Protamine expression in somatic cells condenses chromatin and disrupts transcription without altering DNA methylation

Protamines play a crucial role in nuclear condensation during spermiogenesis, a process associated with significant chromatin remodeling and replacement of histones by protamines. While much research has focused on the function of protamines in sperm development and fertility, their effects in non-sperm cells remain largely unexplored. In this study, we investigated the impact of overexpressing murine and human protamine 1 and 2 (PRM1 and PRM2) on nuclear architecture, histone eviction, DNA methylation, and transcription in HEK293T cells and mesenchymal stromal cells (MSCs). Overexpression of protamines resulted in nuclear condensation and particularly PRM1 showed notable enrichment in nucleoli and cycle abnormalities. Immunofluorescence staining indicated a significant reduction in specific histone modifications (H3K9me3, H3K4me1, and H3K27Ac) in response to protamine expression, especially in MSCs. Interestingly, despite these changes in nuclear organization, the methylome remained largely stable upon protamine expression. However, particularly expression of protamines significantly diminished transcription, especially of the ribosomal genes upon PRM1 expression. Our studies indicate that PRM1 and PRM2 condense distinct genomic regions in somatic cells, resulting in widespread silencing of gene expression. Overall design: HEK293T cells were transfected with empty vector, human PRM1-EFGP, or human PRM2-EGFP. Three days after transfection cells were sorted and high EGFP cells were used to isolate total RNA and perform RNA-Seq analysis.