Investigating the role of enhancer–promoter proximity in regulating gene expression

The development of an organism requires the expression of each gene to be precisely orchestrated in space and time. This spatiotemporal control is partly achieved through enhancers: cis-regulatory sequences that can modulate transcription at their cognate promoters, even from millions of base pairs away. Despite the important regulatory role of enhancers, the mechanisms by which enhancers and promoters interact to ex- change regulatory information is still debated. Specifically, it is unclear whether close spatial proximity between regulatory elements is required for transcription. Adding another layer of complexity, recent studies have revealed multiway enhancer-promoter (E-P) interactions that appear to drive cell type-specific gene expression. However, the frequency of these multiway interactions in single cells remains under-explored. To address these questions, we combined DNA and RNA fluorescence in situ hybridization (FISH), super-resolution microscopy, and Tri-C to investigate changes in E-P distances during the transition from naive to primed pluripotency in mouse. To this end, we developed NOVA-FISH - a method capable of visualizing small regulatory elements in close genomic proximity. We then used NOVA-FISH, together with Oligopaint, to examine pairwise and multiway E-P interactions for five genes that are differentially expressed during the naive-to-primed transition: Nanog, Dppa3, Sox2, Dnmt3a and Prdm14. For Nanog and Dppa3, we additionally correlated E-P distance with nascent transcription. Despite transcriptional changes of several orders of magnitude, we found that changes in pairwise E-P distances during the naive-to-primed transition are highly locus-dependent. Tri-C data at the Nanog locus revealed a weak enrichment of multiway contacts when Nanog was highly expressed in the naive state, but not in the primed state, when Nanog was downregulated. As transcription often occurs in transient bursts within a subset of cells, we combined RNA and DNA FISH to identify active alleles. We observed a positive correlation between shorter E-P distances and transcription at the Nanog and Dppa3 loci. Together, our data support models of dynamic contact, in which shorter E–P distances are transiently stabilized during transcriptional initiation, and multiway hubs may contribute to regulating cell type–specific gene expression

Phasenkontrastbildgebung der Brust mittels Talbot-Lau-Interferometrie

Die Phasenkontrastbildgebung der Brust ist als projektives mammographisches Verfahren sowie als dreidimensionale Computertomographie durchführbar. Die Phänomene Absorption, Phasenverschiebung und Mikrostreuung werden dabei bildgebend genutzt und führen zum simultan erstellten Absorptions,- Phasenkontrast,- und Dunkelfeldbild. Im vorgelegten Habilitationsprojekt wurde ein Talbot-Lau-Interferometer mit konventioneller polychromatischer Röntgenröhre genutzt. Ex vivo Studien am mammographischen Aufbau zeigten klinisch relevante Zusatzinformationen durch die Phasenkontrastbildgebung und insbesondere durch das Dunkelfeldbild. Präparatuntersuchungen am CT-Aufbau zeigten eine deutlich verbesserte Beurteilung der Präparatränder. Bildgebende Merkmale bei gutartigen Zysten und Fibroadenomen der Brust konnten mittels Dunkelfeldbildgebung komplementär zum Absortionsbild dargestellt und zugeordnet werden. Die derzeitige Weiterentwicklung der Phasenkontrast-CT lässt erwarten, dass klinische Untersuchungen an Geräteprototypen zukünftig möglich sein werden.Phase contrast breast mammography and breast CT can be safely performed at a Talbot-Lau-Interferometer using a conventional X-ray source. Absorption, refraction and microscattering of X-rays passing an object are used by this method to obtain absorption, phase contrast and dark field images simultaneously by one single akquisition. The presented ex vivo mammography studies show an improved depiction of different breast lesions, especially in dark field imaging. Phase contrast CT of intraoperative samples show a better margin depiction, especially in areas with in situ carcinoma. Specific imaging features of benign breast lesions like cysts and fibroadenomas revealed additional complementary information compared to absorption imaging. Current work on phase contrast chest CT prototypes is expected to allow clinical phase contrast studies in the near future

Study of microbiome in liver transplantation

Introduction: Biliary complications are major contributors to morbidity after liver transplantation (LT). Recent evidence suggests that the biliary tract harbors a resident microbiome, yet its clinical relevance in LT remains unclear. This study aimed to investigate the biliary microbiome at the time of transplantation and explore its relationship to post-transplant outcomes. Methods: In this prospective observational study, 31 LT cases were enrolled. Paired donor bile fluid and biliary mucosa samples were collected intraoperatively. Active bacterial communities were profiled using RNA-based 16S rRNA sequencing. Microbiome composition, diversity, and associations with clinical parameters were statistically analyzed. Results: The biliary tract exhibited a distinct, non-sterile microbiome. Microbiome composition remained largely stable intraoperatively. Lower microbial richness in donor bile was linked to ITBL, while higher diversity was observed in cholangitis. Opportunistic and biofilm-forming genera were enriched in cases with biliary leaks or strictures. Donor antibiotic exposure reduced microbial diversity, whereas corticosteroid treatment was associated with increased richness. Discussion: This study represents the first RNA-based analysis to characterize the biliary microbiome in LT. The findings reveal that microbial profiles present at transplantation are associated with subsequent biliary complications, suggesting their potential as early biomarkers. Donor treatment regimens influence microbiome structure, highlighting opportunities for targeted interventions. These insights expand current understanding of microbiota-host interactions in transplantation and may inform future strategies to mitigate post-LT complications

Modulation of lysosomal proteases in FTD-GRN

One of the most common genetic risk factors associated with frontotemporal lobar degeneration (FTLD) is GRN haploinsufficiency. However, inclusions of TAR-DNA binding protein 43 (TDP-43) are the major pathological hallmark of progranulin (PGRN) related FTLD (Grn-FTLD). Recently, the lysosomal protease legumain (LGMN) has been proposed to mechanistically link the loss of PGRN to TDP-43 pathology in FTLD. LGMN is capable of generating pathological TDP-43 fragments and upregulation of LGMN activity has been detected in brains of GRN-FTLD patients with TDP-43 pathology. Besides TDP-43, several proteins involved in various neurodegenerative diseases such as tau, amyloid precursor protein (APP) and α-synuclein are also cleaved by LGMN. Hence, modulation of LGMN activity turned out to be a promising new therapeutic target. This work provides two different approaches for the modulation of LGMN activity. One strategy was to analyze the effect of the endogenous cysteine protease inhibitors cystatin-3 and cystatin-7 on maturation and activity of LGMN. The other approach focused on restoring PGRN, a protein which has been shown to dampen LGMN maturation and proteolytic activity. In addition to in vitro studies, the effects of PGRN, cystatin-3 and cystatin-7 were analyzed in the mouse brain, an environment resembling the situation in the human brain. Therefore, transgene expression of the three proteins was induced by ultrasound-guided intraventricular adeno-associated virus (AAV) injection into neonatal mice. In this thesis, an AAV9 capsid containing the respective gene of interest was used. 3 months post injection, strong and widespread protein expression was detected. Areas around the lateral ventricles exhibited the highest expression and the hSyn promoter enabled pronounced expression in neurons. Cystatin-3 as well as cystatin-7 reduced LGMN activity in vitro and in vivo. Incubation of the respective recombinant inhibitors and LGMN revealed that cystatin-7 delayed maturation of LGMN much more efficiently than cystatin-3. For half-maximal inhibition of active LGMN lower concentrations of cystatin-7 were needed whereas cystatin-3 appeared to be better suited for complete LGMN inhibition. In contrast to the mainly unaffected cystatin-3 treated mice, cystatin-7 overexpressing mice showed enlarged lysosomes and minor signs of gliosis combined with compromised motor skills. To assess further effects of a cystatin treatment, the influence of both cystatins on other lysosomal proteases, in particular cathepsins was analyzed. Overexpression of cystatin-3 and -7 in HEK 293T cells and C57BL/6J mouse brains differentially changed the activity and maturation of cathepsins D, B and L next to LGMN. In summary, cystatin-3 appeared as a more favorable candidate for future studies with the aim of modulating LGMN activity. Since GRN-FTLD is caused by GRN haploinsufficiency, boosting PGRN expression seems a logical treatment strategy. Various studies support this idea with positive results obtained from expressing PGRN in Grn knockout mice. In this study, Grn-/-/Tmem106b-/- mice, a model for GRN-FTLD with accelerated pathology concerning lysosomal function, proteostasis, gliosis and motor function, were used for AAV-mediated PGRN delivery. At an age of 3 months, untreated Grn-/-/Tmem106b-/- mice exhibited pathological hallmarks including microgliosis, astrogliosis, impaired autophagy, increased activity of lysosomal proteases and beginning TDP-43-like pathology. Intraventricular AAV-Grn injection in neonatal Grn-/-/Tmem106b-/- mice could not rescue pathology but exacerbated motor dysfunction. Furthermore, overexpression of PGRN in wild type mice induced gliosis, impaired autophagy and lead to the upregulation of lysosomal proteases. Thinning of the cortex dorsal of the injection site was accompanied by severely impaired motor function. Therefore, my findings emphasize the importance of further studies on the dose-dependency and the therapeutic window of a PGRN treatment. In conclusion, modulation of LGMN activity as a strategy for the treatment of GRN-FTLD with TDP-43 pathology is worth further research. Different approaches such as boosting endogenous proteins like cystatin-3, cystatin-7 and PGRN, respectively, or the use of small-molecule inhibitors highly specific for LGMN should be compared to determine a powerful method of treatment for this devastating disease