LDB1 regulates gene expression and chromatin structure in pluripotency and lineage differentiation [Cut&Tag]

Chromatin organization is a pivotal factor in stem cell pluripotency and differentiation. However, despite considerable study, the role of enhancer looping protein LDB1 in these processes has not been explored. Here, we generated Ldb1(-/-) embryonic stem cells (ESC) using CRISPR/Cas9 editing to understand the significance of LDB1 during development. Ldb1(-/-) ESC exhibited a reduction in key stem cell factors SOX2 and KLF4. Embryoid bodies (EB) derived from Ldb1(-/-) ESC displayed reduced expression of lineage-specific markers and impaired ability to undergo terminal differentiation to erythroblasts. Transcriptome analysis revealed differential gene expression between WT and Ldb1(-/-) ESC and EB but altered gene expression was most pronounced after differentiation to erythroblasts. The Lin28-mediated stem cell self-renewal pathway was dysregulated in Ldb1(-/-) cells. Our data reveal that LDB1 occupies super enhancers of pluripotency genes in ESC together with pluripotency factors. LDB1 loss resulted in a global decrease in chromatin accessibility in ESC and EB. Conditional LDB1-deficient mice showed reduced hematopoietic stem cell markers on bone marrow cells, and dysregulation of the Lin28/Let-7/Hmga2 pathway. We conclude LDB1 function is critical for ESC and EB development and becomes progressively more important during differentiation to erythroblasts.