Chromosome conformation capture

Chromosome conformation capture techniques (often abbreviated to 3C technologies or 3C-based methods) are a set of molecular biology methods used to analyze the spatial organization of chromatin in a cell. These methods quantify the number of interactions between genomic loci that are nearby in 3-D space, but may be separated by many nucleotides in the linear genome. Such interactions may result from biological functions, such as promoter-enhancer interactions, or from random polymer looping, where undirected physical motion of chromatin causes loci to collide. Interaction frequencies may be analyzed directly, or they may be converted to distances and used to reconstruct 3-D structures. The chief difference between 3C-based methods is their scope. For example, when using PCR to detect interaction in a 3C experiment, the interactions between two specific fragments are quantified. In contrast, Hi-C quantifies interactions between all possible pairs of fragments simultaneously. Deep sequencing of material produced by 3C also produces genome-wide interactions maps. Historically, microscopy was the primary method of investigating nuclear organization, which can be dated back to 1590. In 1879, Walther Flemming coined the term chromatin. In 1883, August Weismann connected chromatin with heredity. In 1884, Albrecht Kossel discovered histones. In 1888, Sutton and Boveri proposed the theory of continuity of chromatin during the cell cycle In 1889, Wilhelm von Waldemeyer created the term "chromosome". In 1928, Emil Heitz coined the terms heterochromatin and euchromatin. In 1942, Conrad Waddington postulated the epigenetic landscapes. In 1948, Rollin Hotchkiss discovered DNA methylation. In 1953, Watson and Crick reported the double helix structure of DNA based on Rosalind Franklin's X-ray diffraction images. In 1961, Mary Lyon postulated the principle of X-inactivation. In 1973/1974, chromatin fiber was discovered. In 1975, Pierre Chambon coined the term nucleosomes. In 1982, Chromosome territories were discovered. In 1984, John T.

Amplitude and phase reconstruction for Low-Energy Electron Holography of individual proteins

Hannah Julia Ochner

Single-molecule imaging methods are of importance in structural biology, and specifically in the imaging of proteins, since they can elucidate conformational variability and structural changes that might be lost in imaging methods relying on averaging processes. Low-energy electron holography (LEEH) is a promising technique for imaging individual proteins with negligible radiation damage, which can be combined with a sample preparation process by native electrospray ion beam deposition (native ES-IBD) ensuring the creation of chemically pure samples suitable for holographic imaging.The central step in the analysis of data measured by LEEH is the numerical reconstruction of the object from the experimentally acquired holograms.Full information about the imaged object consists of both amplitude and phase information imprinted on the scattered wave during the interaction of the electron beam with the molecule and encoded in the hologram created by the interference of the scattered wave and the transmitted incident wave. A propagation-based algorithm with the goal of reconstructing the wave field in the plane of the object is presented and applied to holograms of individual proteins prepared by ES-IBD and measured with a low-energy electron holography microscope. The information retrieved from the reconstruction of the amplitude distribution in the object plane is discussed by analysing holograms of antibodies, demonstrating that the inherent conformational variability of these molecules can be mapped by LEEH. The influence of the sample preparation process on the surface conformations is tracked by tuning the landing energy of the proteins during deposition.To complement the amplitude data, an iterative phase retrieval algorithm is implemented to reconstruct the phase distribution in the object plane along with the amplitude distribution. The algorithm's performance and robustness is thoroughly evaluated and simulations regarding multiple scattering effects and element-dependent variations in scattering strength are carried out to provide a reference for the interpretation of phase data retrieved from experimentally acquired holograms. The iterative phase retrieval algorithm is then applied to protein data, indicating that both molecular density and charges can be related to features in the phase reconstructions, while the presence of metals does not correlate with specific phase signals at the current resolution obtainable from the experimental data.Since proteins are inherently three-dimensional, approaches towards three-dimensional reconstruction schemes are discussed, which will be the focus of future work.

EPFL2022

The role and transcriptional regulation of FAM46A in adipocyte differentiation

Ann-Kristin Hov

Obesity is currently a leading global health concern. Treatments include undergoing a weight-loss regimen. However, successful weight-loss is variable, where the heritability of obesity is considered part of the cause. This patient variability to weight-loss was addressed by Carayol et al. (2017), where protein plasma level changes was associated to genetic variability and changes in BMI under a low-calorie diet intervention. A highly significant association region was found to regulate downstream gene FAM46A; coding for a nucleotidyl-transferase protein. Loss of function mutations in FAM46A have previously been discovered in multiple human diseases, such as osteogenesis imperfecta. However, there is a lack of understanding of FAM46Aâs function, especially in the context of adipose tissue and metabolism. This thesis aimed to follow up on the Carayol et al. study by investigating the transcriptional regulation of FAM46A and how the associated region is influencing this regulation, as well as how FAM46A is involved in adipose tissue function. When mapping the physical DNA interaction network around the associated region using chromosome conformation capture (3C) in human subcutaneous adipocytes (SGBS cells), no interactions between the FAM46A promoter and selected loci of the associated region were found. Interestingly, significant interaction peaks downstream of the promoter overlapped with regions of open chromatin and were enriched for transcription factor (TF) motifs that are involved in stem cell differentiation and TGFÎ² signalling. SMAD4, part of the TGFÎ² signalling pathway, binds to this region, indicating a potential regulator of FAM46A. To address FAM46Aâs potential role in adipocyte function, FAM46A expression was disrupted in vitro in differentiating adipocytes. Reduction of FAM46A expression in SGBS cells resulted in decreased expression of PPARG and CEBPA, the master regulators of adipogenesis, complemented by a reduction in lipid accumulation. Follow up studies in murine mesenchymal stem cells where Fam46a was over-expressed were controversial as this led to a decrease in adipogenic markers (Adipoq and PPARðŸ). Overall, disruption of FAM46A expression led to opposing changes in adipogenic potential, suggesting the requirement for a more stable cell system to study FAM46A in vitro. To further characterise the role of Fam46a, metabolic phenotyping of a Fam46a knockout (KO) mouse model under hyper-caloric stress was performed in collaboration with the IMG in Prague. This model revealed a significantly smaller mouse, and increased plasma alkaline phosphatase, as previously observed. When fed a high-fat diet, KO mice had reduced overall fat mass, improved glucose clearance and reduced adipocyte size in both subcutaneous and visceral white adipose tissue (subWAT/visWAT) depots. Transcriptomic analysis of these two depots revealed an up-regulation of genes expressed in ribosome related pathways in the subWAT, whereas the visWAT showed the up-regulation of fatty acid metabolism pathways, suggesting that the KO adipose tissue is more metabolically active. Other down-regulated pathways involved stem cell differentiation and BMP/TGFÎ²-signalling. In conclusion, this study found the possible regulation of the FAM46A promoter by TFs involved in adipogenesis regulation, such as SMAD proteins, as well as strengthening its possible functional involvement in TGFÎ² signalling by enrichment of this pathway in KO studies.

EPFL2021

Notch1-driven T-ALL chromatin landscape is regulated by Tcf1 in hematopoietic progenitors

Mateusz Waldemar Antoszewski

Notch1 receptor signaling is essential for T cell fate specification and physiological thymic T lymphocyte development. Its dysregulation and oncogenic activation are detected in almost 80% of pediatric patients suffering from T cell acute lymphoblastic leukemia (T-ALL). T-ALL is a hematological neoplasm arising from immature thymocytes. T cell factor 1 (Tcf1) is a crucial well-known transcription factor regulating the early stages of thymocyte maturation and Notch1 directly drives the expression of Tcf1 during normal T cell development. Here we assessed the role of Tcf1 in modulating chromatin topology which enables the initiation of T-ALL at the level of early hematopoietic progenitors. This allows for the discovery of stage-setting epigenetic regulators establishing a leukemia-prone chromatin landscape during Notch1-driven disease initiation. We found Tcf1 to be an essential mediator of Notch1 signaling during T-ALL induction. Inactivation of Tcf1 prevented leukemogenesis despite oncogenic Notch1 activation. Genomic analysis of hematopoietic progenitors revealed T cell lineage specification to be essential in T-ALL initiation prior to the appearance of phenotypic features of T cells. The expression of genes associated with the T cell lineage was dependent on Notch1 and Tcf1. Chromatin accessibility, chromosome conformation capture and ChIP-seq analysis revealed the co-regulatory epigenetic function of Notch1 and Tcf1 in imprinting a leukemic chromatin landscape. Epigenetic modulation of distal enhancers which regulate leukemic oncogenes has been well described in patient-derived T-ALL cells. The Notch1-Myc enhancer region was shown to be indispensable in the process of leukemogenesis. We have identified a novel regulatory locus, dependent on Tcf1 and Notch1, essential for the expression of Myc in pre-T-ALL cells. This genomic region termed Tcf1-regulated Myc enhancer (TMe) was highly conserved across species. TMe was essential for the transition of pre-leukemic cells to a transplantable full-blown T-ALL but was dispensable for the physiological development of T cells. After activation of the enhancer in hematopoietic progenitors, this regulatory site remained accessible and bound by Tcf1 in murine and human T-ALL cells. Tcf1 is therefore required for shaping the epigenetic landscape of hematopoietic progenitors overexpressing oncogenic Notch1, and thus regulates the expression of genes involved in leukemogenesis.

EPFL2021

Amplitude and phase reconstruction for Low-Energy Electron Holography of individual proteins

Hannah Julia Ochner

EPFL2022

The role and transcriptional regulation of FAM46A in adipocyte differentiation

Ann-Kristin Hov

EPFL2021

Notch1-driven T-ALL chromatin landscape is regulated by Tcf1 in hematopoietic progenitors

Mateusz Waldemar Antoszewski

EPFL2021