Computational study of transcription factor binding sites

Any living organism contains a whole set of instructions encoded as genes on the DNA. This set of instructions contains all the necessary information that the organism will ever need, from its development to a mature individual to environment specific responses. Since all these instructions are not needed at the same time, the gene expression needs to be regulated.

Eukaryotic genomes are stored inside nuclei as chromatin. The chromatin is the association of DNA with dedicated storage proteins - the histones - and the necessary machinery to regulate and express genes (RNA polymerases or RNAPs).

In the nuclei, histones are assembled into octamers around which are wrapped ~148bp of DNA. This structure is known as the nucleosome. The repetition of nucleosomes along the genome allows to drastically compact the genome, eventually allowing to fit it inside the nucleus. However, this comes at the cost of rendering the DNA sequence inaccessible to DNA readers, such as the RNAPs and transcription factors (TFs).

TFs are a class of proteins that have the remarkable property of recognizing and binding specific DNA sequences. More striking, each TF can recognize a multitude of different - but similar - DNA sequences providing TFs with a wide sequence specificity range. Eventually, this allows the cell to recruit TFs at dedicated locations in the genome called regulatory elements (REs).

The action of TFs at REs is crucial to gene expression. Indeed, TFs are involved in many processes such as the opening of the chromatin structure or the recruitment of RNAPs. However if TFs can influence the chromatin structure, the opposite is also true as histones impede TF binding on DNA. Thus the regulation of genes relies on a subtle and complex crosstalk between the chromatin and TFs.

To better understand how TFs and chromatin interact together to regulate gene expression, I lead several projects prospecting TF binding specificity and the chromatin structure at REs in human.

First, I used ENCODE next generation sequencing (NGS) data to explore how TF binding influences the nearby nucleosome organization and the propensity of some TFs to bind together. The results suggest that regular nucleosome arrays are found near all TFs. They also point out two special cases. When CTCF binds with the cohesin complex, it seems to drive the nucleosome organization, which is a unique feature among all TFs investigated. Additionally I present evidence supporting that EBF1 is a pioneer factor - a special class of TFs able to bind nucleosome.

Secondly, I developed several clustering algorithms and software to partition genomic regions according to NGS data and/or on their DNA sequences. These methods allow to discover important trends, for instance different nucleosome architectures . I illustrated the usefulness of these methods for the study of chromatin accessibility data and the identification of REs.

Thirdly, I participated to the assessment of SMiLE-seq, a new microfluidic device that generates TF specificity data. The creation of TF specificity models and their comparison with other publicly available models demonstrated the value of SMiLE-seq to study TF specificity.

Finally, I participated in the development of a software that predicts TF binding sites. A careful benchmarking suggested that this software is - at the time of writing - the best available software in terms of speed while showing other performances similar to its competitors.

From polymers to gene regulation

Aleksandre Japaridze

DNA molecule is the fundamental component of every living organism, since it encodes the hereditary information. Although the genetic code of almost 200 species has been sequenced, the direct connection between gene sequence, DNA structure and biological function remains poorly understood. The aim of this thesis was to investigate the connection between DNA function and structure. Throughout the thesis we will discuss experiments testing the link between different physical properties of DNA with its biological function. For this purpose we used atomic force microscopy (AFM), a high resolution imaging technique. The first chapter is devoted to explain the basic concepts of AFM technique, as well as the composition and structure of DNA molecules. We also describe some of the polymer physics models of DNA, defining basic quantities like the persistence and the critical exponent. In chapter 2 we discuss experiments, in which we studied changes in the physical properties of DNA molecules, when bound to the substrate surface with various methods. We show that, when DNA is strongly bound to the surface, it retains its physiological B-form. On contrary, when weakly bound, a conformational B-to-A-form transition takes place. We also discuss a novel experimental method, combining nanometer sized PDMS slits with AFM, for studying DNA in geometrical confinement. When DNA is weakly confined, DNA persistence length increases, molecules elongate and adopt more anisotropic shapes. Under stronger confinement, DNA hairpins form. In chapter 3 we study binding of staining dyes, proteins and RNA polymerase (RNAP) to DNA molecules. First we show that DNA physical properties are significantly affected when stained with dyes. Depending on the dye binding mode, either the contour length, or the persistence length or both simultaneously change. We move further and investigate the role of protein amino acids and DNA sequence in the formation of nucleoprotein complexes. On the example of EspR protein, a key virulence regulator in Mycobacterium tuberculosis (MTB), we show that single amino acid mutations, lead to lower DNA binding affinity. These mutations also impair the formation of higher order protein-DNA complexes, silencing the MTB virulence. Afterwards, on the examples of RNAP and H-NS protein, we show the importance of DNA sequence for the binding and higher order oligomerization. By introducing DNA mutations disrupting the helical phasing between RNAP binding sites in the fis promoter sequence, we significantly lower the RNAP binding affinity. The helical phasing of the binding sites somehow coordinates the cooperative binding of RNAP to the promoter. Finally on the model of H-NS protein, we show how different spatial arrangements of strong binding sites for an architectural protein in the DNA, determine the final 3D structure of the assembled nucleoprotein complex. The final chapter, is fully devoted to the study of a completely new type of DNA organisation, which we call Hyperplectonemes. Hyperplectonemes are very ordered DNA structures, formed by large supercoiled molecules, in the presence of attractive DNA-DNA potential. First, we describe their structure and its dependence on various environmental factors, than their binding to nucleoid associated proteins. We argue that this emerging DNA organisation is the basic structure of the bacterial chromatin, which is further modulated by numerous DNA binding and condensing proteins in vivo.

EPFL2015

Toward a mechanistic understanding of variable chromatin modules

Gerard Llimos Aubach

Our genome is a long sequence of DNA that contains all the information to be able to constitute a living organism like us, similarly to what the letters in a book do to create a story. This sequence, which is a stretch of molecules called nucleotides, is almost identical between organisms of the same species (>99.9% in humans), however it varies in a small percentage due to some nucleotides being different at specific positions of the genome. This variation in the DNA will therefore influence our traits and affect our susceptibility to a certain condition. Thus, their study is highly relevant in genetics and biomedicine since they allow not only to predict the predisposition to a disease, such as cancer, but also to understand the molecular and cellular mechanism behind a certain phenotype. The effect of a genetic variant on a phenotype is given by their capacity to modulate the expression of a gene, either by directly impacting its sequence and thus changing the protein, or by regulating its expression levels. Interestingly, more than 88% of the disease-associated genetic variants present in humans are located outside genes, on the so-called non-coding part of the genome. This poses a challenge to identify what gene the genetic variant affects and to understand how it does so. Genetic variants do not only influence gene expression but they also affect the epigenetics state of the DNA (i.e. DNA methylation, histone marks, transcription factor (TF) binding...) and its 3D conformation, consequently modifying the activity of gene regulatory elements like promoters and enhancers. Previously, it has been shown that some of these regulatory regions located in the same locus exhibit a high level of molecular coordination and interindividual variation, mostly affected by genetic variation or environmental effects. These regions were termed variable chromatin modules (VCMs) or cis-regulatory domains (CRDs) and are thought to be the manifestation of fine-grained regulatory units of the genome. Understanding how a VCM is formed and the molecular basis behind the cooperativity between regulatory elements is an outstanding question in the field. In this work, we aim at resolving part of this puzzle by mechanistically dissecting one VCM that is influenced by a non-coding germline variant in B cells. Using a set of tools such as genetic engineering, TF binding assays and chromosome conformation studies, we demonstrate that this variant creates a de novo binding site for the TF MEF2, which acts as a pioneering factor for dozens of other collaborating TFs. In turn, this actuates a cluster of enhancers spanning a region over 150 kilobases (kb) that regulates AXIN2 gene expression, and is associated with a global compaction of the chromatin. In addition, in line with the known tumor-suppressor properties of AXIN2, we found that this variant influences chronic lymphocytic leukemia (CLL) progression and predisposition. Together, the characterization of the AXIN2 VCM provides an unique example to the community of how a germline variant is able to switch on an entire genomic locus with high degree of cooperativity between regulatory elements, since it captures both the formation and transition behavior of a VCM. Furthermore, given the link with CLL, this focal variant could have translational potential by serving as a patient-stratifying or treatment diagnostic.

EPFL2021

KRAB zinc-finger proteins and their transposable element targets: between antagonism and cooperation

Jonas Caspar De Tribolet-Hardy

There are 377 Krüppel-associated box (KRAB) domain-containing zinc finger proteins (KZFPs) in the human genome, making them the largest family of transcription factors. KZFPs are defined by a N-terminal KRAB domain and several zinc-finger domains arranged in an array at the C-terminus of the proteins. The zinc-finger domains each form sequence specific interactions with double stranded DNA, allowing the zinc-finger array to target specific genomic sequences. The KRAB domain, through its interaction with the protein TRIM28 (also known as KAP1) allows for the stable silencing of transcription in a genomic region. Together these two domains allow KZFPs to bind specific regions of the genome and generally lead to the formation of heterochromatin and silencing of transcription allthough other functions for some KZFPs have been recently reported. KZFPs usually target transposable elements (TEs), mobile genomic elements that can move through the genome either by cut-and-paste or copy-paste mechanisms and make up almost half of the human genome. Different KZFPs bind to specific regions of TEs, limiting their expression and thus mitigating some of the threat they pose to human development, allowing both the TE and its host to survive. In recent years it became apparent that both certain TEs and KZFPs have roles beyond the previously described threat and mitigation of that threat. TEs harbor gene regulatory regions allowing, through their spread, for a dissemination of those regions through the genome and for new gene regulatory networks to form. KZFPs can affect the transcription of genes located in proximity to their binding sites leading to changes in gene regulation as well. A multitude of regulatory roles involving KZFPs and TEs have been described in recent years making them an exciting field of study. A major hurdle in understanding both KZFPs and TEs is to identify the binding sites of KZFPs, as they reveal both the targets of the KZFP and how, if at all, a TE is regulated by KZFPs. To do so, we expanded on previous efforts and aimed to perform chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) on every member of the human KZFP family. Here we present ChIP-seq experiments for 110 new KZFPs, which together with previously published data, allow us to study the binding of almost all human KZFPs (95%). The entirety of the data was analysed together to generate a coherent dataset and results for each KZFP are made available to the public on our web portal (https://tronoapps.epfl.ch/web/krabopedia/). The identified binding sites allowed us to witness the adaptation of the KZFP family to new TEs and showed how targeted sequences shift after segmental gene duplication events. Furthermore, we could corroborate that several KZFPs target the same TE subfamilies in a seemingly redundant fashion and show that unexpectedly these KZFPs arose independently in different genomic locations. These results represent a valuable tool for anyone studying KZFPs and should aid in the pursuit of both understanding KZFPs and TEs.

EPFL2022