Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription

In yeast, ribosome production is controlled transcriptionally by tight coregulation of the 138 ribosomal protein genes (RPGs). RPG promoters display limited sequence homology, and the molecular basis for their coregulation remains largely unknown. Here we identify two prevalent RPG promoter types, both characterized by upstream binding of the general transcription factor (TF) Rap1 followed by the RPG-specific Fhl1/Ifh1 pair, with one type also binding the HMG-B protein Hmo1. We show that the regulatory properties of the two promoter types are remarkably similar, suggesting that they are determined to a large extent by Rap1 and the Fhl1/Ifh1 pair. Rapid depletion experiments allowed us to define a hierarchy of TF binding in which Rap1 acts as a pioneer factor required for binding of all other TFs. We also uncovered unexpected features underlying recruitment of Fhl1, whose forkhead DNA-binding domain is not required for binding at most promoters, and Hmo1, whose binding is supported by repeated motifs. Finally, we describe unusually micrococcal nuclease (MNase)-sensitive nucleosomes at all RPG promoters, located between the canonical +1 and -1 nucleosomes, which coincide with sites of Fhl1/Ifh1 and Hmo1 binding. We speculate that these "fragile'' nucleosomes play an important role in regulating RPG transcriptional output.

International Union of Pharmacology. LXI. Peroxisome proliferator-activated receptors.

Johan Auwerx, Liliane Michalik, Walter Wahli

The three peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors of the nuclear hormone receptor superfamily. They share a high degree of structural homology with all members of the superfamily, particularly in the DNA-binding domain and ligand- and cofactor-binding domain. Many cellular and systemic roles have been attributed to these receptors, reaching far beyond the stimulation of peroxisome proliferation in rodents after which they were initially named. PPARs exhibit broad, isotype-specific tissue expression patterns. PPARalpha is expressed at high levels in organs with significant catabolism of fatty acids. PPARbeta/delta has the broadest expression pattern, and the levels of expression in certain tissues depend on the extent of cell proliferation and differentiation. PPARgamma is expressed as two isoforms, of which PPARgamma2 is found at high levels in the adipose tissues, whereas PPARgamma1 has a broader expression pattern. Transcriptional regulation by PPARs requires heterodimerization with the retinoid X receptor (RXR). When activated by a ligand, the dimer modulates transcription via binding to a specific DNA sequence element called a peroxisome proliferator response element (PPRE) in the promoter region of target genes. A wide variety of natural or synthetic compounds was identified as PPAR ligands. Among the synthetic ligands, the lipid-lowering drugs, fibrates, and the insulin sensitizers, thiazolidinediones, are PPARalpha and PPARgamma agonists, respectively, which underscores the important role of PPARs as therapeutic targets. Transcriptional control by PPAR/RXR heterodimers also requires interaction with coregulator complexes. Thus, selective action of PPARs in vivo results from the interplay at a given time point between expression levels of each of the three PPAR and RXR isotypes, affinity for a specific promoter PPRE, and ligand and cofactor availabilities.

2006

The role of OCT4 and SOX2 in regulating chromatin accessibility and cell fate across the cell cycle in embryonic stem cells

Torgil Elias Friman

Precise spatiotemporal regulation of gene expression is essential for development and homeostasis of complex organisms. This is achieved in large part by sequence-specific transcription factors (TF) that bind to genomic regulatory elements to activate or repress transcription. DNA in eukaryotic nuclei is wrapped around histones into nucleosomes, which are inaccessible to most proteins. However, certain TFs can access their binding sites in the presence of histones and promote nucleosome eviction to increase chromatin accessibility, allowing for binding of other protein. These TFs, called pioneer factors, include OCT4 and SOX2, which are master regulators of embryonic stem (ES) cells. Dividing cells, including stem cells, must achieve proper gene regulation despite the progression of the cell cycle, which imposes several constraints. During S phase, the replication fork passes through the genome to make a copy of each chromosome, which leads to a loss of chromatin accessibility. Furthermore, the substantial chromatin compaction that occurs during mitosis to prepare for chromosome segregation is associated with eviction of most DNA-binding proteins and reduced accessibility at distal cis-regulatory elements including enhancers. How pioneer factors operate in the context of cell cycle progression is unclear. In this thesis, I will present work studying different features of OCT4 and SOX2, including how they interplay to regulate chromatin accessibility in ES cells, the impact of small endogenous fluctuations of their protein levels on cell fate and chromatin accessibility, and how their roles in regulating cell fate and chromatin accessibility is related to different stages of the cell cycle. Using live-cell imaging of fluorescently labeled proteins, we discovered that OCT4 and SOX2 were associated to mitotic chromatin. Using cell-cycle specific degradation strategies, we could show that the presence of SOX2 and OCT4 at the mitosis-G1 (M-G1) transition contributes to maintain the pluripotency of ES cells. We further showed that ES cells with high levels of OCT4 in G1, but not in S phase, are more prone to differentiate towards neuroectoderm and mesendoderm cell fates. Cells with high levels of OCT4 displayed increased chromatin accessibility at enhancers of differentiation genes, suggesting accessibility fluctuates with OCT4 levels and can prime cells for differentiation. To explore potential cell cycle-dependent regulation of chromatin accessibility, we degraded OCT4 at the M-G1 transition, which led to a reduction in accessibility at many OCT4-bound regions. When OCT4 returned later in the cell cycle, chromatin accessibility was recovered, although at many loci this recovery was incomplete, suggesting that OCT4 is required at the M-G1 transition to maintain these regions open. We then rapidly degraded OCT4 at other phases of the cell cycle. Surprisingly, loss of chromatin accessibility was highly similar at all cell cycle phases, showing that OCT4 is constantly required to maintain regulatory elements accessible. To explore the dynamic regulation of accessibility by OCT4, we performed time-course measurements of accessibility upon its degradation. This revealed that loss of accessibility was temporally correlated to the degradation of OCT4. These results suggest that chromatin accessibility is highly dynamic and continuously dependent on the presence of OCT4 across the cell cycle in ES cells.

EPFL2019

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