An Analysis of the Essential Chromatin Factor Zinc Finger Protein 1 (ZFP-1), and a Study of the Involvement of RNAi factors in Histone Processing in Caenorabditis Elegans

The formation of chromatin defines when and where genes will be expressed, from the histone proteins forming the nucleosome and their many post-translational modifications, to the immense number of proteins that bind these modifications. This thesis is comprised of two projects: the first is an analysis of the essential plant homeodomain-containing protein called zinc finger protein 1 (ZFP-1); the second is a study into how RNA interference (RNAi) factors are involved in histone production in C. elegans. I investigate the physical and biological properties of the PHD fingers of ZFP-1 and find that 1) they are essential for viability and 2) they specifically bind to methylated lysine 4 on histone H3. This study has expanded our understanding of the molecular nature of ZFP-1, the C. elegans ortholog of AF10, which has a role in chromosomal translocations promoting leukemia. I also determine that the RNAi factors CSR-1, EGO-1 and DRH-3 are required for proper histone production in C. elegans. Severe histone depletion results from the knockdown of these RNAi pathway components, which explains both the phenotypes of sterility and chromosome segregation defects in early embryos that are associated with mutants of these factors. This discovery explains the well-known, but poorly understood, phenotypes of the RNAi mutants and provides the first evidence for RNAi positively affecting gene expression

Virology under the microscope—a call for rational discourse

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns – conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we – a broad group of working virologists – seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology

Adenovirus protein VII binds the A-box of HMGB1 to repress interferon responses.

Viruses hijack host proteins to promote infection and dampen host defenses. Adenovirus encodes the multifunctional protein VII that serves both to compact viral genomes inside the virion and disrupt host chromatin. Protein VII binds the abundant nuclear protein high mobility group box 1 (HMGB1) and sequesters HMGB1 in chromatin. HMGB1 is an abundant host nuclear protein that can also be released from infected cells as an alarmin to amplify inflammatory responses. By sequestering HMGB1, protein VII prevents its release, thus inhibiting downstream inflammatory signaling. However, the consequences of this chromatin sequestration on host transcription are unknown. Here, we employ bacterial two-hybrid interaction assays and human cell culture to interrogate the mechanism of the protein VII-HMGB1 interaction. HMGB1 contains two DNA binding domains, the A- and B-boxes, that bend DNA to promote transcription factor binding while the C-terminal tail regulates this interaction. We demonstrate that protein VII interacts directly with the A-box of HMGB1, an interaction that is inhibited by the HMGB1 C-terminal tail. By cellular fractionation, we show that protein VII renders A-box containing constructs insoluble, thereby acting to prevent their release from cells. This sequestration is not dependent on HMGB1's ability to bind DNA but does require post-translational modifications on protein VII. Importantly, we demonstrate that protein VII inhibits expression of interferon β, in an HMGB1-dependent manner, but does not affect transcription of downstream interferon-stimulated genes. Together, our results demonstrate that protein VII specifically harnesses HMGB1 through its A-box domain to depress the innate immune response and promote infection

HSV-1 exploits host heterochromatin for nuclear egress

Herpes simplex virus (HSV-1) progeny form in the nucleus and exit to successfully infect other cells. Newly formed capsids navigate complex chromatin architecture to reach the inner nuclear membrane (INM) and egress. Here, we demonstrate by transmission electron microscopy (TEM) that HSV-1 capsids traverse heterochromatin associated with trimethylation on histone H3 lysine 27 (H3K27me3) and the histone variant macroH2A1. Through chromatin profiling during infection, we revealed global redistribution of these marks whereby massive host genomic regions bound by macroH2A1 and H3K27me3 correlate with decreased host transcription in active compartments. We found that the loss of these markers resulted in significantly lower viral titers but did not impact viral genome or protein accumulation. Strikingly, we discovered that loss of macroH2A1 or H3K27me3 resulted in nuclear trapping of capsids. Finally, by live-capsid tracking, we quantified this decreased capsid movement. Thus, our work demonstrates that HSV-1 takes advantage of the dynamic nature of host heterochromatin formation during infection for efficient nuclear egress

APOBEC3A damages the cellular genome during DNA replication

<p>The human APOBEC3 family of DNA-cytosine deaminases comprises 7 members (A3A-A3H) that act on single-stranded DNA (ssDNA). The APOBEC3 proteins function within the innate immune system by mutating DNA of viral genomes and retroelements to restrict infection and retrotransposition. Recent evidence suggests that APOBEC3 enzymes can also cause damage to the cellular genome. Mutational patterns consistent with APOBEC3 activity have been identified by bioinformatic analysis of tumor genome sequences. These mutational signatures include clusters of base substitutions that are proposed to occur due to APOBEC3 deamination. It has been suggested that transiently exposed ssDNA segments provide substrate for APOBEC3 deamination leading to mutation signatures within the genome. However, the mechanisms that produce single-stranded substrates for APOBEC3 deamination in mammalian cells have not been demonstrated. We investigated ssDNA at replication forks as a substrate for APOBEC3 deamination. We found that APOBEC3A (A3A) expression leads to DNA damage in replicating cells but this is reduced in quiescent cells. Upon A3A expression, cycling cells activate the DNA replication checkpoint and undergo cell cycle arrest. Additionally, we find that replication stress leaves cells vulnerable to A3A-induced DNA damage. We propose a model to explain A3A-induced damage to the cellular genome in which cytosine deamination at replication forks and other ssDNA substrates results in mutations and DNA breaks. This model highlights the risk of mutagenesis by A3A expression in replicating progenitor cells, and supports the emerging hypothesis that APOBEC3 enzymes contribute to genome instability in human tumors.</p

Virology under the Microscope—a Call for Rational Discourse

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology