Transcription Factor Homeobox D9 Drives the Malignant Phenotype of HPV18-Positive Cervical Cancer Cells via Binding to the Viral Early Promoter

Persistent infections with two types of human papillomaviruses (HPV), HPV16 and HPV18, are the most common cause of cervical cancer (CC). Two viral early genes, E6 and E7, are associated with tumor development, and expressions of E6 and E7 are primarily regulated by a single viral promoter: P97 in HPV16 and P105 in HPV18. We previously demonstrated that the homeobox D9 (HOXD9) transcription factor is responsible for the malignancy of HPV16-positive CC cell lines via binding to the P97 promoter. Here, we investigated whether HOXD9 is also involved in the regulation of the P105 promoter using two HPV18-positive CC cell lines, SKG-I and HeLa. Following the HOXD9 knockdown, cell viability was significantly reduced, and E6 expression was suppressed and was accompanied by increased protein levels of P53, while mRNA levels of TP53 did not change. E7 expression was also downregulated and, while mRNA levels of RB1 and E2F were unchanged, mRNA levels of E2F-target genes, MCM2 and PCNA, were decreased, which indicates that the HOXD9 knockdown downregulates E7 expression, thus leading to an inactivation of E2F and the cell-cycle arrest. Chromatin immunoprecipitation and promoter reporter assays confirmed that HOXD9 is directly associated with the P105 promoter. Collectively, our results reveal that HOXD9 drives the HPV18 early promoter activity to promote proliferation and immortalization of the CC cells.

Human papillomavirus 16 L1 gene methylation as a potential biomarker for predicting anal intraepithelial neoplasia in men who have sex with men (MSM)

The human papillomavirus (HPV) 16 early promoter and L1 gene methylation were quantitatively measured using pyrosequencing assay in anal cells collected from men who have sex with men (MSM) to determine potential biomarkers for HPV-related anal cancer. The methylation patterns of HPV16 genes, including the early promoter (CpG 31, 37, 43, 52, and 58) and L1 genes (CpG 5600, 5606, 5609, 5615, 7136, and 7145), were analyzed in 178 anal samples. The samples were diagnosed as normal, anal intraepithelial neoplasia (AIN) 1, AIN2, and AIN3. Low methylation levels of the early promoter (< 10%) and L1 genes (< 20%) were found in all detected normal anal cells. In comparison, medium to high methylation (≥ 20–60%) in the early promoter was found in 1.5% (1/67) and 5% (2/40) of AIN1 and AIN2-3 samples, respectively. Interestingly, slightly increased L1 gene methylation levels (≥ 20–60%), especially at the HPV16 5’L1 regions CpGs 5600 and 5609, were demonstrated in AIN2-3 specimen. Moreover, a negative correlation between high HPV16 L1 gene methylation at CpGs 5600, 5609, 5615, and 7145 and a percentual CD4 count was found in AIN3 HIV positive cases. When comparing the methylation status of AIN2-3 to that of normal/AIN1 lesions, the results indicated the potential of using HPV16 L1 gene methylation as a biomarker for HPV-related cancer screening.

A pioneer transcription factor and type I nuclear hormone receptors synergistically activate the bovine herpesvirus 1 infected cell protein 0 (ICP0) early promoter.

Following bovine herpesvirus 1 (BoHV-1) acute infection of ocular, oral or nasal cavities, sensory neurons within trigeminal ganglia are an important site for latency. Stress, as mimicked by the synthetic corticosteroid dexamethasone, consistently induces reactivation from latency. Expression of two key viral transcriptional regulatory proteins, infected cell protein 0 (bICP0) and bICP4, are regulated by sequences within the immediate early promoter (IEtu1). A separate early promoter also drives bICP0 expression, presumably to ensure sufficient levels of this important transcriptional regulatory protein. Productive infection and bICP0 early promoter activity are cooperatively transactivated by Krüppel like factor 4 (KLF4) and a Type I nuclear hormone receptor (NHR), androgen receptor, glucocorticoid receptor, or progesterone receptor. The bICP0 early promoter contains 3 separate transcriptional enhancers that mediate cooperative transactivation. In contrast to the IEtu1 promoter, the bICP0 early promoter lacks consensus Type I NHR binding sites. Consequently, we hypothesized KLF4 and Sp1 binding sites are essential for Type I NHR and KLF4 to transactivate the bICP0 promoter. Mutating KLF4 and Sp1 binding sites in each enhancer domain significantly reduced transactivation by KLF4 and a Type I NHR. Chromatin immunoprecipitation (ChIP) studies demonstrated occupancy of bICP0 early promoter sequences by KLF4 and Type I NHR is significantly reduced when KLF4 and/or Sp1 binding sites were mutated. These studies suggest cooperative transactivation of the bICP0 E promoter by Type I NHRs and a stress induced pioneer transcription factor (KLF4) promote viral replication and spread in neurons or non-neural cells in reproductive tissue. IMPORTANCE Understanding how stressful stimuli and changes in cellular milieu mediate viral replication and gene expression in the natural host is important for developing therapeutic strategies that impair virus transmission and disease. For example, bovine herpesvirus 1 (BoHV-1) reactivation from latency is consistently induced by the synthetic corticosteroid dexamethasone, which mimics the effects of stress. Furthermore, BoHV-1 infection increases the incidence of abortion in pregnant cows suggesting sex hormones stimulate viral growth in certain tissue. Previous studies revealed Type I nuclear hormone receptors (androgen, glucocorticoid, or progesterone) and the pioneer transcription factor, Krüppel like factor 4 (KLF4), cooperatively transactivate the BoHV-1 infected cell protein 0 (bICP0) early promoter. Transactivation was mediated by Sp1 and/or KLF4 consensus binding sites within the 3 transcriptional enhancers. These studies underscore the complexity by which BoHV-1 exploits Type I NHR fluctuations to enhance viral gene expression, replication, and transmission in the natural host.

Potential biomarker of human papillomavirus 16 L1 methylation for prediction of anal intraepithelial neoplasia in men who have sex with men (MSM)

Quantitative measurement of human papillomavirus (HPV) 16 early promoter and L1 genes methylation were analyzed in anal cells collected from men who have sex with men (MSM) to determine the potential biomarker for screening of HPV related anal cancer. The methylation patterns of the HPV16 genes including early promoter (CpG 31, 37, 43, 52 and 58) and L1 gene (CpG 5600, 5606, 5609, 5615, 7136 and 7145) were analyzed in 178 anal samples with histology diagnosed as normal, anal intraepithelial neoplasia (AIN) 1, AIN2 and AIN3 by pyrosequencing assay. Low methylation levels of early promoter (<10%) and L1 genes (<20%) were found in all detected normal anal cells, while medium to high methylation (>20-60%) in early promoter was found 1.5% (1/67) and 5% (2/40) in AIN1 and AIN2-3 samples, respectively. Interestingly, slightly increased L1 gene methylation level (>20-60%) especially at HPV16 5'L1 regions CpGs 5600 and 5609 from normal to AIN3 were demonstrated. Moreover, negative correlation between high HPV16 L1 gene methylation at CpGs 5600, 5609, 5615 and 7145 and low percentage of CD4+ was found in AIN3 HIV positive cases. When compared methylation status of AIN2-3 to those of the normal/AIN1 lesion, the results indicated potential of using HPV16 L1 gene methylation as a biomarker for HPV related cancer screening.