Chromatin Fiber Invasion and Nucleosome Displacement by the Rap1 Transcription Factor

Pioneer transcription factors (pTFs) bind to target sites within compact chromatin, initiating chromatin remodeling and controlling the recruitment of downstream factors. The mechanisms by which pTFs overcome the chromatin barrier are not well understood. Here, we reveal, using single-molecule fluorescence, how the yeast transcription factor Rap1 invades and remodels chromatin. Using a reconstituted chromatin system replicating yeast promoter architecture, we demonstrate that Rap1 can bind nucleosomal DNA within a chromatin fiber but with shortened dwell times compared to naked DNA. Moreover, we show that Rap1 binding opens chromatin fiber structure by inhibiting inter-nucleosome contacts. Finally, we reveal that Rap1 collaborates with the chromatin remodeler RSC to displace promoter nucleosomes, paving the way for long-lived bound states on newly exposed DNA. Together, our results provide a mechanistic view of how Rap1 gains access and opens chromatin, thereby establishing an active promoter architecture and controlling gene expression.

Computational study of transcription factor binding sites

Romain Fernand Pietro Groux

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.

EPFL2020

Developement of a gateway compatible system for speciﬁc DNA integration in mouse embryonic stem cells and evaluation of the effect of nucleosome positioning on gene expression

Julien Bryois

An emerging model views gene regulation as a competition for regulatory sequences between transcription factors and nucleosomes at thermodynamic equilibrium. A computational model of the DNA affinity of nucleosomes was recently developed allowing to predict the nucleosome occupancy of DNA sequences. The aim of this project was to investigate the effect of different predicted nucleosome occupancies of an important transcription factor binding site on mammalian gene expression. To this end, a two site and three site gateway compatible system allowing integration of DNA at a speciﬁc locus of two mouse embryonic stem cell lines was developed. An important POU5F1 (previously known as OCT4)/SOX2 binding site located in the Nanog promoter was chosen to be the transcription factor binding site of interest as it was shown to play a major role for Nanog expression. A 2kb sequence from the Nanog proximal promoter (-1585 to +415) was cloned upstream of two different minimal promoters and a GFP reporter using three site gateway cloning. These constructs were integrated in both cell lines and expressed low to medium levels of GFP. Next, several variants of a 1kb sequence from the Nanog promoter (-933 to +53), previously shown to be functional, were constructed. 35bp surrounding the POU5F1/SOX2 binding site were altered resulting in differential predicted nucleosome occupancy of the binding site. These promoters were linked to GFP with or without the addition of a kozak consensus sequence and a SV40 polyadenylation signal. Surprisingly, all these variants showed low GFP expression after transfection. This was not caused by biases introduced by the gateway system as a gateway construct containing the CMV promoter linked to GFP was shown to be functional. Furthermore, direct cloning of the 1kb Nanog promoter in a GFP expressing vector without gateway showed similar GFP expression as gateway cloning. Results indicate that both 2kb and 1kb Nanog promoters express GFP at low levels and that the constructs produced may be useful to measure differences in GFP expression using ﬂow cytometry

2010

In Vivo Studies on the Control of Gene Expression and Protein Degradation

Karolina Bojkowska

Cellular homeostasis is maintained by tightly regulated gene networks, controlled notably at the levels of transcription, mRNA processing, and protein post-translational modification and turnover. This thesis focuses on two of these regulatory steps, namely gene transcription and protein stability. I first explored the impact of the KRAB-ZFP/KAP1 gene regulation system on liver function. KRAB-ZFPs constitute the largest family of transcription factors encoded by mouse and humans, yet their physiological roles and gene targets remain mostly elusive. Conversely, their molecular mechanism of action has been rather well characterized, as they can recognize DNA in a sequence-specific manner and recruit the universal co-factor KAP1 (also known as TRIM28), which in turn serves as a scaffold for various heterochromatin-inducing effectors. Several studies have shed some light on the roles played by this system in embryonic tissues, but its functions in the adult remain almost completely unknown. As chromatin regulation has been shown to be a major factor in the control of metabolism, we decided to investigate the role of KRAB-ZFP/KAP1 in the liver, an organ central to this process. For this, we generated conditional liver-specific Kap1 KO mice, the study of which revealed a strikingly sex-specific phenotype, with early-onset steatosis and age-related tumorigenesis restricted to males, contrasting with the mild metabolic disturbances recorded in Kap1-deleted females. Underlying this phenotype, our combined transcriptome and chromatin analyses revealed that KAP1 controls sexually dimorphic genes notably involved in hormone, drug and xenobiotic metabolism. Finally, by using a recently developed technique for direct RNA quantification, we established the range of KRAB-ZFPs expressed in the liver and likely responsible for at least part of the observed effects. This study thus demonstrates the central role of the KRAB/KAP1 system in control of sexual dimorphism, responses to xenobiotic stress and metabolic control in the liver, and further links disturbances in these processes with hepatic carcinogenesis. In parallel, in an effort to pursue a multidisciplinary training combining skills in biology and chemistry, I worked towards the development of a method to study the half-life of proteins in vivo. Protein turnover critically influences many biological functions, yet methods have been lacking to assess this parameter in vivo. Capitalizing on an in vitro technology previously developed in Kai Johnsson's laboratory, I demonstrated how chemical labeling can be used to measure the half-life of resident intracellular and extracellular proteins in living mice. Our approach is based on labeling of SNAP-tag-fusion proteins with fluorescent probes. First, we verified that SNAP-tag substrates have wide bio-availability in mice and can be used for the specific in vivo labeling of SNAP-tag fusion proteins. We then applied near-infrared probes to perform non-invasive imaging of in vivo labeled tumors. Finally, we used SNAP-mediated chemical pulse-chase labeling to measuring in vivo the half-life of different extra- and intracellular proteins, including KAP1. Importantly, this tagging technology can be applied to any protein amenable to expression as a fusion partner, whether cell-associated or secreted. Furthermore, because the SNAP-tag chemical labeling allows for a large array of probes also suited for experiments aimed at manipulating and monitoring protein function in vivo, such as cross-linking, pull-down or FRET, this methodology opens broad perspectives for studying protein function in living animals.

EPFL2011