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. 2009 Jun 23;106(25):10141-6.
doi: 10.1073/pnas.0900437106. Epub 2009 Jun 3.

LSD1-mediated epigenetic modification is required for TAL1 function and hematopoiesis

Xin Hu  1 , Xingguo Li, Kristell Valverde, Xueqi Fu, Constance Noguchi, Yi Qiu, Suming Huang
Affiliations

LSD1-mediated epigenetic modification is required for TAL1 function and hematopoiesis

Xin Hu et al. Proc Natl Acad Sci U S A. .

Abstract

TAL1 is a critical transcription factor required for hematopoiesis. However, perturbation of its activity often leads to T cell leukemia. Whether and how its transcriptional activities are regulated during hematopoiesis remains to be addressed. Here, we show that TAL1 is associated with histone demethylase complexes containing lysine-specific demethylase 1 (LSD1), RE1 silencing transcription factor corepressor (CoREST), histone deacetylase 1 (HDAC1), and histone deacetylase 2 in erythroleukemia and T cell leukemia cells. The enzymatic domain of LSD1 plays an important role in repressing the TAL1-directed transcription of GAL4 reporter linked to a thymidine kniase minimal promoter. Furthermore, we demonstrate that the TAL1-associated LSD1, HDAC1, and their enzymatic activities are coordinately down-regulated during the early phases of erythroid differentiation. Consistent with the rapid changes of TAL1-corepressor complex during differentiation, TAL1 recruits LSD1 to the silenced p4.2 promoter in undifferentiated, but not in differentiated, murine erythroleukemia (MEL) cells. Finally, shRNA-mediated knockdown of LSD1 in MEL cells resulted in derepression of the TAL1 target gene accompanied by increasing dimeH3K4 at the promoter region. Thus, our data revealed that histone lysine demethylase LSD1 may negatively regulate TAL1-mediated transcription and suggest that the dynamic regulation of TAL1-associated LSD1/HDAC1 complex may determine the onset of erythroid differentiation programs.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
TAL1 associates with LSD1 complex in erythroid and T cell leukemia cells. (A) A partial list of TAL1-associated polypeptides identified by LC-MS/MS is indicated. (B) The Flag-purified complexes pulled down from Flag-Tal1 and mock-transduced Jurkat cells were analyzed by WB. (C) Nuclear extract from MEL cells was precipitated with TAL1 antibody and analyzed by WB using LSD1 and HDAC1 antibodies. (D) Nuclear extracts from Jurkat and K562 cells were precipitated with LSD1 antibody and analyzed by WB using TAL1 antibody. (E) Schematic representation of GST-TAL1 fusion proteins used in a GST pull-down assay. (F) 35S-labeled LSD1 was incubated with GST and GST-TAL1 fusion proteins preabsorbed to glutathione-Sepharose beads. (Upper) Bound LSD1 was visualized by fluorography after SDS/PAGE. (Lower) Coomassie-stained gel shows the relative protein loading.
Fig. 2.
Fig. 2.
TAL1 forms 2 distinct complexes with LSD1. (A) Nuclear extracts from Jurkat cells expressing Flag-Tal1 were fractionated through the Sephacryl S-300 HR column. The fractions were collected and analyzed by WB. (B) The peak fractions from 1.8 MDa (I), 650 kDa (II), 300 kDa (III), and <50 kDa (IV) were collected. The pooled fractions were precipitated with Flag antibody and analyzed by WB. (C) The peak fractions from 1.8 MDa (I), 650 kDa (II), 300 kDa (III), and <50 kDa (IV) were incubated with radiolabeled hyperacetylated histone. The amount of [3H]acetate released was quantitated by liquid scintillation counting. (D) Demethylase activity of LSD1 is required to repress TAL1-mediated transcription. HeLa cells were transfected with a Gal4-TK-luc reporter, an expression vector for GAL4-TAL1, and increased amounts of LSD1 or LSD1ΔC. A CMV-driven renilla luciferase plasmid was used as a transfection control. Transfected cells were cultured for 48 h and lysed for measurement of luciferase activity.
Fig. 3.
Fig. 3.
TAL1-associated LSD1 complex, HDM, and HDAC activities were subjected to regulation during erythroid differentiation. (A) Nuclear extracts from MEL cells treated with DMSO for the indicated periods of time were analyzed by WB. (B) The fold difference of the expressed proteins in MEL cells was determined from 4 independent experiments using image analysis of exposed X-ray films. (C) The same nuclear extracts were precipitated with TAL1 antibody and analyzed for the presence of LSD1, CoREST, and HDAC1 in the complex. (D) The fold difference of the immunoprecipitated proteins was determined from 3 independent experiments using image analysis of exposed X-ray films by ImageQuant 400. (E) The TAL1 complexes purified from MEL cells treated with DMSO for the indicated times were incubated with 1 μg of chicken core histone in the HDM buffer. The changes of diMeH3K4 and AcH3K9/K14 were analyzed by WB.
Fig. 4.
Fig. 4.
TAL1 recruits LSD1 to the p4.2 promoter in undifferentiated, but not differentiated, MEL cells. (A) Schematic representation of the mouse P4.2 gene locus. The primers are indicated. (B) mRNAs from WT or TAL1-transduced MEL cells treated with 1.5% DMSO were purified and analyzed by Northern blotting using the P4.2 probe. (C and D) Cross-linked chromatin from MEL cells in the presence and absence of 1.5% DMSO was precipitated with TAL1 antibody. The TAL1-chromatin complexes were reversed and the selected DNA was analyzed by PCR (C). (D) TAL1-DNA complexes from the first TAL1 ChIP were subjected to a second IP with anti-LSD1. The cross-links of these double IP protein/DNA complexes were reversed, and the DNA was analyzed by PCR. (E) Cross-linked chromatin from MEL cells in the presence and absence of 1.5% DMSO was precipitated with TAL1 and LSD1 antibodies. The selected chromatin complexes were reversed and analyzed by quantitative PCR. (F) The cross-linked chromatin from MEL cells treated with or without DMSO was precipitated with dimeH3K4 antibody. The precipitated DNA fragments were amplified by using primers specific for −6 Kb, the P4.2 promoter, and 3′ UTR regions by quantitative PCR.
Fig. 5.
Fig. 5.
The KD of LSD1 derepresses the expression of P4.2 gene. (A) Schematic representation of the mouse P4.2 gene locus. The primers are indicated. (B) WB analysis of whole-cell extracts from WT, pSuper vector control, and LSD1 KD MEL cells. (C) The RNA from the pSuper vector or LSD1 KD (shLSD1) MEL cells was isolated and quantitative RT-PCR was performed. (D) The cross-linked chromatin from the vector control and LSD1 KD MEL cells were precipitated with dimeH3K4 antibody. The precipitated DNA fragments were amplified by using primers specific for the −6 Kb, the p4.2 promoter, and 3′ UTR regions.
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