Biology subjects

Abrahams, R. R.

Publications and source records attributed to Abrahams, R. R..

3 recordsLinked to original sources

bioRxiv2026

CTCF regulates wild-type and recombinant AAV gene expression by shaping viral chromatin

Adeno-Associated Viruses (AAVs) are powerful platforms for delivering therapeutic transgenes via recombinant AAV (rAAV) vectors. However, a limited understanding of the regulation of AAV gene expression has narrowed the ability to efficiently express therapeutic transgenes from rAAV vectors. Since rAAVs retain only the wtAAV inverted terminal repeats (ITR), we hypothesized that regulatory elements outside the ITR that govern wild-type AAV (wtAAV) gene expression can be used to modify rAAV genomes to enhance vector performance. Through in silico analysis, biochemical pulldowns, and high-throughput sequencing, we have identified that the host architectural protein CCCTC-binding Factor (CTCF) associates with the wtAAV type 2 (wtAAV2) genome but is absent from rAAV vectors. Global knockdown and site-specific deletion revealed that the CTCF binding element (CBE) on the wtAAV2 genome, located upstream of the viral P5 promoter, regulates expression of the viral Rep68/78 genes. We have re-engineered new rAAV vectors expressing a GFP reporter transgene to contain the wtAAV2-CBE upstream of the vector promoter. Our results show that CTCF binding dramatically increased rAAV transduction efficiency and GFP expression by up to four-fold across multiple cell types. This enhancement was independent of the AAV capsid serotype used for packaging rAAV vectors. CUT&RUN analysis revealed that this CBE was necessary and sufficient to regulate the chromatin landscape of wtAAV2 and rAAV2. Finally, we observed that CTCF-mediated chromatin remodeling of rAAV2 led to increased production of nascent RNA transcripts from the vector genome. Based on our findings, we propose that CTCF supports wtAAV2/rAAV gene expression by shaping the local chromatin landscape. SIMPLE ABSTRACTRecombinant Adeno-Associated Viruses (rAAV) gene therapy vectors have been engineered from wild-type AAV (wtAAV) by inserting the viral telomeres (that serve as replication and packaging signals) on either side of therapeutic transgenes. However, efficient expression of transgenes using current rAAV technologies require high doses, which can lead to sporadic toxic side effects. We hypothesized that uncharacterized regulatory elements in the wtAAV2 genome drive efficient viral gene expression and are absent from the current generation of rAAV vectors. Using in-silico analysis combined with biochemical pulldowns, high-throughput sequencing, and mutant viral systems, we have identified a novel cis-acting element bound by the cellular architectural protein CCCTC-binding Factor (CTCF). This CTCF binding element is necessary for wtAAV2 gene expression and is sufficient to enhance the rAAV vectors ability to express reporter transgenes. This CTCF-binding element regulates the chromatin landscape of the virus and its vectors. Our discovery that adding the 19-bp AAV2 CTCF-binding element enhances transgene expression without affecting vector production efficiency presents a promising new rAAV gene therapy platform that is likely to reduce clinical doses and minimize toxicity in therapeutic applications.

microbiology↗

bioRxiv2026

The interaction between virus-bound KLF4 and host-bound PARP1 directs the localization of Adeno-Associated Virus Type 2 (wtAAV2) to cellular sites of DNA damage

Wild-type adeno-associated virus type 2 (wtAAV2) is a small, non-pathogenic DNA virus within the family Parvoviridae that is modified to engineer recombinant AAV (rAAV) vectors used in gene therapy. wtAAV2 genomes localize to nuclear domains enriched in DNA Damage Response (DDR) factors through mechanisms that remain unknown. We have discovered that the host transcription factor KLF4 (Kruppel-like Factor 4) and the DDR protein PARP1 (poly-ADP ribose polymerase 1) are key effectors regulating wtAAV2 nuclear trafficking. In-silico analysis revealed that wtAAV2-associated genomic sites are enriched in KLF4 binding motifs. Confocal imaging demonstrated a close spatial association between KLF4 and wtAAV2 nuclear reservoirs, consistent with KLF4s known ability to form nuclear condensates. Mutation of the KLF4 binding site within the wtAAV2 genome and RNAi-mediated knockdown of KLF4 globally reduced the expression of viral Rep68/78 genes and attenuated the localization of the viral genome to cellular DDR sites. Chemical inhibition of the KLF4-interacting protein PARP1 with Olaparib decreased the ability of wtAAV2 genomes to localize to cellular DDR sites and transcribe viral genes. Ectopic expression of wild-type PARP1, but not its KLF4-binding-deficient mutant, rescued wtAAV2 gene expression in PARP1-deficient cells. These findings define a novel mechanism by which wtAAV2 exploits the interaction between host transcription factors and DNA repair machinery to establish a persistent nuclear niche. Insertion of KLF4-binding elements into recombinant AAV2 (rAAV2) gene therapy vectors is sufficient to enhance transduction of target cells, providing a framework for engineering vectors with improved nuclear targeting and transcriptional activity. IMPORTANCEWild-type Adeno-associated virus type 2 (wtAAV2) has emerged as the preferred platform for engineering gene therapy vectors due to its non-pathogenic nature and ability to persist in host cells long-term. However, limited understanding of how wtAAV2 genomes navigate the nuclear environment to establish viral reservoirs has hindered the development of efficient recombinant AAV (rAAV) vectors. We demonstrate that the protein KLF4 bound to wtAAV2 genome recruits the virus to cellular KLF4 sites bound by PARP1. Disruption of either KLF4 binding, PARP1 activity or KLF4-PARP1 interaction significantly impairs wtAAV2 localization and transcription, highlighting the importance of their function in the non-replicative wtAAV2 life cycle. KLF4 binding sites are sufficient to improve the expression of transgenes from rAAV vectors and increase their association with cellular DDR proteins. This study advances our understanding of wtAAV2-host interactions and opens new avenues for improving rAAV gene therapy platforms.

microbiology↗

bioRxiv2021

PASTA kinase-dependent control of peptidoglycan synthesis via ReoM is required for cell wall stress responses, cytosolic survival, and virulence in Listeria monocytogenes

Pathogenic bacteria rely on protein phosphorylation to adapt quickly to stress, including that imposed by the host during infection. Penicillin-binding protein and serine/threonine- associated (PASTA) kinases are signal transduction systems that sense cell wall integrity and modulate multiple facets of bacterial physiology in response to cell envelope stress. The PASTA kinase in the cytosolic pathogen Listeria monocytogenes, PrkA, is required for cell wall stress responses, cytosolic survival, and virulence, yet its substrates and downstream signaling pathways remain incompletely defined. We combined orthogonal phosphoproteomic and genetic analyses in the presence of a {beta}-lactam antibiotic to define PrkA phosphotargets and pathways modulated by PrkA. These analyses synergistically highlighted ReoM, which was recently identified as a PrkA target that influences peptidoglycan (PG) synthesis, as an important phosphosubstrate during cell wall stress. We find that deletion of reoM restores cell wall stress sensitivities and cytosolic survival defects of a {Delta}prkA mutant to nearly wild-type levels. While a {Delta}prkA mutant is defective for PG synthesis during cell wall stress, a double {Delta}reoM {Delta}prkA mutant synthesizes PG at rates similar to wild type. In a mouse model of systemic listeriosis, deletion of reoM in a {Delta}prkA background almost fully restored virulence to wild-type levels. However, loss of reoM alone also resulted in attenuated virulence, suggesting ReoM is critical at some points during pathogenesis. Finally, we demonstrate that the PASTA kinase/ReoM cell wall stress response pathway is conserved in a related pathogen, methicillin-resistant Staphylococcus aureus. Taken together, our phosphoproteomic analysis provides a comprehensive overview of the PASTA kinase targets of an important model pathogen and suggests that a critical role of PrkA in vivo is modulating PG synthesis through regulation of ReoM to facilitate cytosolic survival and virulence. AUTHOR SUMMARYMany antibiotics target bacterial cell wall biosynthesis, justifying continued study of this process and the ways bacteria respond to cell wall insults, especially in the context of infection. Penicillin-binding protein and serine/threonine-associated (PASTA) kinases are master regulators of cell wall stress responses in bacteria and are conserved in several major pathogens, including Listeria monocytogenes, Staphylococcus aureus, and Mycobacterium tuberculosis. We previously showed that the PASTA kinase in L. monocytogenes, PrkA, is essential for the response to cell wall stress and virulence. PrkA is also necessary for adaptation to the cytosol, which L. monocytogenes requires during its infectious lifecycle. In this work, we combined orthogonal phosphoproteomic and genetic screens to identify PrkA substrates in L. monocytogenes. We show that phosphoregulation of one candidate from both screens, ReoM, increases peptidoglycan synthesis and that this regulation is required for cytosolic survival and virulence. We also demonstrate that the PASTA kinase-ReoM pathway regulates cell wall stress responses in another significant pathogen, methicillin-resistant S. aureus. Additionally, we uncover a PrkA-independent role for ReoM in vivo in L. monocytogenes, suggesting a need for nuanced modulation of peptidoglycan synthesis during infection. Cumulatively, this study provides new insight into how bacterial pathogens control cell wall synthesis during infection.

microbiology↗