Recruitment to the nuclear periphery enhances repression of H3K9me2-marked genes and transposons to shape cell fate [CUT&RUN]

Heterochromatic loci marked by histone H3 lysine 9 dimethylation (H3K9me2) are enriched at the nuclear periphery in metazoans, but the functional importance of this organization is unknown. Both the nuclear lamins and the nuclear membrane protein lamin B receptor (LBR) have been linked to heterochromatin positioning in mammals, yet embryonic stem cells (mESCs) lacking all lamins retain both LBR and H3K9me2 at the nuclear periphery. We ablated LBR in this context and show that heterochromatin detaches from the nuclear periphery in lamin + LBR quadruple knockout (QKO) cells. QKO mESCs sustain naïve pluripotency and maintain H3K9me2 at normal levels across the genome but cannot repress H3K9me2-marked genes or transposons. Further, their capacity to differentiate into epiblast-like cells (EpiLCs) is impaired. While normal EpiLCs expand H3K9me2 across the genome, QKO EpiLCs exhibit abnormal patterning of this mark. QKO cells can exit naïve pluripotency and activate epiblast-stage genes but also express markers of alternative fates including the primitive endoderm. These results establish that recruitment of H3K9me2 to the nuclear periphery controls the spatial position, dynamic remodeling, and repressive capacity of this mark to shape cell fate decisions.