Biology subjects

Heinzer, D.

Publications and source records attributed to Heinzer, D..

3 recordsLinked to original sources

bioRxiv2020

HIV-1 promoter is gradually silenced when integrated into BACH2

The persistence of the latent HIV-1 reservoir is a major obstacle to cure HIV-1 infection. HIV-1 integrates into the cellular genome and some targeted genomic loci are frequently detected in clonally expanded latently HIV-1 infected cells, for instance, the gene BTB domain and CNC homology 2 (BACH2). We investigated HIV-1 promoter activity after integration into specific sites in BACH2. The HIV-1-based vector LTatCL[M] contains two fluorophores: 1.) Cerulean, which reports the activity of the HIV-1 promoter, and 2.) mCherry driven by a constitutive promotor and flanked by genetic insulators. This vector was inserted into introns 2 and 5 of BACH2 of Jurkat T-cells via CRISPR/Cas9 technology in the same and convergent transcriptional orientation of BACH2, and into the genomic safe harbour AAVS1. Single cell clones representing active (Cerulean+/mCherry+) and inactive (Cerulean-/mCherry+) HIV-1 promoters were characterized. Upon targeted integration of the 5.3 kb vector LTatCL[M] into BACH2, active HIV-1 promoters were gradually silenced as reflected by decrease in Cerulean expression over a period of 162 days in culture. Silenced HIV-1 promoters could be reactivated by TNF- and Romidepsin. This observation was independent of the targeted intron and the transcriptional orientation. BACH2 mRNA and protein expression was not impaired by mono-allelic integration of LTatCL[M]. Our results show that the HIV-1 promoter is silenced when integrated into BACH2 without impairing BACH2 mRNA and protein expression. This might contribute to HIV-1 persistence, enabling infected T-cells to complete differentiation into a memory phenotype, persist, and clonally expand over time.

microbiology↗

bioRxiv2020

Genome-wide transcriptomics identifies an early preclinical signature of prion infection

The clinical course of prion diseases is accurately predictable despite long latency periods, suggesting that prion pathogenesis is driven by precisely timed molecular events. We constructed a searchable genome-wide atlas of mRNA abundance, splicing and editing alterations during the course of disease in prion-inoculated mice. Prion infection induced transient changes in mRNA abundance and processing already at eight weeks post inoculation, well ahead of any neuropathological and clinical signs. In contrast, microglia-enriched genes displayed an increase simultaneous with the appearance of clinical symptoms, whereas neuronal-enriched transcripts remained unchanged until the very terminal stage of disease. This suggests that glial pathophysiology, rather than neuronal demise, represents the final driver of disease. The administration of young plasma attenuated the occurrence of early mRNA abundance alterations and delayed symptoms in the terminal phase of the disease. The early onset of prion-induced molecular changes might thus point to novel biomarkers and potential interventional targets.

neuroscience↗

bioRxiv2019

An integrated genomic approach to dissect the genetic landscape regulating the cell-to-cell transfer of a-synuclein

Neuropathological and experimental evidence suggests that the cell-to-cell transfer of a-synuclein has an important role in the pathogenesis of Parkinsons disease (PD). However, the mechanism underlying this phenomenon is not fully understood. We undertook an siRNA, genome-wide high throughput screen to identify genes regulating the cell-to-cell transfer of a-synuclein. We transiently transfected HEK cells stably overexpressing a-synuclein with a construct encoding GFP-2a-aSynuclein-RFP. The cells expressing a-synuclein-RFP through transfection were double positive for GFP and RFP fluorescence, whereas the cells receiving it through transfer were positive only for RFP fluorescence. The amount of a-synuclein transfer was quantified by high content microscopy. A series of unbiased screens confirmed the involvement of 38 genes in the regulation of a-synuclein-RFP transfer. One of those hits was ITGA8, a candidate gene recently identified through a large PD genome wide association study (GWAS). Weighted gene co-expression network analysis (WGCNA) and weighted protein-protein network interaction analysis (WPPNIA) showed that the hits clustered in networks that included known PD Mendelian and GWAS risk genes more frequently than expected than random chance. Given the genetic overlap between a-synuclein transfer and PD, those findings provide supporting evidence for the importance of the cell-to-cell transfer of a-synuclein in the pathogenesis of PD, and expand our understanding of the mechanism of a-synuclein spread.

neuroscience↗