*2.7. ChIP Using JARID2 or EZH2 Antibody Reveals Other Co-Factors of PRC2 Complex*

*2.7. ChIP Using JARID2 or EZH2 Antibody Reveals Other Co-Factors of PRC2 Complex*  In order to delineate the alterations in the binding partners of EZH2 in the PF treated In order to delineate the alterations in the binding partners of EZH2 in the PF treated cells, ChIP was performed using either JARID2 or EZH2 antibodies followed by ChiPqPCR promoter array of genes associated with polycomb and trithorax complexes in cells

cells, ChIP was performed using either JARID2 or EZH2 antibodies followed by ChiP-

treated with or without PF. Table 1A provides a list of focused gene panel involved in polycomb and trithorax complex activity, that were differentially expressed in endo PF treated cells when compared to control PF after IP by either JARID2 or EZH2 antibodies. The enrichment of EZH2 after JARID2 IP was lower in endo PF-treated cells compared to control cells. However, EZH1, a polycomb enzyme which is responsible for mono-, di-, or tri-methylation of H3K27, showed an enrichment after JARID2 IP in endo treated cells when compared to control PF treated cells but this enrichment was not significant. In contrast, the enrichment of JARID2 after EZH2 IP was over 5-fold higher in endo PF treated cells compared to control PF treated cells. ARID1A, a subunit of the SWI/SNF complex, with an antagonistic relationship with EZH2 [51], showed enrichment after both JARID2

When comparing the genes involved in polycomb and trithorax complex activity,

pulled down by the two antibodies, (Table 1B), JARID2 IP compared to EZH2 IP showed

in mean compared to endo PF. Density of protein bands obtained was normalized to β-actin or H3.

IP and even higher after EZH2 IP.

treated with or without PF. Table 1A provides a list of focused gene panel involved in polycomb and trithorax complex activity, that were differentially expressed in endo PF treated cells when compared to control PF after IP by either JARID2 or EZH2 antibodies. The enrichment of EZH2 after JARID2 IP was lower in endo PF-treated cells compared to control cells. However, EZH1, a polycomb enzyme which is responsible for mono-, di-, or tri-methylation of H3K27, showed an enrichment after JARID2 IP in endo treated cells when compared to control PF treated cells but this enrichment was not significant. In contrast, the enrichment of JARID2 after EZH2 IP was over 5-fold higher in endo PF treated cells compared to control PF treated cells. ARID1A, a subunit of the SWI/SNF complex, with an antagonistic relationship with EZH2 [51], showed enrichment after both JARID2 IP and even higher after EZH2 IP.

**Table 1.** ChIP-qPCR of PRC2 complex proteins in PF-treated cells. Chromatin Immunoprecipitation (ChIP) was used to analyze interactions between JARID2 and EZH2 and genes associated with the polycomb and trithorax complexes, normalized to IgG. **A**. Fold change values represent the ratio of enrichment/binding of JARID2 or EZH2 to various genes in endo PF-treated cells (*n* = 3) to enrichment in control PF treated cells (*n* = 3). Genes with a *p*-value < 0.05 are shown as bold and italicized. EZH1 and EZH2 are also shown but did not have a significant *p*-value for either JARID2 or EZH2 when comparing the two cell treatments. **B**. Fold change values representing the ratio of enrichment/binding in EZH2 precipitated cells (*n* = 3) to enrichment in JARID2 precipitated cells (*n* = 3) for both cell treatments. *p*-values < 0.05 are shown as bold and italicized along with EZH1, EZH2, and JARID2 which did not show significant *p*-values for either treatment.


When comparing the genes involved in polycomb and trithorax complex activity, pulled down by the two antibodies, (Table 1B), JARID2 IP compared to EZH2 IP showed upregulation of 4 genes with *p*-values < 0.05 in control PF treated cells. While no significant *p*-values were seen for any genes in the endo PF treated cells, when JARID2/EZH2 ratio was calculated, all but 7 genes were shown to be downregulated in these cells suggesting that ChIP by EZH2 in endo PF treated cells has more of an effect on the pull-down expression of these genes compared to JARID2. Any *p*-values > 0.05 may be due in part to the smaller sample size tested.
