| Backgroud: LSD1,the first well-characterized H3K4 demethylase, plays an important role in hematopoiesis. It can specifically catalyzes the demethylation of mono- and di-methylated histone H3 lysine 4 through a FAD-dependent oxidative reaction. LSD1 can regulate the expression of target genes by occupation with co-regulators such as TAL1. Transcription factors form an elaborate regulatory network to maintain homeostasis within the hematopoietic system. We hypothesize that LSD1 mediated epigenetic modification regulates the switching of GATA factors during hematopoietic differentiation. It has been reported that GATA2 is expressed in hematopoietic stem cells(HSCs) and its expression is gradually decreased upon erythroid differentiation, while GATA1 is absent in HSC stage and is upregulated upon differentiation. The interplay between GATA1 and GATA2 is critical for maintaining homeostasis of cells during HSC differentiation. GATA1 can regulate the expression of GATA2, while GATA2 can also activate GATA1’s expression in early stage of erythroid differentiation.Objective: From a new perspective, we expected to explore the effect of histone modification during hematopoietic differentiation. We hoped to make further explanation about the mechanism for regulation of erythroid differentiation. GATA1 and GATA2 both play important roles in hematopoietic. And epigenetic modification mediated by LSD1 regulates expression of multiple target genes. Through inducing erythroid differentiation by using K562 and MEL cell line, we analysis the influence of epigenetic modification mediated by LSD1 on regulation of GATA switch mechanisms, as well as the function of LSD1 in hematopoietic differentiation. We studied GATA switch from the perspective of the histone modification, which helps us further explore the hematopoietic system differentiation process. The study of GATA switch mechanism provided theoretical basis and a new research direction for us. In this study we clarified epigenetic changes in diseases including leukemia and laid a foundation for the molecular mechanism of gene transcription regulation.Method: We identified components of LSD1 complexs interacting with GATA2 by immunopresipitation(IP). We compared the interaction among LSD1, GATA2, TAL1 in early stage and late stage of erythroid differentiation. To characterize which domain of LSD1 was involved in the interaction with GATA2, LSD1’s truncated forms were expressed as GST fusion proteins(GST-LSD1N-term, GST-LSD1N-term+SWIRM, GST-LSD1 SWIRM, GST-LSD1 AO, GST-LSD1SWIRM+AO). We employed GST-pull down assay to characterize which domain of LSD1 was involved in the interaction with GATA2. We also tested the influence of LSD1-KD on expression of GATA1 and GATA2. Chromatin- Immunoprecipitation(Ch IP) assay was performed to demonstrate the binding sites of LSD1 during hematopoiesis differentiation. LSD1 regulated erythroid differentiation through an epigenetic mechanism.Results: We employed GST-pull down assay, immunopresipitation(IP) and chromatin immunopresipitation(Ch IP) to confirm SWIRM domain of LSD1 determined interaction between LSD1 and GATA2. We successfully investigated that GATA2 interacted with LSD1 complex and targeted LSD1 to the GATA binding sites of GATA1 gene, which can repress GATA1 in early stage of hematopoiesis. The inhibition of GATA1 expression upon erythroid differentiation is dependent on LSD1 and its mediated histone demethylation. Upon erythroid differentiation, the interaction between GATA2 and LSD1 was decreased, consistent with a de-repression of GATA1 expression. Meanwhile, the interaction between TAL1 and LSD1 was increased, which forms a complex that efficiently suppressed GATA2 expression. In addition, we understood the functional effects of LSD1 during erythroid differentiation and found that LSD1 ablation impairs HSCs homeostasis and erythroid differentiation by disrupting the normal expression pattern of GATA1 and GATA2.Conclusion: The experiments have provided a new insight into epigenetic mechanism by which LSD1 regulates the GATA switch during hematopoiesis. Our study revealed a key role of LSD1 in regulating hematopoietic differentiation. |
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