## 3 Results
### CpxoxRNA Interacts with the CTCF/Colesin Complex to Act in Trans to Regulate Erythropoiesis  
Although Cpxox encodes the sixth enzyme in the heme synthesis pathway, it appears to be dispensable for erythropoiesis [71; 72]. Additionally, other genes that loop with the  
Figure 4: _CpxoxRNA_ is critical for survival and proliferation during terminal erythropoiesis. (**A**) The enucleation rate in the control and _CpxoxRNA_ knockdown cells. Levels of the differentiation marker TER19 (fluorescent immunolabeling) vs. DNA content (Hoechst staining) of shRNA-transduced cells after 48 h of ex vivo culture were detected by Flow cytometry. (**B**) May–Grunwald Giemsa staining of shRNA-transduced mouse E14.5 fetal liver erythroblasts fixed after 48 h of ex vivo differentiation. Arrow shows enucleated cells. (**C**,**D**) Growth curve shows the cell proliferation rate in the differentiated WT MEL cell (iMEL) after _CpxoxRNA_ knockout by CRISPR-Cas9 (**C**) and after transfection with a shRNA target to _CpxoxRNA_ (shCPOXeRNA-1) or an empty vector (shCTRL) (**D**). ***: \(p\leq 0.001\).  
_CpoxeRNA_ locus (Figure 3A and Supplementary Figure S3A) are not directly related to erythropoiesis [71].  
Since cohesin can mediate the action of eRNAs in trans to regulate genes on other chromosomes, we investigated whether _CpoxeRNA_ might also act in trans. We performed ChIP-seq with two sets of probes targeting _CpoxeRNA_ (Figure 5A). The circos plot illustrates the 149 loci across different chromosomes that are occupied by _CpoxeRNA_ (Figure 5B). _CpoxeRNA_ occupancy was detected at four sites on chromosome 16 in addition to the _CpoxeRNA_ region, but none were within the _CpoxeRNA_/_Cpox_ TAD. Most _CpoxeRNA_ binding sites are in intergenic regions, although approximately 16% of the 149 loci are in promoter regions (<3 kb) (Figure 5C). We tested 8 genes, and 3 of the 23 genes bound by _CpoxeRNA_ at the promoter were downregulated following _CpoxeRNA_ knockdown (Figure 5D). These genes are crucial for chromatin remodeling, with _Erc1_ being additionally important for stress erythropoiesis and cell proliferation [73].  
Figure 5: _CpoxeRNA_ acts in trans to regulate genes important for erythropoiesis. **(A)** qRT-PCR and qPCR results showing RNA and DNA pull-down efficiency of ChIP experiment. **(B)** Circos plot showing the genome-wide binding sites of _CpoxeRNA_ identified by ChIP-seq experiment. **(C)** Pie chart  
showing the genomic distribution of _CpoxreRNA_ binding sites identified from ChIP-seq analysis. (**D**) Top image presents IGV tracks showing the ENCODE ChIP-seq data for CTCF, RAD21, and SMC3 binding at target genes loci in undifferentiated MEL cells and _CpoxreRNA_ ChIP-seq peaks at target genes, while the bottom image presents the qRT-PCR result showing the expression level of target interacting genes identified by the ChIP experiment in the control and _CpoxreRNA_ knockdown cells. Two biological replicates with three technical replicates each (\(n=6\)), one-tailed unpaired \(t\)-test.  
In summary, these findings indicate that _CpoxreRNA_ interacts with the CTCF/cohesin complex to act in trans, contributing to the regulation of erythropoiesis.