Fig 2: The expression levels of HDAC1 and Klf4 are significantly associated in human leukemia patients. (a) Analysis of the expression levels of Class I HDACs (HDAC1, HDAC2, HDAC3, and HDAC8), HDAC11, and Klfs (Klf1, KLf3, Klf4, and Klf7) in human leukemia patients with M2, M3, M4, M5, and M6. The expression levels were detected using qRT-PCR analysis with GAPDH as the internal control and compared with normal controls. *, **, and *** indicate P<0.05, P<0.01, and P<0.001, respectively. (b) Analysis of the expression levels of Class I HDACs (HDAC1, HDAC2, HDAC3, and HDAC8), HDAC11, and Klfs (Klf1, KLf3, Klf4, and Klf7) in CD34+ leukemia patients compared with CD34- leukemia patients. ** and *** indicate P<0.01 and P<0.001, respectively. (c) Analysis of the expression levels of Class I HDACs (HDAC1, HDAC2, HDAC3, and HDAC8), HDAC11, and Klfs (Klf1, KLf3, Klf4, and Klf7) in leukemia patients with different cytogenetic risks (M, intermediate risk; H, high risk) compared with low-risk (L) of leukemia patients. * and ** indicate P<0.05 and P<0.01, respectively. (d) The Kaplan–Meier Curve showing the effects of HDAC1 on overall survival and disease-free survival function in human leukemia patients

Fig 3: Differentially expressed genes are coregulated by KLF4 and STAT3.Significantly up-regulated genes identified in Klf4?Pod glomeruli compared to Klf4fl/fl glomeruli were cross-matched with published ChIP-seq studies on KLF4 and STAT3 from Chen et al. (20). (A) Scaled Venn diagram showing overlap of TFBS. Of 506 significantly up-regulated genes, 218 contain KLF4 TFBS (P = 9.15 × 10-7) and 156 contain STAT3 TFBS (P = 1.03 × 10-6), with 101 genes containing both. Top enriched pathways from KEGG, WikiPathways, and Reactome databases of genes up-regulated in Klf4?Pod mice that contain (B) KLF4 TFBS (218 genes) and (C) STAT3 TFBS (156 genes). MAPK, mitogen-activated protein kinase. (D) Top enriched PPI hub proteins of 101 genes significantly up-regulated in Klf4?Pod glomeruli containing both KLF4 and STAT3 TFBS. (E) Bar plot showing the number of genes passing each TF-gene association score threshold. Higher TF-gene association scores indicate TFBS closer to TSS. TF-gene association scores > 0.8 indicate presence of TFBS within 1 kb of TSS. (F) Top enriched pathways from KEGG, WikiPathways, and Reactome databases of genes up-regulated in Klf4?Pod mice that have STAT3 or KLF4 TF-gene association score > 0.8.

Fig 4: ZNF750 activates or represses transcription with different interacting proteins. (A) Heat map of combined ZNF750, KLF4, RCOR1, and KDM1A ChIP-seq peaks, selected for KLF4 or KDM1A enrichment in ZNF750- and RCOR1-binding regions. Heat map shows ChIP-seq signal over 6-kb regions around peak center. (B) Log2 fold change during keratinocyte differentiation of genes near the top 500 ZNF750, KLF4, and RCOR1 peaks or the ZNF750, RCOR1, and KDM1A peaks. (*) P < 0.05. (C) Sequence-specific motif identified in KLF4 ChIP-seq peaks, compared with canonical KLF4 motif. (D) Frequency of distances between pairs of ZNF750 and KLF4 motifs in ChIP-seq peaks. (E) Example ZNF750, KLF4, RCOR1, and KDM1A ChIP-seq tracks near two differentiation genes (left) and two progenitor genes (right), showing locations of motifs for ZNF750 (green) and KLF4 (purple). (Black bars) MACS-called peaks. (F) Change in luciferase activity of reporters near differentiation genes (top) or progenitor genes (bottom) during calcium-induced keratinocyte differentiation. t-test: (*) P < 0.05; (**) P < 0.01; (***) P < 0.001, compared with –Ca2+. (G) Change in luciferase activity of each reporter with ectopic ZNF750 expression in progenitor keratinocytes compared with empty vector control (CTL). (**) P < 0.01, compared with CTL. (H) Luciferase activity of reporters with control knockdown (CTLi) compared with depletion of ZNF750, KLF4, RCOR1, KDM1A, or CTBP1/2. (*) P < 0.05; (**) P < 0.01, compared with CTLi. See also Supplemental Figure S5 and Supplemental Tables S2–S6.

Fig 5: Identification of functional ZNF750-interacting proteins. (A) Silver stain gel of Flag-HIS ZNF750 or tag-only control (CTL) tandem affinity-purified from differentiated keratinocytes. (B) Network of ZNF750-interacting proteins identified by mass spectrometry, grouped by functional category. Red lines indicate novel ZNF750 interactions, and black lines indicate published interactions. (C) Table of spectral counts and SAINT scores for 13 ZNF750-interacting proteins tested for phenotypic overlap with ZNF750 depletion. (D) Heat map of gene expression changes with depletion of ZNF750-interacting proteins by siRNA in keratinocytes induced to differentiate for 3 d with calcium, shown as number of genes with >1.5-fold expression change by qPCR in the same direction as ZNF750 depletion. Proteins above the dotted line were considered for further analysis. (E) Heat map of gene expression changes with depletion of ZNF750, RCOR1, CTBP1/2, KDM1A, or KLF4 in differentiated keratinocytes by qPCR. (F) Effects of depletion of ZNF750-interacting proteins on differentiation proteins loricrin (LOR; red) and filaggrin (FLG; green) and proliferation (Ki67; red) in regenerated organotypic epidermal tissue. Epidermal basement membrane is noted with dashed lines, and white arrowheads denote suprabasal Ki67-positive cells. Bar, 25µm. See also Supplemental Figure S2.