Campos-Leon, K. | Search bioRxiv

bioRxiv2022

The guardian of the genome meets a viral master gene regulator at a biomolecular condensate

As guardian of the genome, p53 exerts its tumor suppressor activity by modulating the expression of several hundreds of genes and by interacting with a large number of proteins. However, p53 can also repress viral replication and it is targeted by a variety of viral proteins to allow viral replication to proceed. p53 can repress human papillomavirus replication by binding to the viral E2 master gene regulator. Here we show how full-length p53 can spontaneously form phase separated liquid-like droplets that evolve to amyloid-like aggregates in a time-dependent manner, highlighting the fact that homotypic condensation is on the path to aggregation as observed in several protein aggregopathies. The DNA binding domain of HPV E2 (E2C) triggers heterotypic liquid-liquid phase separation with p53 with a precise 1 p53 : 2 E2C stoichiometry at the onset for demixing, yielding large regular spherical droplets that increase in size with E2C concentration. Moreover, E2C is able to slowly reshape time-evolved p53 aggregates into regular heterotypic liquid droplets. Using in situ sub-cellular fractionation, we show that E2 and wild-type p53 co-localize to the nucleus with a grainy pattern, and E2 can re-localize p53 into chromatin associated foci, a function independent of the DNA binding capacity of p53. A small DNA duplex containing the specific binding site for p53 deforms and dissolves both homotypic and heterotypic condensates at a 1 p53 : 1 DNA stoichiometry, whereas a [~]1000 base pair DNA fragment instead reshaped the condensates into distinct amorphous condensates containing p53, E2C and DNA, reminiscent of what we observe bound to chromatin. We conclude that p53 is a scaffold for liquid-liquid phase separation in line with its structural and functional features, in particular as a hub that binds multiple cellular protein partners as well as nucleic acids. Moreover, the capacity of E2C to rescue p53 from the amyloid aggregation route impacts on p53-rescuing drugs cancers where p53 mutation leads to loss of function.

biophysics↗

bioRxiv2021

The chromatin insulator CTCF regulates HPV18 transcript splicing and differentiation-dependent late gene expression

The ubiquitous host protein, CCCTC-binding factor (CTCF), is an essential regulator of cellular transcription and functions to maintain epigenetic boundaries, stabilise chromatin loops and regulate splicing of alternative exons. We have previously demonstrated that CTCF binds to the E2 open reading frame (ORF) of human papillomavirus (HPV) 18 and functions to repress viral oncogene expression in undifferentiated keratinocytes by co-ordinating an epigenetically repressed chromatin loop within HPV episomes. Cellular differentiation, which is necessary for HPV life cycle completion disrupts CTCF-dependent chromatin looping of HPV18 episomes inducing enhanced activity of the HPV18 early promoter P105 and increased viral oncogene expression. To further characterise CTCF function in HPV transcription control we utilised direct, long-read Nanopore RNA-sequencing which provides information on the structure and abundance of full-length transcripts. Nanopore analysis of primary human keratinocytes containing HPV18 episomes before and after synchronous differentiation allowed quantification of viral transcript species in these cultures, including the identification of low abundance novel transcripts. Comparison of transcripts produced in wild type HPV18 genome-containing cells to those identified in CTCF-binding deficient genome-containing cells (HPV18-{Delta}CTCF) identifies CTCF as a key regulator of differentiation-dependent late promoter activation, required for efficient E1^E4 and L1 protein expression. Furthermore, our data show that CTCF binding at the E2 ORF of HPV18 promotes usage of the downstream weak splice donor (SD) sites SD3165 and SD3284, to the dominant E4 splice acceptor site at nucleotide 3434. These findings demonstrate importance of CTCF-dependent transcription regulation at multiple stages of the HPV life cycle. IMPORTANCEOncogenic human papillomavirus (HPV) infection is the cause of a subset of epithelial cancers of the uterine cervix, other anogenital areas and the oropharynx. HPV infection is established in the basal cells of epithelia where a restricted programme of viral gene expression is required for replication and maintenance of the viral episome. Completion of the HPV life cycle is dependent on the maturation (differentiation) of infected cells which induces enhanced viral gene expression and induction of capsid production. We previously reported that the host cell transcriptional regulator, CTCF, is hijacked by HPV to control viral gene expression. In this study, we use long-read mRNA sequencing to quantitatively map the variety and abundance of HPV transcripts produced in early and late stages of the HPV life cycle and to dissect the function of CTCF in controlling HPV gene expression and transcript processing.

microbiology↗