Lysine-specific Demethylase 1 Represses THP-1 Monocyte-to-macrophage Differentiation△

Rui-feng Yang, Guo-wei Zhao, Shu-ting Liang, Hou-zao Chen, and De-pei Liu

National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China

MONOCYTES are generated from bone marrow hematopoietic stem cells, released into peripheral blood, and circulate for couple of days. After migrating into tissues, monocytes give rise to a variety of tissue-dependent macrophages and dendritic cells, which participate in host defense and tissue protection.1Monocyte-to-macrophage differentiation and macrophage activation are two steps to generate functional macrophages. Epigenetic regulation of macrophage activation has been demonstrated. The studies in this area initially focused on NF-κB recruitment to its downstream genes promoters. NF-κB plays key roles in inflammatory gene induction during the process of macrophage activation. Phosphorylation of histone 3 at serine 10 (H3S10) at interleukin (IL)-6, an important macrophage-specific cytokine, and IL-12 promoters have a gene-specific role in NF-κB recruitment.2-4In other studies, lysine-specific demethylase 2 (LSD2) is involved in NF-κB target gene activation by H3K9 demethylation.5Histone demethylase Jmjd3 has been reported to cause H3K27 demethylation and induce inflammatory gene expression.6-8However, the epigenetic regulations of monocyte differentiation into macrophage are largely unclear. Recently, studies on whole genomic histone modification have been performed in monocytes and monocyte-derived macrophages. The results provide a basis for further functional correlations between gene expression and histone modifications.9In addition, Pham et al10found that several macrophage-specific transcription factors were related to epigenetic changes during macrophage differentiation. However, the detailed mechanisms underlying dynamic regulation of the factors that are capable of epigenetic modification need to be investigated further.

This study selectively detected several enzymes (LSD1, LSD2, UTX, JMJD3, JMJD1C, JARID1B, and JHDM2A) involved in histone methylation in THP-1 monocytes and THP-1-derived macrophages. Except LSD1, the others showed no effects on THP-1 monocytes and THP-1-derived macrophages. Therefore the effect of LSD1 on THP-1 monocyte-to-macrophage differentiation and the possible mechanism of the effect was explored.

MATERIALS AND METHODS

Cell culture and treatment

Human monocytic THP-1 cells (provided by Michalle Weiss Laboratory in the Children's Hospital of Philadelphia, PA, USA) were maintained in RPMI 1640 supplemented with 10% fetal bovine serum. The cells were incubated with 200 nmol/L phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) to induce monocyte-to-macrophage differentiation at 0, 4, 8, and 12 hours. THP-1-derived macrophages were obtained by treating THP-1 cells with TPA for 24 hours.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

Total cellular RNA was extracted using RNeasy mini kit (Qiagen, Valencia, CA, USA). Total RNA at 1 ug was reversely transcribed with SuperScript First-Strand synthesis kit (Invitrogen, Carlsbad, CA, USA). qPCR was performed with SYBR green dye in a Real-time PCR system (Bio-Rad, Hercules, CA, USA). The primer pairs for the expression of histone demethylases, including LSD1, LSD2, UTX, JMJD3, JMJD1C, JARID1B, and JHDM2A, are listed in Table 1.

RNA interference and retroviral infection

LSD1 hairpin sequences were designed and synthesized according to LSD1 shRNA sequences on Open Biosystem website. The oligonucleotides were annealed and cloned into PIG retroviral vector.11The recombinant retrovirus was generated by transfecting 293T cells with the recombinant plasmid and helper plasmids pCGP and pVSV-G with Fugene 6 (Roche, Brandford, CT, USA). Spin infection of THP-1 cells with virus was performed as described previously.12The THP-1 cells infected with retrovirus expressing LSD1 shRNAs or luciferase shRNA were named shLSD1-1, shLSD1-2, and shLUC cells respectively. The knockdown efficiency was confirmed with Western blotting. After infection, the stable cell lines were obtained after selection with puromycin for 5 days.

Antibodies

Antibody specific for LSD1 (Cell Signaling Technology, Danvers, MA, USA), antibody against H3K4me2 (Millipore, Billerica, MA, USA), antibody against GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA), and antibody against β-actin (Sigma, St. Louis, MO, USA) were used for Western blotting and chromatin immunoprecipitation (ChIP) assay. Antibody specific for Mac1 (BD Bioscience, San Jose, CA, USA) was used for flow cytometry.

Western blotting

THP-1 cells and the incubated THP-1 cells were lysed in 2×Laemmli sample buffer (Sigma). Proteins were resolved on 12% NuPage Bis-Tris gels using MOPS running buffer (Invitrogen) and transferred to 0.45 umol/L polyvinylidene fluoride membranes (Thermo Scientific, Hudson, NH, USA). The membranes were incubated with specific primary antibodies and horse radish peroxidase-conjugated secondary antibodies. The blots were developed using enhanced chemiluminescence substrate (Thermo Scientific).

ChIP assay

ChIP was performed with Chromatin Immunoprecipitation Kits (Active Motif, Carlsbad, CA, USA) according to manufacturer's instructions. Briefly, cells were fixed after various treatments by TPA. Histone modification H3K4me2 and LSD1 occupancy at IL-6 gene promoter were examined. The results were quantified with qPCR. Signals were normalized to isotype control IgG. Six pairs of primers were employed, which covered 1kb range of IL-6 promoter region. ChIP primer sequences are listed in Table 1.

Flow cytometry

Fluorescence-activated flow cytometry (FACS) was performed with Calibur (BD Bioscience). The antibody specific for Mac1, a macrophage-specific marker, was used to monitor the percentage of macrophages differentiated from THP-1 cells.

Statistical analysis

The experimental results were analyzed using SPSS 13.0. Each experiment was performed for at least 3 times. The quantitative data were expressed as means±SD. Compari- sons were analyzed using two tailed student t-test. One-way analysis of variance was applied to multiple group analysis. P＜0.05 was considered statistically significant.

RESULTS

LSD1 expression during monocyte-to-macrophage differentiation

The results of qRT-PCR showed that only LSD1 was significantly repressed among the candidate histone demethylases (P＜0.01, Fig. 1A). In THP-1 cells treated with TPA at different time points, LSD1 expression decreased in a time-dependent manner at mRNA level (Fig. 1B). The results of Western blotting showed the decreased protein expression of LSD1 after TPA treatment (Fig. 1C). Meanwhile, IL-6 was significantly increased after TPA treatment (Fig. 1B).

LSD1 occupancy at IL-6 promoter during monocyte-to- macrophage differentiation

ChIP assay showed that LSD1 was highly concentrated between -417 bp and 120 bp at IL-6 promoter in THP-1 cells (Fig. 2A). No obvious occupancy sites of LSD1 binding to IL-6 promoter were detected after TPA treatment (Fig. 2B). Increased levels of H3K4 methylation were observed in a time-dependent manner in TPA-treated THP-1 cells at different time points (Fig. 2C).

Effect of LSD1 knockdown on THP-1 monocyte-to- macrophage differentiation

LSD1 was efficiently silenced in both shLSD1-1 and shLSD1-2 cells compared with shLUC cells (Fig. 3A). LSD1 occupancy was significantly decreased and H3K4 methylation increased in shLSD1 cells compared with shLUC cells after TPA treatment at different time points (Fig. 3B and C). IL-6 mRNA expression level was also increased with LSD1 knockdown (Fig. 3D).

More adhesive cells were observed in culture after LSD1 knockdown. According to the results of flow cytometry, the percentage of Mac1+cells in shLUC, shLSD1-1, and shLSD1-2 cells was 12.02%, 20.91%, and 20.49%, respectively, with that in shLUC cells being the lowest (both P＜0.05). That percentage in the 3 types of cells treated with TPA for 8 hours was 27.87%, 34.77%, and 34.95%, respectively (Fig. 4).

Figure 1. Expression of lysine-specific demethylase 1 (LSD1) and interleukin-6 (IL-6) during THP-1 monocyte-to-macrophage differentiation. A. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) result of enzymes capable of histone demethylation in THP-1 monocytes and THP-1-derived macrophages. THP-1-derived macrophages were obtained by treating THP-1 cells with 12-O- tetradecanoylphorbol-13-acetate (TPA) for 24 hours. B. qRT-PCR analysis of IL-6 and LSD1 expression in THP-1 cells treated with TPA for 0, 4, 8, and 12 hours. C. Western blotting of LSD1 in THP-1 cells treated with TPA for 0, 4, 8, and 12 hours. GADPH was used as an internal control. *P＜0.01 compared with THP-1 monocytes； #P＜0.05, ##P＜0.01 compared with THP-1 cells treated with TPA for 0 hour.

Figure 2. Chromatin immunoprecipitation (ChIP) assay results show LSD1 occupancy and H3K4 methylation during THP-1 monocyte- to-macrophage differentiation. A and B. ChIP analysis of LSD1 occupancy at IL-6 promoter in THP-1 cells and THP-1-derived macrophages. THP-1 cells and THP-1-derived macrophages were collected, lysed, and immunoprecipitated with antibody against LSD1 and IgG (negative control). The precipitated protein was then eluted and DNA extracted for PCR analysis. C. ChIP assay of H3K4 dimethylation (H3K4me2) at IL-6 promoter in THP-1 cells upon TPA treatment at different time points, with IgG as a negative control.

Figure 3. Knockdown of LSD increased the expression of IL-6 and the level of H3K4 methylation. A. THP-1 cells were infected with retroviruses expressing shRNA targeting luciferase (shLUC cells) and LSD1 (shLSD1-1 and shLSD1-2 cells). The cells were then collected for analysis of LSD1 expression with Western blotting. β-actin was used as a control. B. ChIP analysis of LSD1 occupancy at IL-6 promoter in TPA-treated shLUC and shLSD1 cells, with IgG as negative control. C. ChIP analysis of H3K4me2 at IL-6 promoter in TPA-treated shLUC and shLSD1 cells, with IgG as negative control. D. qRT-PCR analysis of IL-6 expression in shLUC and shLSD1 cells upon TPA treatment. *P＜0.05, **P＜0.01 compared with shLUC cells.

Figure 4. Fluorescence-activated flow cytometry analysis reveals the percentage of Mac1+ cells in shLUC and shLSD1 cells upon TPA treatment for 0 and 8 hours. The experiment was repeated in three independent times. *P＜0.05 compared with shLUC cells.

DISCUSSION

In the present study, the enzymes capable of histone demethylation during the monocyte-to-macrophage differentiation were analyzed. The results show that only LSD1 expression was repressed, resulting in increased H3K4 methylation at IL-6 promoter. In addition, no change was observed in the expression of H3K9 and H3K27 demethylases, including LSD2 and Jmjd3, which contribute to macrophage activation by decreasing H3K9 methylation at IL-12b promoter and H3K27 methylation at PcG target gene promoters respectively.5,6This finding indicates that monocyte-to-macrophage differentiation requires different histone epigenetic marks to regulate gene expression. IL-6 is one of the important cytokines that is primarily expressed in macrophages and secreted during macroph- age activation.13In this study, during THP-1 monocyte-to- macrophage differentiation, decreased LSD1 occupancy and increased H3K4 methylation at IL-6 promoter were noticed, along with increased IL-6 mRNA level, suggesting that LSD1-mediated H3K4 demethylation blocks the activation of IL-6 transcription before monocyte-to- macrophage differentiation. This is further evidenced by the results that LSD1 knockdown increased H3K4 methylation at IL-6 promoter and released IL-6 transcription. Moreover, knockdown of LSD1 triggers THP-1 monocyte-to-macrophage differentiation and increases the proportion of macrophages even without TPA treatment.

In this study, we reported LSD1 as the first demethylase involved in establishing the epigenetic signature of macrophage- specific genes such as IL-6 during monocyte-to-macrophage differentiation. However, how LSD1 is recruited to or deprived from IL-6 promoter and/or other macrophage-specific promoters is still an open question. This is probably dependent on the recruitment of sequence-specific transcription factors.14-17Pham et al10recently identified the novel, de novo-derived, macrophage-specific enhancer signature which associates with epigenetic changes. These enhancer elements include a subset of motif corresponding transcription factors PU.1, C/EBPβ, and EGR2, which may provide clues to the factors related to LSD1 recruitment to macro- phage-specific gene promoters. In conclusion, H3K4 methy- lation is established along with repression of LSD1 expression at IL-6 promoter during THP-1 monocyte-to- macrophage differentiation. Knockdown of LSD1 facili- tates THP-1 monocyte-to-macrophage differentiation by increasing the level of H3K4 methylation.

1. Volkman A, Gowans JL. The origin of macrophages from bone marrow in the rat. Br J Exp Pathol 1965； 46:62-70.

2. Saccani S, Pantano S, Natoli G. p38-Dependent marking of inflammatory genes for increased NF-kappa B recruitment. Nat Immunol 2002； 3:69-75.

3. Anest V, Hanson JL, Cogswell Pc, et al. A nucleosomal function for IkappaB kinase-alpha in NF-kappaB-dependent gene expression. Nature 2003； 423:659-63.

4. Yamamoto Y, Verma UN, Prajapati S, et al. Histone H3 phosphorylation by IKK-alpha is critical for cytokine-induced gene expression. Nature 2003； 423:655-9.

5. van Essen D, Zhu Y, Saccani S. A feed-forward circuit controlling inducible NF-kappaB target gene activation by promoter histone demethylation. Mol Cell 2010； 39: 750-60.

6. De Santa F, Totaro MG, Prosperini E, et al. The histone H3 lysine-27 demethylase Jmjd3 links inflammation to inhibition of polycomb-mediated gene silencing. Cell 2007； 130:1083-94.

7. Das ND, Jung KH, Choi MR, et al. Gene networking and inflammatory pathway analysis in a JMJD3 knockdown human monocytic cell line. Cell Biochem Funct 2012； 30:224-32.

8. De Santa F, Narang V, Yap ZH, et al. Jmjd3 contributes to the control of gene expression in LPS-activated macroph- ages. EMBO J 2009； 28:3341-52.

9. Tserel L, Kolde R, Rebane A, et al. Genome-wide promoter analysis of histone modifications in human monocyte- derived antigen presenting cells. BMC Genomics 2010； 11:642.

10. Pham TH, Benner C, Lichtinger M, et al. Dynamic epigenetic enhancer signatures reveal key transcription factors associated with monocytic differentiation states. Blood 2012； 119:e161-71.

11. Hemann MT, Fridman JS, Zilfou JT, et al. An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo. Nat Genet 2003； 33:396-400.

12. Yu D, dos Santos CO, Zhao G, et al. miR-451 protects against erythroid oxidant stress by repressing 14-3-3zeta. Genes Dev 2010； 24:1620-33.

13. Sica A, Schioppa T, Mantovani A, et al. Tumour-associated macrophages are a distinct M2 polarised population promoting tumour progression: potential targets of anti-cancer therapy. Eur J Cancer 2006； 42:717-27.

14. Saleque S, Kim J, Rooke HM, et al. Epigenetic regulation of hematopoietic differentiation by Gfi-1 and Gfi-1b is mediated by the cofactors CoREST and LSD1. Mol Cell 2007； 27:562-72.

15. Yamane K, Toumazou C, Tsukada Y, et al. JHDM2A, a JmjC-containing H3K9 demethylase, facilitates transcrip- tion activation by androgen receptor. Cell 2006； 125: 483-95.

16. Metzger E, Wissmann M, Yin n, et al. LSD1 demethylates repressive histone marks to promote androgen-receptor- dependent transcription. Nature 2005； 437:436-9.

17. Wissmann M, Yin N, Müller JM, et al. Cooperative demethylation by JMJD2C and LSD1 promotes androgen receptor-dependent gene expression. Nat Cell Biol 2007； 9:347-53.