Chromatin in 3D distinguishes dMes-4/NSD and Hypb/dSet2 in protecting genes from H3K27me3 silencing

Cell type-specific barcoding of genomes requires the establishment of hundreds of heterochromatin domains where heterochromatin-associated repressive complexes hinder chromatin accessibility thereby silencing genes. At heterochromatin–euchromatin borders, regulation of accessibility not only depends on the delimitation of heterochromatin but may also involve interplays with nearby genes and their transcriptional activity, or alternatively on histone modifiers, chromatin barrier insulators, and more global demarcation of chromosomes into 3D compartmentalized domains and topological-associating domain (TADs). Here, we show that depletion of H3K36 di- or tri-methyl histone methyltransferases dMes-4/NSD or Hypb/dSet2 induces reproducible increasing levels of H3K27me3 at heterochromatin borders including in nearby promoters, thereby repressing hundreds of genes. Furthermore, dMes-4/NSD influences genes demarcated by insulators and TAD borders, within chromatin hubs, unlike transcription-coupled action of Hypb/dSet2 that protects genes independently of TADs. Insulator mutants recapitulate the increase of H3K27me3 upon dMes-4/NSD depletion unlike Hypb/dSet2. Hi-C data demonstrate how dMes-4/NSD blocks propagation of long-range interactions onto active regions. Our data highlight distinct mechanisms protecting genes from H3K27me3 silencing, highlighting a direct influence of H3K36me on repressive TADs.