy (SMLM, 5) to report chromatin mobility by tracking histone H2B-Halo and found that global chromatin movement is increased in primed pluripotent cells lacking MLL2, supporting a role for MLL2 in tethering chromatin contacts. These observations address the MLL enigma, which has been focused on H3K4 methylation rather than their abilities, as extremely large multidomain proteins, to tether distant cis-elements and thereby secure lineage commitment decisions.

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  5. 5. Basu et al (2023) Nat Struct Mol Biol,30, 1628-39.