Transcriptome Analysis of Human Cancer Cells with Depleted or Overexpressed STELLA Proteins [RNA-seq]
UHRF1 maintains DNA methylation by recruiting DNA methyltransferases (DNMT's) to chromatin. These dynamics are well defined for mouse STELLA (mSTELLA) but poorly characterized for human STELLA (hSTELLA). Herein, we demonstrate that hSTELLA is defective, while mSTELLA is fully proficient in associating with UHRF1 and inhibiting the abnormal DNA methylation and oncogenic functions of UHRF1 in human cancer cells. We illustrate, in structural studies a region of low sequence homology between the above STELLA orthologs, allows mSTELLA but not hSTELLA to bind tightly to the essential histone binding domains of UHRF1, thus mediating their above functional differences. The ortholog-specific binding modes of STELLA proteins with UHRF1 prompt us to explore a rationale for designing UHRF1 inhibitors for cancer therapy. For this, we use a lipid nanoparticle (LNP)-mediated mRNA delivery approach demonstrating the short mSTELLA, but not hSTELLA regions are required to reverse cancer-specific DNA methylation abnormalities and impair CRC tumor growth. Overall design: To investigate the role of hSTELLA versus mSTELLA in regulating abnormal DNA hypermethylation associated tumor suppressor genes (TSGs) silencing, we established hSTELLA-knockout isogenic cell clones in BeWo and NCCIT cells. The transcriptome changes induced by hSTELLA-knockout were studied on the cell clones transduced with control sgRNAs (sgSCR) and hSTELLA-targeting sgRNAs. Next, we stably over-expressed both hSTELLA and mSTELLA in both HCT116 and RKO cells, and compared the transcriptome differences (relative to EV) between cells with hSTELLA overexpression and cells with mSTELLA overexpression To define the key regions mediating the functional differences between hSTELLA and mSTELLA, we designed two chimeric mutants for C-terminal mSTELLA which replace the corresponding sequences from hSTELLA. The two mutants were designated as mSTELLA-Swap1 and mSTELLA-Swap2, respectively. We compared the transcriptome changes induced by these mutants with those induced by mSTELLA and hSTELLA, upon their stable overexpression in UHRF1-deficient HCT116 cells (HCT116_UHRF1_def or HCT116-UHRF1+/-). Based on the above findings, we utilized a lipid nanoparticle (LNP) approach for delivering mRNAs encoding mSTELLA and mSTELLA-Swap1 into HCT116 cells. Cells treated with LNP-mSTELLA and LNP-mSTELLA-Swap1 were collected at Days 2, 6 and 10, and the transcriptome differences for each group were compared at each time point.