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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 5. — Yeats4 recruits the Dot1l–RNA Pol II complex onto the Lmo4 promoter through recognizing H3K27Ac. (A) Pull-downs were performed in BM cells from Yeats4^Flag knockin mice using anti-Flag or IgG. Eluted fractions were resolved by SDS-PAGE, followed by silver staining and mass spectrometry. (B) Flag-Yeats4 precipitated Dot1l in BM cell lysates. IP, immunoprecipitation. (C) Yeats4 and Dot1l were visualized in α₄β₇⁺ CLPs from Yeats4^fl/fl mice by immunofluorescence staining. Red, Yeats4; green, Dot1l. Nuclei were counterstained by DAPI. Scale bar, 5 µm. (D) Dot1l enrichment on Lmo4 promoter was analyzed via ChIP-qPCR analysis. (E) Dot1l enrichment on Lmo4 promoter was analyzed in α₄β₇⁺ progenitors (Lin⁻CD127⁺c-Kit^intSca-1^intα₄β₇⁺) from Yeats4^fl/fl and Yeats4^fl/flVav-Cre mice. (F) α₄β₇⁺ CLPs cells from Yeats4^fl/fl and Yeats4^fl/flVav-Cre mice were in situ hybridized with probes against the Lmo4 promoter, followed by staining with antibodies against Yeats4 and Dot1l. Arrowheads indicate Lmo4 promoters colocalized with Dot1l. Scale bar, 5 µm. (G) Enrichment of H3K79me3 on the Lmo4 promoter was tested using ChIP-qPCR analysis. (H) Enrichment of H3K79me3 on the Lmo4 promoter in WT, Yeats4-deficient, or Dot1l-deficient α₄β₇⁺ progenitor (Lin⁻CD127⁺c-Kit^intSca-1^intα₄β₇⁺) cells was examined. (I) Enrichment of RNA Pol II Ser2P on the Lmo4 promoter in WT, Yeats4-deficient or Dot1l-deficient α₄β₇⁺ progenitor (Lin⁻CD127⁺c-Kit^intSca-1^intα₄β₇⁺) cells was analyzed. (J) BM cells from Yeats4^fl/fl and Yeats4^fl/flVav-Cre mice were lysed and treated with 1% formaldehyde for cross-linking. Then, anti-Dot1l antibody was incubated with treated lysates for ChIP assays, followed by size fractionation with sucrose gradient ultracentrifugation. Eluate gradients were examined by Western blotting and PCR assays. (K) Dot1l^−/− or control α₄β₇⁺ progenitors (Lin⁻CD127⁺c-Kit^intSca-1^intα₄β₇⁺) were subjected to nuclear run-on assay, followed by RT-PCR analysis for Lmo4 transcription. (L) Dot1l-overexpressing or control α₄β₇⁺ progenitors (Lin⁻CD127⁺c-Kit^intSca-1^intα₄β₇⁺) were subjected to nuclear run-on assay, followed by RT-PCR analysis for Lmo4 transcription. (M and N) Relative mRNA (M) and protein (N) levels of Lmo4 in Yeats4-deficient, Dot1l-deficient, or control α₄β₇⁺ progenitors (Lin⁻CD127⁺c-Kit^intSca-1^intα₄β₇⁺) were analyzed. ***, 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.