Figure 5. Transcriptional and Post-transcriptional Regulation of Tet2 by Zfp281. See also Figure S5.

(A) Relative expression of Tet2 by qPCR showing upregulation of Tet2 in Zfp281 KO relative to WT ESCs cultured during 48-hour pluripotent state transitions.

(B) Enrichment of Zfp281 at the regulatory locus of Tet2 by ChIP-qPCR validating Tet2 as a direct transcriptional target of Zfp281.

(C) ChIP-qPCR analysis of relative enrichment of HDAC2 and H3K27ac at the Zfp281 binding Tet2 loci as indicated in (B).

(D) Chemical inhibition of HDAC activity increases Tet2 expression in Zfp281 WT, but not KO cells. Expression analysis was determined by qPCR in Zfp281 KO and WT ESCs treated with VPA (0.5 mM) for 24 hours.

(E) Schematic depiction of seed sequences for miR-302/367 cluster on Tet1 and Tet2 genes. Seed match sequences of miR-302b within Tet2 3′-UTR were shown.

(F) Relative expression of mature miR-302b, miR-302d, and miR-367 detected by miRCURY™ LNA Array in Zfp281 WT, HET (heterozygous) and KO ESCs. The color scale bar represents fold change.

(G) Relative expression of pri-miR-302/367 cluster measured by qPCR in Zfp281 KO and WT ESCs at SL and 48 hours at 2iL or FA.

(H) Depiction of miR-302/367 cluster gain- and loss-of-function assays (left), and qPCR analysis of Tet2 expression levels (right).

(I) Zfp281 depletion enhances firefly luciferase activity. Zfp281 KO and WT ESCs were transiently transfected with the luciferase reporter tethering to the 3′-UTR of Tet2. Renilla reporter was included for normalization (F/R ratio).

(J) A model illustrating the transcriptional activation and repression roles of Zfp281 in regulating Tet2 expression.

All error bars in this figure indicate average ± SEM (n=3). P values were determined by Student's unpaired t test (ns, not significant; ***P<0.0001, **P<0.001).