Biology subjects

Beck, T.

Publications and source records attributed to Beck, T..

24 records · Page 2Linked to original sources

bioRxiv2020

Combined fluorescence, optical diffraction tomography and Brillouin microscopy

Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples -- so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epi-fluorescence imaging for explicitly measuring the Brillouin shift, RI and absolute density with specificity to fuorescently labeled structures. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the nucleoplasm of wild-type HeLa cells, we find that it has lower density but higher longitudinal modulus than the cytoplasm. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample -- a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.

biophysics↗

bioRxiv2020

Smchd1 is a maternal effect gene required for autosomal imprinting

Genomic imprinting establishes parental allele-biased expression of a suite of mammalian genes based on parent-of-origin specific epigenetic marks. These marks are under the control of maternal effect proteins supplied in the oocyte. Here we report the epigenetic repressor Smchd1 as a novel maternal effect gene that regulates imprinted expression of 16 genes. Most Smchd1-sensitive genes only show loss of imprinting post-implantation, indicating maternal Smchd1s long-lived epigenetic effect. Sm-chd1-sensitive genes include both those controlled by germline polycomb marks and germline DNA methylation imprints; however, Smchd1 differs to other maternal effect genes that regulate the latter group, as Smchd1 does not affect germline DNA methylation imprints. Instead, Smchd1-sensitive genes are united by their reliance on polycomb-mediated histone methylation marks as germline or secondary imprints. We propose that Smchd1 translates these imprints to establish a heritable chromatin state required for imprinted expression later in development, revealing a new mechanism for maternal effect genes.

genomics↗

bioRxiv2019

Xmas ESC: A new female embryonic stem cell system that reveals the BAF complex as a key regulator of the establishment of X chromosome inactivation

Although female pluripotency significantly differs to male, complications with in vitro culture of female embryonic stem cells (ESC) have severely limited the use and study of these cells. We report a replenishable female ESC system, Xmas, that has enabled us to optimise a protocol for preserving the XX karyotype. Our protocol also improves male ESC fitness. We utilised our Xmas ESC system to screen for regulators of the female-specific process of X chromosome inactivation, revealing chromatin remodellers Smarcc1 and Smarca4 as key regulators of establishment of X inactivation. The remodellers create a nucleosome depleted region at gene promotors on the inactive X during exit from pluripotency, without which gene silencing fails. Our female ESC system provides a tractable model for XX ESC culture that will expedite study of female pluripotency and has enabled us to discover new features of the female-specific process of X inactivation.

cell biology↗

bioRxiv2018

Using long-read sequencing to detect imprinted DNA methylation

Systematic variation in the methylation of cytosines at CpG sites plays a critical role in early development of humans and other mammals. Of particular interest are regions of differential methylation between parental alleles, as these often dictate monoallelic gene expression, resulting in parent of origin specific control of the embryonic transcriptome and subsequent development, in a phenomenon known as genomic imprinting. Using long-read nanopore sequencing we show that, with an average genomic coverage of approximately ten, it is possible to determine both the level of methylation of CpG sites and the haplotype from which each read arises. The long-read property is exploited to characterise, using novel methods, both methylation and haplotype for reads that have reduced basecalling precision compared to Sanger sequencing. We validate the analysis both through comparison of nanopore-derived methylation patterns with those from Reduced Representation Bisulfite Sequencing data and through comparison with previously reported data. Our analysis successfully identifies known imprinting control regions as well as some novel differentially methylated regions which, due to their proximity to hitherto unknown monoallelically expressed genes, may represent new imprinting control regions.

genomics↗

bioRxiv2018

Long-range chromatin interactions on the inactive X and at Hox clusters are regulated by the non-canonical SMC protein Smchd1.

The regulation of higher order chromatin structure is complex and dynamic; however we do not yet understand the full suite of mechanisms governing architecture. Here we reveal the non-canonical SMC protein Smchd1 as a novel regulator of long-range chromatin interactions, and add it to the canon of epigenetic proteins required for Hox gene regulation. The effect of losing Smchd1-dependent chromatin interactions has varying outcomes dependent on chromatin context. At autosomal targets transcriptionally sensitive to Smchd1 deletion, we find increased short-range interactions and ectopic enhancer activation. By contrast, the inactive X chromosome is transcriptionally refractive to Smchd1 ablation, despite chromosome-wide increases in short-range interactions. There we observe spreading of H3K27me3 domains into regions not normally decorated by this mark. Together these data suggest Smchd1 has the capacity to insulate the chromatin, thereby limiting access to other chromatin modifying proteins.

molecular biology↗

bioRxiv2017

Complex rearrangements and oncogene amplifications revealed by long-read DNA and RNA sequencing of a breast cancer cell line

The SK-BR-3 cell line is one of the most important models for HER2+ breast cancers, which affect one in five breast cancer patients. SK-BR-3 is known to be highly rearranged although much of the variation is in complex and repetitive regions that may be underreported. Addressing this, we sequenced SK-BR-3 using long-read single molecule sequencing from Pacific Biosciences, and develop one of the most detailed maps of structural variations (SVs) in a cancer genome available with nearly 20,000 variants present, most of which were missed by prior efforts. Surrounding the important HER2 locus, we discover a complex sequence of nested duplications and translocations, suggesting a punctuated progression. Full-length transcriptome sequencing further revealed several novel gene fusions within the nested genomic variants. Combining long-read genome and transcriptome sequencing enables an in-depth analysis of how SVs disrupt the transcriptome and sheds new light on the complexity of cancer progression.

genomics↗