18 research outputs found
Bioinformatic analyses of the structural and functional complexity in chromosomal interactomes
Evolution requires information storage systems with different demands with respect to
persistence. While the genome provides a mechanism for long term, static and accurate
information storage, it is incapable of mediating adaptation to short term changes in the
environment. Chromatin, however, constitutes a dynamic, reprogrammable memory with
different levels of persistence. Moreover, chromatin states carry information not only in 2D, i.e.
in the structure of the primary chromatin fibre, but also in the 3D organization of the genome in
the nuclear space. The following thesis delves into the new bioinformatic and wet lab protocols
developed to map, quantitative and functionally analyze the 3D architecture of chromatin.
The chromatin insulator protein CTCF is a major factor underlying the 3D organization
of the epigenome. We have uncovered, however, that CTCF binding sites within a regulatory
region have multiple functions that are influenced by the chromatin environment and possibly
the combinatorial usage of the 11 Zn-fingers of CTCF (Paper I). This observation exemplifies
that understanding the function of dynamic and transient chromatin fibre interactions requires
novel technology that enables the detection of 3D chromatin folding with high resolution in single
cells and in small cell populations. We therefore set out to devise a novel method for the
visualization of higher order chromatin structures by combining the strengths of both DNA
Fluorescent In Situ Hybridization (FISH) and In Situ Proximity Ligation Assay (ISPLA)
technologies (Paper II). The resulting Chromatin in Situ Proximity (ChrISP) assay thus takes
advantage of the direct contact detection of ISPLA and the locus-specific nature of FISH and
uncovered the existence of compact chromatin structures at the nuclear envelope with
unprecedented resolution. To complement ChrISP with a high throughput method capable of
quantitatively recovering chromatin fibre contacts in small cell populations, we furthermore
innovated the Nodewalk assay (Paper III). The protocol builds on existing ligation based
chromosome conformation capture methods, but features significant reduction in the random
ligation event frequency, inclusion of negative and positive ligation controls, iterative template
resampling, increased signal to noise ratio and improved sensitivity. Using this technique, we
have uncovered a cancer cell-specific, productive chromatin fibre interactome connecting the
promoter and enhancer of c-MYC to a network of enhancers and super-enhancers. Underpinning
this new protocol, I have developed the Nodewalk Analysis Pipeline (NAP) (Paper IV). This suite
of tools consists of preprocessing, analysis and post-processing modules designed specifically for
the rapid and efficient analysis of Nodewalk datasets through an interactive and user-friendly web
based interface.
Overall the work described in this thesis advances our understanding of the role of CTCF
in nuclear organization and provides innovative wet lab techniques along with specialized
software tools. Moreover, this work is an example of an emerging trend where the challenge of
understanding chromatin dynamics within the 3D nuclear architecture demands a close
synergistic collaboration between the fields of biology, biotechnology and bioinformatics
Immunohistochemical typing of adenocarcinomas of the pancreatobiliary system - data and analysis code
Data and analysis code for study "Immunohistochemical Typing of Adenocarcinomas of the Pancreatobiliary System Improves Diagnosis and Prognostic Stratification
Immunohistochemical typing of adenocarcinomas of the pancreatobiliary system - Reproducible computer environment
Reproducible computer environment as Docker image for study "Immunohistochemical Typing of Adenocarcinomas of the Pancreatobiliary System Improves Diagnosis and Prognostic Stratification
Correction: Immunohistochemical Typing of Adenocarcinomas of the Pancreatobiliary System Improves Diagnosis and Prognostic Stratification.
[This corrects the article DOI: 10.1371/journal.pone.0166067.]