## 3 Results
### CpxceRNA Mediates Chromatin Interactions Between Its Genomic Locus and TAD Boundaries  
PCHiC data [52] show that the _CpxceRNA_ genomic locus interacts with the proximal coding genes _Cpxx_, _Gm813_, and _E330017A01 Rik_ (Supplementary Figure S3A). It also interacts with distal genes _Cldnd1_ and _S13 gal6_. We performed a 3C experiment and found that _CpxceRNA_ mainly interacts with the two flanking TAD boundaries (Figure 3A). The  
Figure 2: _CpxceRNA_ is an erythroid-specific enhancer RNA. **(A)** IGV tracks showing the H3K27 ac signals in mouse CH12. LX, mouse embryonic stem cell (mES), and MEL cells. H3K9 ac and H3K4 me1 signals in MEL cells. G1E cell and Erythroblast ATAC signal, Mouse E14.5 super enhancer, and RNA-seq from mouse E14.5 fetal liver erythroid progenitor cells (FLC PROG) and erythroid blast cells (FLC ERY). ChIP-seq data from ENCODE. RNA-seq data from [PERSON] et al. [24]. **(B)** qRT-PCR result showing the expression level of _CpxceRNA_ in ex vivo differentiated mouse E14.5 fetal liver cells. Two biological replicates with three technical replicates each, \(n=6\), two-tailed unpaired \(t\)-test. **(C)** Cell fractionation qRT-PCR result showing the distribution of _CpxceRNA_. **(D)** Immunofluorescence RNA FISH data showing the localization of _CpxceRNA_. _CpoxeRNA_ genomic locus had the highest interaction frequency with _Cpox_, while for other genes, the interaction frequencies were more than 600-fold lower (Figure 3A). We next asked whether the _CpoxeRNA_ eRNA transcript itself participated in regulating chromatin looping. We performed _CpoxeRNA_ knockdown using shRNA in differentiated MEL cells (iMEL) and ex vivo differentiated E14.5 fetal liver cells (Supplementary Figure S3B,C). After the knockdown of _CpoxeRNA_, the interaction frequency of _CpoxeRNA_ with the genes located within this TAD (_St3 gal6_, _Gm813_, and _E330017A01 Rik_) and outside the TAD (_Clndnd1_) were not affected. Surprisingly, we observed that a loss of _CpoxeRNA_ significantly decreased the interaction between its genomic locus and both flanking TAD boundaries (Figure 3A). Figure 3: _CpoxeRNA_ interacts with CTCF/cohesin complex. (**A**) 3C experiment shows the interactions between _CpoxeRNA_ and flanking regions in the control and _CpoxeRNA_ knockdown iMEL cells. _CpoxeRNA_ locus acts as an anchor. TAD boundary regions are highlighted in red. Three biological replicates with two technical replicates each, \(n=6\), one-tailed unpaired \(t\)-test. *: \(p\leq 0.05\); ***: \(p\leq 0.001\). (**B**). RT-qPCR results showing _Cpox_ expression in the control and shCPOXeRNA knockdown iMEL and ex vivo differentiated E14.5 fetal liver cells. \(n=3\) three technical replicates. Unpaired one-tailed \(t\)-test. (**C**) HiC maps derived from Mouse E14.5 fetal liver [52], ESC [52], and C2C12 cells [61] showing that _CpoxeRNA_ is located in an erythroid-specific TAD. Bottom tracks show the Gene annotation track and ENCODE CTCF narrow peak in C2C12 and MEL cells and RAD21 narrow peak in MEL cells. Red arrows indicate the left TAD boundary and blue arrows indicate the CTCF/RAD21 peaks overlapping with _CpoxeRNA_. (**D**). RNA pull-down experiments showing _CpoxeRNA_ interacts with the CTCF/cohesin complex. The _CpoxeRNA_ probe with RNaseA treatment (_CpoxeRNA_ + RNaseA) was used as the negative control sample. ACT was used as the negative control. AR RNA and polyA RNA were the positive RNA control and negative RNA control for the pull-down experiment procedure, and HUR was used to confirm whether the procedure was working as it binds to AR RNA but not polyA RNA. Since _Cpx_ overlaps with the right TAD boundary, we next checked whether its expression was affected after _CpxexRNA_ knockdown, which reduced their interaction. As shown in Figure 3B, the knockdown of _CpxexRNA_ by shRNA in iMEL cells and ex vivo differentiated E14.5 fetal liver cells consistently downregulated _Cpx_.