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. 2015 Feb 6;10(2):e0117773.
doi: 10.1371/journal.pone.0117773. eCollection 2015.

Identification of transcription factor AML-1 binding site upstream of human cytomegalovirus UL111A gene

Xiaoqun Zheng  1 , Yan Gao  2 , Qi Zhang  3 , Yanqing Liu  3 , Ying Peng  4 , Miao Fu  5 , Yanhong Ji  6
Affiliations

Identification of transcription factor AML-1 binding site upstream of human cytomegalovirus UL111A gene

Xiaoqun Zheng et al. PLoS One. .

Abstract

Human cytomegalovirus (HCMV) interleukin-10 (hcmvIL-10), encoded by HCMV UL111A gene, is a homolog of human IL-10. It exerts immunomodulatory effects that allow HCMV to evade host defense mechanisms. However, the exact mechanism underlying the regulation of hcmvIL-10 expression is not well understood. The transcription factor acute myeloid leukemia 1 (AML-1) plays an important role in the regulation of various genes involved in the differentiation of hematopoietic lineages. A putative AML-1 binding site is present within the upstream regulatory region (URR) of UL111A gene. To provide evidence that AML-1 is involved in regulating UL111A gene expression, we examined the interaction of AML-1 with the URR of UL111A in HCMV-infected human monocytic THP-1 cells using a chromatin immunoprecipitation assay. HcmvIL-10 transcription was detected in differentiated THP-1 cells, but not in undifferentiated ones. Furthermore, the URR of UL111A showed a higher intensity of AML-1 binding, a higher level of histone H3 acetyl-K9, but a lower level of histone H3 dimethyl-K9 in differentiated THP-1 cells than undifferentiated cells. Down-regulation of AML1 by RNA interference decreased the expression of the UL111A gene. Our results suggest that AML-1 may contribute to the epigenetic regulation of UL111A gene via histone modification in HCMV-infected differentiated THP-1 cells. This finding could be useful for the development of new anti-viral therapies.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. HCMV genome maintenance and transcription in infected THP-1 cells.
(A) Microscopic examination of THP-1 cells infected with HCMV-Towne and grown in suspension (original magnification ×200). (B) Real-time qPCR analysis revealed the maintenance but lack of accumulation of the HCMV genome in infected THP-1 cells. Each sample was analyzed in triplicate. (C) Expression of HCMV transcripts (UL123, UL83, and LUNA mRNA) in infected THP-1 cells over a 10-day course of infection analyzed by RT-PCR. RT− represents RNA without prior reverse transcription, employed as a negative control. Dpi: day post infection.
Fig 2
Fig 2. Induction of viral transcription in infected THP-1 cells with PMA treatment.
(A) Microscope image showing differentiation of HCMV-infected THP-1 cells into an adherent and macrophage-like phenotype following stimulation with PMA (100 ng/μl) (original magnification ×200). (B) Viral DNA loads over time in HCMV-infected THP-1 cells following PMA treatment. Each sample was assayed in triplicate by qPCR. (C) Expression of viral transcripts in PMA-treated THP-1 cells infected with HCMV. RT− represents the RNA without prior reverse transcription, employed as a negative control.
Fig 3
Fig 3. Transcription of the UL111A gene and expression of transcription factors in PMA-treated and untreated THP-1 cells infected with HCMV.
(A) Expression levels of the hcmvIL-10 and LAcmvIL-10 transcripts in differentiated and undifferentiated HCMV-infected THP-1 cells, analyzed by RT-PCR. (B) Quantitative analysis of the data in (A) from three independent experiments. RNA transcripts were normalized to cellular GAPDH. (C) Western blots showing the expression level of AML-1, GATA-1, and C/EBP β proteins in differentiated and undifferentiated HCMV-infected THP-1 cells. (D) Quantitative analysis of the data in (C) from three independent experiments. Proteins were normalized to cellular β-actin. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 4
Fig 4. Identification of transcription factor binding sites in the URR of the UL111A gene.
(A) Potential binding sites for the myeloid transcription factors AML-1, C/EBP β, and GATA-1 in the URR of the UL111A gene predicted using the MATCH program and TRANSFAC database. (B) ChIP analysis of three transcription factors binding to their corresponding sites. The lanes are designated as: “input”—PCR amplification of input DNA, “anti-TF”—PCR amplification of chromatin DNA fragments precipitated by antibodies against transcription factors, “isotype”—a control for non-specific reactions. (C) Comparison of transcription factor binding between undifferentiated and differentiated THP-1 cells. Quantitative analysis of the data is from three independent experiments. *** P < 0.001.
Fig 5
Fig 5. Epigenetic modification status in the URR of UL111A from undifferentiated and differentiated HCMV-infected THP-1 cells.
(A) ChIP analysis showing the association of the transcription factors binding sites with histone H3 acetylated at K9 position (H3 acetyl-K9) and dimethylated at K9 position (H3 dimethyl-K9). The lanes are designated as: “input”—PCR amplification of input DNA, “anti-acetyl”—PCR amplification of the chromatin DNA fragments precipitated by antibody against H3 acetyl-K9, “anti-dimethyl”—PCR-amplification of the chromatin DNA fragments precipitated by antibody against H3 dimethyl-K9, “isotype”—a negative control for non-specific reactions. (B) Quantitative analysis of the data in (A) from three independent ChIP experiments. The results were normalized to the input signal, which was set to 1. (C) Methylation status of the CpG sites in the URR of the UL111A gene in undifferentiated and differentiated infected THP-1 cells infected with HCMV. The methylation status of individual CpG dinucleotides is indicated as: ○ and ●, which represent unmethylated and methylated cytosine, respectively. (D) Quantitative analysis of the total methylation data in (C) from three independent BSP experiments. **P < 0.01.
Fig 6
Fig 6. AML-1 suppression affects the UL111A gene expression.
(A) FACS analysis of transfection efficiency. The infection efficiency was measured by FACS analysis in HCMV-infected THP-1 cells transfected with LV-shRNA-AML1 or LV-shRNA-NC at 72 h post-transfection. (B) Western blot was detected in HCMV-infected THP-1 cells transfected with LV-shRNA-AML1 or LV-shRNA-NC. **P<0.01 Verse LV-shRNA-NC. (C) HCMV-infected THP-1 cells stimulated with PMA were transfected with LV-shRNA-AML1 or LV-shRNA-NC for 72h. RT-PCR was taken to analyze the change of hcmvIL-10. *P<0.05. Verse LV-shRNA-NC.
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