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RNA modulation of structure and function of the Drosophila MSL complex in vitro
Get PDFGene expression is highly regulated in eukaryotes by histone modifications and corresponding reader-writer proteins. In the fruit fly Drosophila melanogaster the gene expression from the single male X chromosome is doubled, matching the transcriptional output from the two female X chromosomes. Absence of this gene dosage compensation results in male fly lethality. The molecular machinery involved is called dosage compensation complex (DCC), which consists of five proteins and a long non-coding RNA. The proteins of the DCC are the histone acetyltransferase MOF, the male-specific lethal proteins (MSL1, MSL2, MSL3) and the helicase MLE. At least one of two long non-coding RNAs roX1 or roX2 are critical for male fly viability in vivo. How the lncRNA impacts the function of the DCC remains hitherto unknown.
A hypothesis states that roX incorporation leads to structural changes of the DCC and potentially enhances one of its enzymatic activities, particularly its acetylation activity. To date, only limited information on the structure of the complete complex is available. Thus, I applied crosslinking mass spectrometry (XL-MS) to analyze structural interfaces within the DCC in absence or presence of roX RNA. Novel MSL-MSL protein interactions were found and previously reported contact sites were confirmed. Addition of roX2 RNA subtly changed XL patterns in MLE and the MSL1-MSL2 submodule of the MSL complex, which could hint towards a conformational change upon roX2 integration.
Transcriptional upregulation of the male X chromosome is molecularly linked to H4K16 acetylation by the HAT MOF. Hypothetically the activity and the specificity of MOF could be regulated by roX2 RNA. To address this hypothesis, I reconstituted nucleosome arrays in vitro and used purified MSL complexes for acetylation assays. These assays were evaluated by mass spectrometry, which allows for accurate site-specific acetylation identification and quantification. In absence of RNA MOF within the MSL complex is active, however not selective for H4K16ac. At longer incubation times the complete H4 tail is acetylated, starting at H4K16 and progressing outwards to H4K12, K8 and K5. This zipper-like processive behavior is supported by mathematical modelling. Upon addition of roX2 RNA or unrelated long RNA the oligo-acetylation of the H4 tail is suppressed, even at prolonged incubation times. If this effect can be linked to roX RNA in vivo or can be ensured as well by heterogeneous nuclear RNA (hnRNA) remains to be elucidated. Finally, dTIP60, which is a HAT complex of the same enzymatic family, does not show the processive oligo-acetylation mechanism and is not impacted by RNA.
In conclusion, the lncRNA roX2 induces subtle conformational changes in MLE and the MSL1-MSL2 submodule of the MSL complex. Moreover, it increases the specificity of the HAT MOF towards H4K16ac in nucleosome arrays. The possible connection between these roles via a shared allosteric mechanism awaits further investigation
The interlinkages of design and effectiveness of governing bodies in the United Nations development system
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Stable isotope analyses of human skeletal remains
Get PDFThe transition from Roman times to the Middle Ages in Europe is characterized by profound changes in all spheres of life, related to the decline of the Western Roman Empire and the emergence of medieval kingdoms. It is often assumed that the migration of various Germanic gentes played an important role in this transition. However, little is known about the local history of the Roman frontier province of Raetia secunda (Raetia II) in present-day southern Bavaria due to the lack of historical sources, so it can be referred to as a "Dark Age." Therefore, the anthropological study of human skeletal remains from this period is of particular importance to gain more insight into historical events and developments.
The stable isotope analyses of human bones and teeth carried out in this thesis investigate the different origins, diets, and weaning practices of the population living in Raetia II between Late Antiquity and the Early Middle Ages.
The results suggest that a large number of migrants came to Raetia II in the second half of the 5th century. The emerging differences in mobility between men and women, as well as the available evidence for multiple distinct regions of origin, suggest that immigration and mobility in South Bavaria after the fall of the Roman Empire were extremely complex. In particular, it has been noted that the tracked consumption of food sources atypical of the Bavarian region, such as marine resources and millet, may indicate a non-local origin of individuals.
It is implied that the Bavarian population subsisted on a mixture of different types of C3 plants and terrestrial animals. There is separate evidence that economic practices changed during the Early Middle Ages, but further analysis is needed to confirm this.
The observed breastfeeding and weaning practices vary considerably, even within local communities. Most individuals are weaned by the time they reach the age of three. However, in some migrants, probably from a population with a different cultural background and lifestyle, different weaning practices with longer breastfeeding periods are observed. Furthermore, analysis of physiological stress during and after breastfeeding shows that childhood stress increases when there is little or no breastfeeding.
Overall, the results of this study have contributed to enlightening the "Dark Ages" in South Bavaria
Identification of in vitro model systems capable of capturing the polygenic basis of mental illness
Get PDFLarge genome wide association studies have identified thousands of genetic variants associated with psychiatric diseases. These variants are likely to act in a highly condition and cell type specific fashion. However, at this point their cellular context and developmental of action remains poorly understood. Moreover, it remains un- clear to what extent in vivo and in vitro model systems can approximate the human cellular conditions where polygenic psychiatric disease risk is operational.
One of the application scenarios of genome wide association studies results, is the calculation of polygenic risk scores to identify subjects at risk of developing disease or to stratify a cohort with a given trait or disease, in this thesis I performed genomic imputation and traditional polygenic risk score calculation of two case/control cohorts for schizophrenia at different P-value thresholds used to subset the GWAS derived SNP associations considered in the scoring. The power of polygenic risk scores in the clinical setting for psychiatric disorders is limited, the fact that the hundreds of loci that contribute to disease liability are likely to act in a cell type specific manner, supports the need to develop cell type specific polygenic risk scores, for this purpose it is crucial to understand how the disease risk affects specific cell types.
One of the objectives of this thesis was to identify which cell types are vulnera- ble to psychiatric disease associated polygenic risk, I did this by using stratified LD score regression for partitioning heritability from GWAS summary statistics while accounting for linkage disequilibrium, this allowed me to identify which cell type groups are enriched for psychiatric disorders heritability. I performed this partitioned heritability analysis in the transcriptomic and chromatin accessibility profiles of 10 neuronal and non neuronal cerebral cell types derived from human post-mortem brain tissue of the prefrontal cortex.
LD score regression was performed using the identified active elements and GWAS derived summary statistics for various psychiatric disorders as well as control traits. Cell types including excitatory neurons of the cortical layer 2-3, corticothalamic neurons and inhibitory neurons as well as microglial cells and oligodendrocyte progenitor cells were significantly enriched for schizophrenia and bipolar disorder, many more significant positive associations were found between some of these cell types and traits corresponding to psychiatric disorders and Central Nervous System traits. All of this in line with previous research where these cell types had been found to be relevant to susceptibility to variants associated to the above mentioned traits. I performed the same analyses on ATACseq and RNAseq having ATACseq yielding finer and more specific results than RNAseq, which failed to identify some relevant cell types.
ATACseq was used in assessing the validity of different model systems to see if they could capture the polygenic architecture of psychiatric diseases, this model systems included iPSC derived neurons, cerebral organoids, post-natal mouse cortical cells and fetal cortical neurons. The results were consistent with the postmortem tissue findings.
These results allowed us to benchmark the used in vitro models that maintain the heritability enrichment of postmortem tissues, opening the possibility for a robust use of scATACseq data derived from these models to reliably identify cell type specific elements relevant for disease and leverage this information in building better predictive tools like cell-type specific polygenic risk scores
