DAXX-ATRX regulation of p53 chromatin binding and DNA damage response.

DAXX and ATRX are tumor suppressor proteins that form a histone H3.3 chaperone complex and are frequently mutated in cancers with the alternative lengthening of telomeres (ALT). Here, we show that DAXX and ATRX knock-out (KO) U87-T cells that have acquired ALT-like features have defects in p53 chromatin binding and DNA damage response. RNA-seq analysis revealed that p53 pathway is among the most perturbed. ChIP-seq and ATAC-seq revealed a genome-wide reduction in p53 DNA-binding and corresponding loss of chromatin accessibility at many p53 response elements across the genome. Both DAXX and ATRX null cells showed a depletion of histone H3.3 and accumulation of γH2AX at many p53 sites, including subtelomeres. These findings indicate that loss of DAXX or ATRX can compromise p53 chromatin binding and p53 DNA damage response in ALT-like cells, providing a link between histone composition, chromatin accessibility and tumor suppressor function of p53.

TERRA G-quadruplex RNA interaction with TRF2 GAR domain is required for telomere integrity.

Telomere dysfunction causes chromosomal instability which is associated with many cancers and age-related diseases. The non-coding telomeric repeat-containing RNA (TERRA) forms a structural and regulatory component of the telomere that is implicated in telomere maintenance and chromosomal end protection. The basic N-terminal Gly/Arg-rich (GAR) domain of telomeric repeat-binding factor 2 (TRF2) can bind TERRA but the structural basis and significance of this interaction remains poorly understood. Here, we show that TRF2 GAR recognizes G-quadruplex features of TERRA. We show that small molecules that disrupt the TERRA-TRF2 GAR complex, such as N-methyl mesoporphyrin IX (NMM) or genetic deletion of TRF2 GAR domain, result in the loss of TERRA, and the induction of γH2AX-associated telomeric DNA damage associated with decreased telomere length, and increased telomere aberrations, including telomere fragility. Taken together, our data indicates that the G-quadruplex structure of TERRA is an important recognition element for TRF2 GAR domain and this interaction between TRF2 GAR and TERRA is essential to maintain telomere stability.

Elevated telomere dysfunction in cells containing the African-centric Pro47Ser cancer-risk variant of TP53.

Subtelomeric transcription and chromatin can have a significant impact on telomere repeat maintenance and chromosome stability. We have previously found that tumor suppressor protein p53 (TP53) can bind to retrotransposon-like elements in a majority of human subtelomeres to regulate TERRA transcription and telomeric histone acetylation in response to DNA damage. TP53 also prevents the accumulation of γH2AX DNA-damage signaling at telomeres. We now show that the inherited TP53 polymorphism Pro47Ser (hereafter S47), which is enriched in populations of African descent, is associated with elevated marks of telomere dysfunction. We found that human and mouse cells carrying the S47 variant show increased γH2AX DNA-damage signals at telomeres, as well as reduced TERRA transcription and subtelomeric histone acetylation in response to DNA damage stress. Cell-lines containing inducible genes for P47 or S47 versions of p53, as well mouse embryo fibroblasts (MEFs) reconstituted with human p53, showed elevated telomere-induced DNA damage foci and metaphase telomere signal loss in cells with S47. Human lymphoblastoid cell lines (LCLs) derived from individuals homozygous for S47, show increased accumulation of subtelomeric γH2AX and unstable telomere repeats in response to DNA damage relative to age matched LCLs homozygous for P47. Furthermore, LCLs with S47 had reduced replicative lifespan. These studies indicate that the naturally occurring S47 variant of p53 can affect telomeric chromatin, telomere repeat stability, and replicative capacity. We discuss the potential evolutionary significance of the S47 variant to African populations with respect to telomere regulation and the implications for inherited health disparities.

Chlamydia trachomatis impairs host base excision repair by downregulating polymerase ß.

Chlamydia trachomatis infections have been associated with ovarian cancer by several epidemiological studies. Here, we show that C. trachomatis-infected primary human ovarian epithelial cells display elevated oxidative DNA damage. Base excision repair, an important cellular mechanism to repair oxidative DNA lesions, was impaired in infected primary ovarian and in several other types of cells. Polymerase ß was downregulated in infected cells associated with upregulation of microRNA-499a (miR-499a). Stabilising polymerase ß by inhibiting miR-499a significantly improved repair. Moreover, downregulation of tumour suppressor p53 also resulted in attenuated repair in these cells. Thus, our data show that downregulation of polymerase ß by direct inhibition through miR-499a and downregulation of p53 debilitate the host-cell base excision repair during C. trachomatis infection.

HHV-6 encoded small non-coding RNAs define an intermediate and early stage in viral reactivation.

Human herpesvirus 6A and 6B frequently acquires latency. HHV-6 activation has been associated with various human diseases. Germ line inheritance of chromosomally integrated HHV-6 makes viral DNA-based analysis difficult for determination of early stages of viral activation. We characterized early stages of HHV-6 activation using high throughput transcriptomics studies and applied the results to understand virus activation under clinical conditions. Using a latent HHV-6A cell culture model in U2OS cells, we identified an early stage of viral reactivation, which we define as transactivation that is marked by transcription of several viral small non-coding RNAs (sncRNAs) in the absence of detectable increase in viral replication and proteome. Using deep sequencing approaches, we detected previously known as well as a new viral sncRNAs that characterized viral transactivation and differentiated it from latency. Here we show changes in human transcriptome upon viral transactivation that reflect multiple alterations in mitochondria-associated pathways, which was supported by observation of increased mitochondrial fragmentation in virus reactivated cells. Furthermore, we present here a unique clinical case of DIHS/DRESS associated death where HHV-6 sncRNA-U14 was abundantly detected throughout the body of the patient in the presence of low viral DNA. In this study, we have identified a unique and early stage of viral activation that is characterized by abundant transcription of viral sncRNAs, which can serve as an ideal biomarker under clinical conditions.

Active HHV-6 Infection of Cerebellar Purkinje Cells in Mood Disorders.

Early-life infections and associated neuroinflammation is incriminated in the pathogenesis of various mood disorders. Infection with human roseoloviruses, HHV-6A and HHV-6B, allows viral latency in the central nervous system and other tissues, which can later be activated causing cognitive and behavioral disturbances. Hence, this study was designed to evaluate possible association of HHV-6A and HHV-6B activation with three different groups of psychiatric patients. DNA qPCR, immunofluorescence and FISH studies were carried out in post-mortem posterior cerebellum from 50 cases each of bipolar disorder (BPD), schizophrenia, 15 major depressive disorder (MDD) and 50 appropriate control samples obtained from two well-known brain collections (Stanley Medical Research Institute). HHV-6A and HHV-6B late proteins (indicating active infection) and viral DNA were detected more frequently (p < 0.001 for each virus) in human cerebellum in MDD and BPD relative to controls. These roseolovirus proteins and DNA were found less frequently in schizophrenia cases. Active HHV-6A and HHV-6B infection in cerebellar Purkinje cells were detected frequently in BPD and MDD cases. Furthermore, we found a significant association of HHV-6A infection with reduced Purkinje cell size, suggesting virus-mediated abnormal Purkinje cell function in these disorders. Finally, gene expression analysis of cerebellar tissue revealed changes in pathways reflecting an inflammatory response possibly to HHV-6A infection. Our results provide molecular evidence to support a role for active HHV-6A and HHV-6B infection in BPD and MDD.

Peptidase Inhibitor 15 (PI15) Regulates Chlamydial CPAF Activity.

Obligate intracellular pathogenic Chlamydia trachomatis express several serine proteases whose roles in chlamydial development and pathogenicity are not completely understood. The chlamydial protease CPAF is expressed during the replicative phase of the chlamydial developmental cycle and is secreted into the lumen of the Chlamydia-containing vacuole called inclusion. How the secreted protease is activated in the inclusion lumen is currently not fully understood. We have identified human serine peptidase inhibitor PI15 as a potential host factor involved in the regulation of CPAF activation. Silencing expression as well as over expression of PI15 affected normal development of Chlamydia. PI15 was transported into the chlamydial inclusion lumen where it co-localized with CPAF aggregates. We show that PI15 binds to the CPAF zymogen and potentially induces CPAF protease activity at low concentrations. However, at high concentrations PI15 inhibits CPAF activity possibly by blocking its protease domain. Our findings shed light on a new aspect of chlamydial host co-evolution which involves the recruitment of host cell proteins into the inclusion to control the activation of bacterial proteases like CPAF that are important for the normal development of Chlamydia.

Chromosomal integration of HHV-6A during non-productive viral infection.

Human herpesvirus 6A (HHV-6A) and 6B (HHV-6B) are two different species of betaherpesviruses that integrate into sub-telomeric ends of human chromosomes, for which different prevalence rates of integration have been reported. It has been demonstrated that integrated viral genome is stable and is fully retained. However, study of chromosomally integrated viral genome in individuals carrying inherited HHV-6 (iciHHV-6) showed unexpected number of viral DR copies. Hence, we created an in vitro infection model and studied retention of full or partial viral genome over a period of time. We observed an exceptional event where cells retained viral direct repeats (DRs) alone in the absence of the full viral genome. Finally, we found evidence for non-telomeric integration of HHV-6A DR in both cultured cells and in an iciHHV-6 individual. Our results shed light on several novel features of HHV-6A chromosomal integration and provide valuable information for future screening techniques.

Possible chromosomal and germline integration of human herpesvirus 7.

Human herpesvirus 7 (HHV-7) is a betaherpesvirus, and is phylogenetically related to both HHV-6A and HHV-6B. The presence of telomeric repeat sequences at both ends of its genome should make it equally likely to integrate into the human telomere as HHV-6. However, numerous studies have failed to detect germline integration of HHV-7, suggesting an important difference between the HHV-6A/-6B and HHV-7 genomes. In search of possible germline integrated HHV-7, we developed a sensitive and quantitative real-time PCR assay and discovered that primers designed against some parts of the HHV-7 genome can frequently miss HHV-7 positive clinical samples even though they work efficiently in cell-culture-derived HHV-7 positive materials. Using a primer pair against the U90 ORF of HHV-7, we identified a possible case of germline integration of HHV-7 with one copy of viral genome per cell in both peripheral blood cells and hair follicles. Chromosomal integration of HHV-7 in these individuals was confirmed by fluorescence in situ hybridization analysis. Germline integration of HHV-7 was further confirmed by detection of ~2.6 copies of HHV-7 in the hair follicles of one of the parents. Our results shed light on the complex nature of the HHV-7 genome in human-derived materials in comparison to cell-culture-derived materials and show the need for stringent criteria in the selection of primers for epidemiological HHV-7 studies.

Anti-herpesviral effects of a novel broad range anti-microbial quaternary ammonium silane, K21.

We have created a novel quaternary ammonium silane, K21 through sol-gel chemistry, using an ethoxylated version of an organosilane quaternary ammonium compound and TetraEthyl Ortho Silicate (TEOS) as precursors. Previous studies using the precursor molecule quaternary ammonium compounds (QACs) and a methacryloxy version of K21, primarily designed for use in dental healthcare, have shown inhibited growth properties against several types of gram-positive and gram-negative bacteria including Escherichia coli, Streptococcus mutans, Actinomyces naeslundii and Candida albicans etc. Here we tested the effect of K21 on HSV-1, HHV-6A and HHV-7 in in vitro cell culture infection models. Our results show growth inhibitory effect of K21 on HSV-1, HHV-6A and HHV-7 infection.

GP96 interacts with HHV-6 during viral entry and directs it for cellular degradation.

CD46 and CD134 mediate attachment of Human Herpesvirus 6A (HHV-6A) and HHV-6B to host cell, respectively. But many cell types interfere with viral infection through rapid degradation of viral DNA. Hence, not all cells expressing these receptors are permissive to HHV-6 DNA replication and production of infective virions suggesting the involvement of additional factors that influence HHV-6 propagation. Here, we used a proteomics approach to identify other host cell proteins necessary for HHV-6 binding and entry. We found host cell chaperone protein GP96 to interact with HHV-6A and HHV-6B and to interfere with virus propagation within the host cell. In human peripheral blood mononuclear cells (PBMCs), GP96 is transported to the cell surface upon infection with HHV-6 and interacts with HHV-6A and -6B through its C-terminal end. Suppression of GP96 expression decreased initial viral binding but increased viral DNA replication. Transient expression of human GP96 allowed HHV-6 entry into CHO-K1 cells even in the absence of CD46. Thus, our results suggest an important role for GP96 during HHV-6 infection, which possibly supports the cellular degradation of the virus.