Unveiling Ido1-Ido2 Imprinting Regulation in the Placenta: Key Players of Maternal-Fetal Immune Tolerance

Neha Biju¹, Madeline Oconnell², Phillip Spinelli², Martha Susiarjo^1,2.

1.Department of Biomedical Genetics, University of Rochester Medical Centre, Rochester, New York

2.Department of Environmental Medicine, University of Rochester Medical Centre, Rochester, New York

Abstract Text:

Genomic imprinting is an epigenetic phenomenon in which genes exhibit allele-specific expression based on parental origin. Imprinted genes are often found in clusters and share a common regulatory element known as the imprinting control region (ICR). ICRs are marked by parent-of-origin specific DNA methylation patterns that are established during gametogenesis. In some imprinted loci, these methylation patterns at ICRs influence the allele-specific binding of CCCTC-binding factor (CTCF), which in turn regulates enhancer-promoter interactions and chromatin architecture, ultimately driving allele-specific gene expression within the cluster. Imprinted genes and ICRs are critical for fetal and placental development and imprinting dysregulation is linked to growth, metabolic and neurobehavioral disorders. Our lab previously reported that two adjacent genes, Ido1 and Ido2, are imprinted and maternally expressed in the mouse placenta. Ido1 and Ido2 encode indoleamine-2,3 dioxygenase (IDO)1 and 2 that catalyze the rate-limiting step of tryptophan catabolism, converting tryptophan to kynurenine. Placental IDO-mediated tryptophan catabolism is crucial for establishing maternal immune tolerance to the allogeneic fetus. Tryptophan catabolites, kynurenine and kynurenic acid, promote the expansion of immunosuppressive regulatory T-cells, protecting the fetus from maternal pro-inflammatory responses. Pharmacological inhibition of mouse IDO leads to loss of allogeneic pregnancies. In humans, pregnancy loss is associated with lower placental IDO gene and protein expression and higher plasma tryptophan levels compared to healthy pregnancies, indicating the importance of IDO1 and IDO2 in pregnancy maintenance. Given their role in maternal-fetal tolerance, it is imperative to understand the regulatory mechanisms of placental Ido1-Ido2 imprinting. Our lab previously identified a novel differentially methylated region (DMR) at the Ido1 promoter, which is hypermethylated in the paternal allele and hypomethylated in the maternal allele in E9.5 mouse placenta. This region is hypermethylated in sperm and unmethylated in oocytes, suggesting it’s a primary DMR with potential role in Ido1-Ido2 imprinting. DNA methylation analysis of human sperm and uncomplicated placenta also suggest similar methylation patterns to mouse. Human placentas from recurrent miscarriage exhibited higher methylation levels, suggesting a correlation with pregnancy outcome. Despite these findings, the precise mechanism regulating Ido1-Ido2 imprinting remains unclear. Imprinting regulation involves interplay of multiple cis-regulating elements including DMRs, enhancers and promoters. The current study investigates the Ido1-Ido2 genomic locus for additional putative cis-regulatory elements involved in imprinting. Using publicly available ENCODE datasets, we identified four CTCF-binding regions (CBRs) in the ~120 kb Ido1-Ido2 locus, exhibiting features similar to ICRs. These regions include CTCF-binding motifs containing CpG sites which, if differentially methylated on parental alleles, can lead to allele-specific CTCF binding. These CTCF motifs exhibit opposing orientations and co-localize with cohesin subunits (RAD21 and SMC3), suggesting long-range DNA interactions and architectural functions, which are key regulators of some imprinted loci. Interestingly, publicly available micro-C data show that these CBRs participate in two long-range interactions (~55 kb and ~140 kb), suggesting they are chromatin loop boundaries potentially regulating enhancer-promoter interactions in the Ido1-Ido2 locus. Bisulfite pyrosequencing analysis of one of these CBRs revealed partial methylation (~30%) at 9 CpG sites suggesting possibility of allele-specific methylation. Currently, we are determining if these CBRs are imprinting regulators by investigating for allele-specific DNA methylation, CTCF binding and chromatin architecture using bisulfite cloning sequencing, CUT&TAG and Capture-C sequencing, respectively. This research will elucidate the Ido1-Ido2 imprinting mechanism, providing insights into how its disruption may impact Ido1-Ido2 expression, maternal-fetal tolerance, and pregnancy outcomes.