Long-distance interaction of the integrated HPV fragment with MYC gene and 8q24.22 region upregulating the allele-specific MYC expression in HeLa cells

Abstract

Human papillomavirus (HPV) infection is the most important risk factor for cervical cancer development. In HeLa cell line, the HPV viral genome is integrated at 8q24 in one allele of chromosome 8. It has been reported that the HPV fragment integrated in HeLa genome can cis-activate the expression of proto-oncogene MYC, which is located at 500 kb downstream of the integrated site. However, the underlying molecular mechanism of this regulation is unknown. A recent study reported that MYC was highly expressed exclusively from the HPV-integrated haplotype, and a long-range chromatin interaction between the integrated HPV fragment and MYC gene has been hypothesized. In this study, we provided the experimental evidences supporting this long-range chromatin interaction in HeLa cells by using Chromosome Conformation Capture (3C) method. We found that the integrated HPV fragment, MYC and 8q24.22 was close to each other and might form a trimer in spatial location. When knocking out the integrated HPV fragment or 8q24.22 region from chromosome 8 by CRISPR/Cas9 system, the expression of MYC reduced dramatically in HeLa cells. Interestingly, decreased expression was only observed in three from eight cell clones, when only one 8q24.22 allele was knocked out. Functionally, HPV knockout caused senescence-associated acidic β-gal activity in HeLa cells. These data indicate a long-distance interaction of the integrated HPV fragment with MYC gene and 8q24.22 region, providing an alternative mechanism relevant to the carcinogenicity of HPV integration.

Keywords: HPV; MYC; cervical cancer; integration; long-distance interaction.

Figure 1

Physical proximity of HPV fragment and MYC gene detected by 3C assays. (a) Schematic diagram of 3C procedure. (b) HPV‐MYC ligation fragments detected by 3C assays. The PCR products were obtained from cross‐linked HeLa cells with three pairs of primers: HPV F + MYC F(410 bp), HPV F + MYC R(506 bp) and HPV R + MYC R(266 bp), respectively (upper). The sequencing of these fragments identified HPV‐MYC piecing sequences mediated by TaqI enzyme site (lower). ACTB‐MYC ligation(320 bp) and ACTB gene(277 bp) were used as specificity and loading control, respectively. Three independent experiments were performed for each 3C assay. M, DNA Marker. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

Physical proximity of HPV‐8q24.22 and MYC‐8q24.22 detected by 3C assays. (a) HPV‐8q24.22 ligation fragments were amplified and sequenced in cross‐linked HeLa cells using primer pairs of HPV F + Alu(430 bp) (upper) and HPV F + 8q24.22 F (450 bp) (lower). (b) MYC‐8q24.22 ligation fragments were amplified and sequenced as (A) by using primers of MYC R + 8q24.22 F (376 bp), MYC R + 8q24.22 R (476 bp). ACTB‐MYC ligation and ACTB gene were used as specificity and loading control, respectively. Three independent experiments were performed for each 3C assay. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

Long‐distance interaction among HPV, MYC and 8q24.22 via constituting a trimer in spatial location. (a) Proposed structure of the chromosome 8 locus containing HPV‐18 integration in HeLa cells. The top panel represented the rearranged haplotype with HPV‐18 insertion on chromosome 8q24.2 region. The bottom panel showed the partial HPV‐18 genome and corresponding genes. Red arrows referred to the location of two designed gRNAs in HPV genome, which were expected to cut about a 4,120 bp fragment. Blue arrows represented the primers used to detect the knockout HPV fragment. (b) HPV knock out in HeLa single clone strains detected by PCR assay (left panel) and DNA sequencing (right panel). (c) Effects of HPV knockout on the spatial co‐location of MYC locus and 8q24.22 region. The 3C assays were performed by using primers of MYC R and 8q24.22 F. (d) 8q24.22 double knockout in HeLa single clone strains detected by PCR assay (left panel) and DNA sequencing (right panel). (e) Effects of 8q24.22 double knockout on the spatial co‐location of integrated HPV and MYC locus. The 3C assays were performed by using primers of HPV F and MYC F. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

Effect of HPV and 8q24.22 knock‐out on MYC expression. (a, b) The efficiency of HPV DNA knockout in two HPV‐ko HeLa cell strains detected by southern blot analysis (a) and real‐time PCR (b). (c) Relative expression of MYC in HPV‐ko HeLa cell strains detected by Real‐time PCR. (d) Relative expression of MYC in 8q24.22 double (#1) or single (#2–9) knock‐out HeLa cell strains detected by Real‐time PCR. (e) The heterozygous SNP (rs4645948, T/C) in MYC DNA and cDNA were detected by PCR and sequencing in wild‐type, HPV‐ko and 8q24.22‐ko HeLa cells. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5

Effect of HPV and 8q24.22 knockout on senescence of HeLa cells. (a) Senescence‐associated acidic β‐gal activity detected by Cell Senescence Assay. Wild‐type HeLa cells and HeLa cells with overexpressed SOX6 were detected as negative and positive controls, respectively. The scale value is 400 μm. (b) The quantified analysis of senescence phenotype in four type of HeLa cells shown in 5a. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 6

Schematic for the long distance regulation among the integrated HPV fragment, MYC and 8q24.22 region.