The haplotype-resolved genome and epigenome of the aneuploid HeLa cancer cell line

Abstract

The HeLa cell line was established in 1951 from cervical cancer cells taken from a patient, Henrietta Lacks. This was the first successful attempt to immortalize human-derived cells in vitro. The robust growth and unrestricted distribution of HeLa cells resulted in its broad adoption--both intentionally and through widespread cross-contamination--and for the past 60 years it has served a role analogous to that of a model organism. The cumulative impact of the HeLa cell line on research is demonstrated by its occurrence in more than 74,000 PubMed abstracts (approximately 0.3%). The genomic architecture of HeLa remains largely unexplored beyond its karyotype, partly because like many cancers, its extensive aneuploidy renders such analyses challenging. We carried out haplotype-resolved whole-genome sequencing of the HeLa CCL-2 strain, examined point- and indel-mutation variations, mapped copy-number variations and loss of heterozygosity regions, and phased variants across full chromosome arms. We also investigated variation and copy-number profiles for HeLa S3 and eight additional strains. We find that HeLa is relatively stable in terms of point variation, with few new mutations accumulating after early passaging. Haplotype resolution facilitated reconstruction of an amplified, highly rearranged region of chromosome 8q24.21 at which integration of the human papilloma virus type 18 (HPV-18) genome occurred and that is likely to be the event that initiated tumorigenesis. We combined these maps with RNA-seq and ENCODE Project data sets to phase the HeLa epigenome. This revealed strong, haplotype-specific activation of the proto-oncogene MYC by the integrated HPV-18 genome approximately 500 kilobases upstream, and enabled global analyses of the relationship between gene dosage and expression. These data provide an extensively phased, high-quality reference genome for past and future experiments relying on HeLa, and demonstrate the value of haplotype resolution for characterizing cancer genomes and epigenomes.

Figure 1. Haplotype-resolved copy number of the HeLa cancer cell line genome

a. Copy-number profile of HeLa split by haplotypes (red = A, blue = B). Links denote likely contiguity and tandem duplications. Boxes indicate marker chromosomes identified by copy number breakpoints (boxes colored by haplotype, or black for unknown); ‘S’ indicates links confirmed by mate-pair sequencing and italics indicate uncertain locations. b. Windowed copy number ratios for HeLa CCL-2 (green and purple, alternating chromosomes) and HeLa S3 (gray), with predicted integer copy number for S3 (black). Notable strain differences are indicated by red arrows (e.g., reduced copy over chromosome 18q). The window containing the HPV insertion and rearrangement is at elevated copy in both strains.

Figure 2. HeLa HPV integration locus

a. Chromosome 8 read depth flanking the HPV integration site (top, blue line), windowed copy-number ratios (purple points) and integer copy states (red bars, middle), and corresponding segments and breakpoints (circled numbers with genomic coordinates, bottom). b. Proposed HPV integration structure: per-segment copy number (upper left), non-rearranged haplotype B (CN=1, upper right), rearranged haplotype A with HPV insertion (CN=3, bottom) carrying ~3 and ~6 tandem copies of repeats ‘R1’ and ‘R2’ respectively. c. The partial HPV-18 genome and corresponding genes (gray and blue, top) with breakpoints highlighted by numbered circles. For reference, the entire HPV-18 genome is shown (bottom).

Figure 3. Gene expression by copy number and haplotype in HeLa S3

a. Transcript abundance (reads per kilobase per million, RPKM, for genes with an RPKM ≥ 1) is positively correlated with gene copy. b. Expression per copy (RPKM / gene copy-number) does not correlate with copy number. c. Fractional contribution of haplotype A to overall expression (RPKM averaged across megabase windows at phased sites) split by haplotype-resolved copy number. Open circles indicate expected fractions. d. Haplotype A-specific expression in HeLa S3 but not CCL-2 across S3-specific LOH on chr18q. e. Haplotype A fractional contribution to expression across the genome, color-coded by underlying haplotype-resolved copy number as in c (point size represents the log₂ total RPKM, gray boxes indicate HeLa S3 LOH).

Figure 4. Haplotype-specific regulation near the HPV integration site

a. Long-range chromatin interactions between the HPV and MYC loci demonstrated by ChIA-PET with the RNA polymerase II signal (top) shown for HeLa S3 and an HPV⁻ cell line (K562). Chromatin interactions (below) are highlighted with a green arrow. Bar graphs show read counts at phased, informative sites in MYC (red = A, blue = B). b. Transcript abundance in HeLa S3 across the MYC locus measured by RNA-Seq. Overall coverage is shown in gray (top) with phased, informative sites highlighted (green ticks; italic indicates non-reference allele). Haplotype contributions at each variant are shown at bottom, as in a.