Joanna L. Parish

The DNA helicase ChlR1 is required for sister chromatid cohesion in mammalian cells

It has recently been suggested that the Saccharomyces cerevisiae protein Chl1p plays a role in cohesion establishment. Here, we show that the human ATP-dependent DNA helicase ChlR1 is required for sister chromatid cohesion in mammalian cells. Localization studies show that ChlR1 diffusely coats mitotic chromatin in prophase and then translocates from the chromatids to concentrate at the spindle poles during the transition to metaphase. Depletion of ChlR1 protein by RNA interference results in mitotic failure with replicated chromosomes failing to segregate after a pro-metaphase arrest. We show that depletion also results in abnormal sister chromatid cohesion, determined by increased separation of chromatid pairs at the centromere. Furthermore, biochemical studies show that ChlR1 is in complex with cohesin factors Scc1, Smc1 and Smc3. We conclude that human ChlR1 is required for sister chromatid cohesion and, hence, normal mitotic progression. These functions are important to maintain genetic fidelity.

E2 proteins from high- and low-risk human papillomavirus types differ in their ability to bind p53 and induce apoptotic cell death

ABSTRACT The E2 proteins from oncogenic (high-risk) human papillomaviruses (HPVs) can induce apoptotic cell death in both HPV-transformed and non-HPV-transformed cells. Here we show that the E2 proteins from HPV type 6 (HPV6) and HPV11, two nononcogenic (low-risk) HPV types, fail to induce apoptosis. Unlike the high-risk HPV16 E2 protein, these low-risk E2 proteins fail to bind p53 and fail to induce p53-dependent transcription activation. Interestingly, neither the ability of p53 to activate transcription nor the ability of p53 to bind DNA, are required for HPV16 E2-induced apoptosis in non-HPV-transformed cells. However, mutations that reduce the binding of the HPV16 E2 protein to p53 inhibit E2-induced apoptosis in non-HPV-transformed cells. In contrast, the interaction between HPV16 E2 and p53 is not required for this E2 protein to induce apoptosis in HPV-transformed cells. Thus, our data suggest that this high-risk HPV E2 protein induces apoptosis via two pathways. One pathway involves the binding of E2 to p53 and can operate in both HPV-transformed and non-HPV-transformed cells. The second pathway requires the binding of E2 to the viral genome and can only operate in HPV-transformed cells.

The histone deacetylase inhibitor LBH589 inhibits expression of mitotic genes causing G2/M arrest and cell death in head and neck squamous cell carcinoma cell lines

Head and neck squamous cell carcinoma represents a complex set of neoplasms arising in diverse anatomical locations. The site and stage of the cancer determine whether patients will be treated with single or multi‐modality therapy. The HDAC inhibitor LBH589 is effective in treating some haematological neoplasms and shows promise for certain epithelial neoplasms. As with other human cancer cell lines, LBH589 causes up‐regulation of p21, G2/M cell cycle arrest, and cell death of human HNSCC cell lines, as measured using flow cytometry and cDNA microarrays. Global RNA expression studies following treatment of the HNSCC cell line FaDu with LBH589 reveal down‐regulation of genes required for chromosome congression and segregation (SMC2L1), sister chromatid cohesion (DDX11), and kinetochore structure (CENP‐A, CENP‐F, and CENP‐M); these LBH589‐induced changes in gene expression coupled with the down‐regulation of MYC and BIRC5 (survivin) provide a plausible explanation for the early mitotic arrest and cell death observed. When LBH589‐induced changes in gene expression were compared with gene expression profiles of 41 primary HNSCC samples, many of the genes that were down‐regulated by LBH589 showed increased expression in primary HNSCC, suggesting that some patients with HNSCC may respond to treatment with LBH589. Copyright

Targeting mitotic chromosomes: a conserved mechanism to ensure viral genome persistence

Viruses that maintain their genomes as extrachromosomal circular DNA molecules and establish infection in actively dividing cells must ensure retention of their genomes within the nuclear envelope in order to prevent genome loss. The loss of nuclear membrane integrity during mitosis dictates that paired host cell chromosomes are captured and organized by the mitotic spindle apparatus before segregation to daughter cells. This prevents inaccurate chromosomal segregation and loss of genetic material. A similar mechanism may also exist for the nuclear retention of extrachromosomal viral genomes or episomes during mitosis, particularly for genomes maintained at a low copy number in latent infections. It has been heavily debated whether such a mechanism exists and to what extent this mechanism is conserved among diverse viruses. Research over the last two decades has provided a wealth of information regarding the mechanisms by which specific tumour viruses evade mitotic and DNA damage checkpoints. Here, we discuss the similarities and differences in how specific viruses tether episomal genomes to host cell chromosomes during mitosis to ensure long-term persistence.

Cohesin: a regulator of genome integrity and gene expression.

Following DNA replication, chromatid pairs are held together by a proteinacious complex called cohesin until separation during the metaphase-to-anaphase transition. Accurate segregation is achieved by regulation of both sister chromatid cohesion establishment and removal, mediated by post-translational modification of cohesin and interaction with numerous accessory proteins. Recent evidence has led to the conclusion that cohesin is also vitally important in the repair of DNA lesions and control of gene expression. It is now clear that chromosome segregation is not the only important function of cohesin in the maintenance of genome integrity.

CCCTC-Binding Factor Recruitment to the Early Region of the Human Papillomavirus 18 Genome Regulates Viral Oncogene Expression

ABSTRACT Host cell differentiation-dependent regulation of human papillomavirus (HPV) gene expression is required for productive infection. The host cell CCCTC-binding factor (CTCF) functions in genome-wide chromatin organization and gene regulation. We have identified a conserved CTCF binding site in the E2 open reading frame of high-risk HPV types. Using organotypic raft cultures of primary human keratinocytes containing high-risk HPV18 genomes, we show that CTCF recruitment to this conserved site regulates viral gene expression in differentiating epithelia. Mutation of the CTCF binding site increases the expression of the viral oncoproteins E6 and E7 and promotes host cell proliferation. Loss of CTCF binding results in a reduction of a specific alternatively spliced transcript expressed from the early gene region concomitant with an increase in the abundance of unspliced early transcripts. We conclude that high-risk HPV types have evolved to recruit CTCF to the early gene region to control the balance and complexity of splicing events that regulate viral oncoprotein expression. IMPORTANCE The establishment and maintenance of HPV infection in undifferentiated basal cells of the squamous epithelia requires the activation of a subset of viral genes, termed early genes. The differentiation of infected cells initiates the expression of the late viral transcripts, allowing completion of the virus life cycle. This tightly controlled balance of differentiation-dependent viral gene expression allows the virus to stimulate cellular proliferation to support viral genome replication with minimal activation of the host immune response, promoting virus productivity. Alternative splicing of viral mRNAs further increases the complexity of viral gene expression. In this study, we show that the essential host cell protein CTCF, which functions in genome-wide chromatin organization and gene regulation, is recruited to the HPV genome and plays an essential role in the regulation of early viral gene expression and transcript processing. These data highlight a novel virus-host interaction important for HPV pathogenicity.

Targeting CTCF to Control Virus Gene Expression: A Common Theme amongst Diverse DNA Viruses.

All viruses target host cell factors for successful life cycle completion. Transcriptional control of DNA viruses by host cell factors is important in the temporal and spatial regulation of virus gene expression. Many of these factors are recruited to enhance virus gene expression and thereby increase virus production, but host cell factors can also restrict virus gene expression and productivity of infection. CCCTC binding factor (CTCF) is a host cell DNA binding protein important for the regulation of genomic chromatin boundaries, transcriptional control and enhancer element usage. CTCF also functions in RNA polymerase II regulation and in doing so can influence co-transcriptional splicing events. Several DNA viruses, including Kaposi’s sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and human papillomavirus (HPV) utilize CTCF to control virus gene expression and many studies have highlighted a role for CTCF in the persistence of these diverse oncogenic viruses. CTCF can both enhance and repress virus gene expression and in some cases CTCF increases the complexity of alternatively spliced transcripts. This review article will discuss the function of CTCF in the life cycle of DNA viruses in the context of known host cell CTCF functions.

Prevalence and Genotyping of High Risk Human Papillomavirus in Cervical Cancer Samples from Punjab, Pakistan

Cervical cancer is the third most common cause of cancer-related death in women worldwide. Infection with high-risk human papillomavirus (HPV) is established as the cause of cervical carcinoma, therefore, high risk HPV detection may have prognostic significance for the women who are at increased risk of disease progression. The paucity of data on the incidence of cervical cancer in Pakistan makes it difficult to determine disease burden. Even less information is available regarding the prevalent HPV strains in cervical specimens collected from this region. Cervical cancer is a neglected disease in Pakistan in terms of screening, prevention, and vaccination. Identification and accurate genotyping of the virus burden in cancer specimens is important to inform intervention policies for future management of HPV associated disease and to potentially stratify patients dependent on HPV status. In this study, detection and genotyping of HPV types 16 and 18 from 77 cervical specimens were carried out. Consensus primers GP5+/GP6+, which detect 44 genital HPV types, and type specific primers (TS16 and TS18) were used in conjunction with newly designed type specific primers. Using a combination of these methods of detection, a total of 94.81% (95% CI ±4.95) of cervical lesions were positive for HPV. Single infections of HPV16 were detected in 24.68% (95% CI ±9.63) of total samples and HPV18 was found in 25.97% (95% CI ±9.79) samples. Interestingly, a high proportion of samples (40.26%, 95% CI ±10.95) was positive for both HPV16 and 18, indicating a higher incidence of co-infection than previously reported for similar ethnic regions. The HPV genotype of 3.90% of HPV positive samples remained undetected, although these samples were positive with the GP5+/GP6+ primer set indicating infection with an HPV type other than 16 or 18. These data indicate that the overall incidence of high risk HPV infection in cervical cancer and intraepithelial neoplasia specimens in Punjab, Pakistan is in line with the worldwide prevalence, but that the incidence of HPV16 and 18 co-infections in our cohort is higher than that previously reported.

In vivo analysis of the cell cycle dependent association of the bovine papillomavirus E2 protein and ChlR1

It has been shown that the genomes of episomally maintained DNA viruses are tethered to host cell chromosomes during cell division, facilitating maintenance in dividing cells. The papillomavirus E2 protein serves this mechanism of viral genome persistence by simultaneously associating with chromatin and the viral genome during mitosis. Several host cell proteins are reported to be necessary for the association of E2 with chromatin including the cohesion establishment factor ChlR1. Here we use fluorescence resonance energy transfer (FRET) technology to confirm the interaction between BPV-1 E2 and ChlR1. Furthermore, we use synchronised live cells to study the temporal nature of this dynamic protein interaction and show that ChlR1 and E2 interact during specific phases of the cell cycle. These data provide evidence that the association of E2 with ChlR1 contributes to a loading mechanism during DNA replication rather than direct tethering during mitotic division.

The human papillomavirus type 16 L1 protein directly interacts with E2 and enhances E2-dependent replication and transcription activation

The human papillomavirus (HPV) E2 protein is a multifunctional protein essential for the control of virus gene expression, genome replication and persistence. E2 is expressed throughout the differentiation-dependent virus life cycle and is functionally regulated by association with multiple viral and cellular proteins. Here, we show for the first time to our knowledge that HPV16 E2 directly associates with the major capsid protein L1, independently of other viral or cellular proteins. We have mapped the L1 binding region within E2 and show that the α-2 helices within the E2 DNA-binding domain mediate L1 interaction. Using cell-based assays, we show that co-expression of L1 and E2 results in enhanced transcription and virus origin-dependent DNA replication. Upon co-expression in keratinocytes, L1 reduces nucleolar association of E2 protein, and when co-expressed with E1 and E2, L1 is partially recruited to viral replication factories. Furthermore, co-distribution of E2 and L1 was detected in the nuclei of upper suprabasal cells in stratified epithelia of HPV16 genome-containing primary human keratinocytes. Taken together, our findings suggest that the interaction between E2 and L1 is important for the regulation of E2 function during the late events of the HPV life cycle.