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. 2019 Aug 21;216(11):2653–2668. doi: 10.1084/jem.20182363

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© 2019 Liu et al.

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Figure 4. — Yeats4 initiates Lmo4 expression in α₄β₇⁺ CLPs. (A) Differentially expressed TFs in α₄β₇⁺ progenitors (Lin⁻CD127⁺c-Kit^intSca-1^intα₄β₇⁺) from Yeats4^fl/fl and Yeats4^fl/flVav-Cre mice by microarray analysis. (B) qRT-PCR analysis for mRNA levels of indicated top 10 down-regulated TFs in α₄β₇⁺ CLPs from Yeats4^fl/fl and Yeats4^fl/flVav-Cre mice. (C) Protein levels of Lmo4 were tested by immunoblotting in α₄β₇⁺ progenitors (Lin⁻CD127⁺c-Kit^intSca-1^intα₄β₇⁺) from Yeats4^fl/fl and Yeats4^fl/flVav-Cre mice. (D) Yeats4 enrichment on Lmo4 promoter was determined. α₄β₇⁺ progenitors (Lin⁻CD127⁺c-Kit^intSca-1^intα₄β₇⁺) were isolated from Yeats4^fl/fl and Yeats4^fl/flVav-Cre mice, followed by ChIP assay. (E) Yeats4 was colocalized with the Lmo4 promoter by FISH assay. Scale bar, 5 µm. (F) EMSA for detection of direct interaction between Yeats4 and the Lmo4 promoter. The Lmo4 promoter was biotin labeled. Positive control probes interacted with nuclear extract (right panel). (G) Pull-downs were performed using Flag-Yeats4 and biotinylated histone peptides with different modifications, followed by immunoblotting with anti-Flag antibody. IP, immunoprecipitation. (H) Colocalization between Yeats4 and H3K27ac in α₄β₇⁺ CLPs was assessed by immunofluorescence staining. Nuclei were stained with DAPI. Scale bar, 5 µm. (I) Analysis of H3K27ac enrichment on Lmo4 promoter using ChIP-qPCR assay. (J) Enrichment of H3K4me3 on Lmo4 promoter in αLPs was analyzed by ChIP analysis. (K) DNase I accessibility assay of Lmo4 promoter in α₄β₇⁺ progenitors (Lin⁻CD127⁺c-Kit^intSca-1^intα₄β₇⁺) from Yeats4^fl/fl and Yeats4^fl/flVav-Cre mice. (L) α₄β₇⁺ progenitors (Lin⁻CD127⁺c-Kit^intSca-1^intα₄β₇⁺) from Yeats4^fl/fl and Yeats4^fl/flVav-Cre mice were subjected to nuclear run-on assay, followed by RT-PCR analysis for Lmo4 transcription. ***, P < 0.001 by two-tailed unpaired Student’s t test. All data are representative of at least three independent experiments and are expressed as mean ± SD.