1,721,065 research outputs found
Protein Engineering Tools to Explore the Function of Protein Post-Translational Modifications for Chromatin and Microtubule Cytoskeletal Biology
No full textProtein post-translational modifications (PTMs) play a crucial role in expanding the protein diversity and are one of the major mechanisms through which cells respond to ever changing environmental cues. The function of two of the most important cellular complexes, chromatin and microtubules, is influenced and very tightly regulated by underlying protein post-translational modifications. The focus of my thesis is to develop tools to investigate the role of protein PTMs in the context of chromatin and microtubules.
Eukaryotic DNA is organized in the form of chromatin whose basic unit is the nucleosome. The nucleosome is composed of 147 base pair of DNA wrapped around four core histones forming H2A, H2B, H3 and H4 forming an octamer. Each histone can be highly post-translationally modified, especially on their N-terminal tails protruding from the nucleosome particle. Histone post-translational modifications (PTMs) work combinatorially to establish chromatin states defined by specific gene expression status known as the Histone Code. Although, each nucleosome carries two copies of each histone, each copy in a single nucleosome can be differently modified resulting in PTM based nucleosome asymmetry. In bivalent domains, a chromatin signature prevalent in embryonic stem cells (ESCs), histone H3 methylated at lysine 4(H3K4me3)- an activating histone PTM mark, coexists with H3K27me3- a repressive histone PTM mark, in asymmetric nucleosomes. In the first project, a general, modular and a traceless synthetic strategy to produce asymmetrically modified nucleosomes is described. Using these asymmetric nucleosomes, I show that in bivalent nucleosomes, H3K4me3 inhibits the activity of the H3K27-specific lysine methyltransferase (KMT) Polycomb Repressive Complex 2 (PRC2) solely on the same histone tail. Whereas H3K27me3 stimulates PRC2 activity via a positive feedback mechanism across tails, thereby partially overriding the H3K4me3-mediated repressive effect.
Microtubules are the largest components of the eukaryotic cytoskeleton and play an import role in maintain cellular organization, intracellular and neuronal transport, cell motility and cell division. Microtubules are dynamically assembled from αβ-tubulin heterodimer, which is the basic repeating unit. The primary sequence and structure of tubulin proteins and microtubules are highly conserved in eukaryotic evolution. Despite this conservation, tubulin is subjected to heterogeneity that arises from differential expression of multiple tubulin isotypes and the vast repertoire of tubulin PTMs, predominantly on the unstructured C-terminal tubulin tails. Together, this gives rise to the Tubulin Code. Lack of access to uniformly modified tubulin presents a major difficulty in study of PTM function. Here, I have developed a method to link synthetic, modified tails to recombinant tubulin by use of a split intein based, protein trans-splicing (PTS) approach. I demonstrate this approach by linking a fluorescently modified tubulin tail to recombinant human tubulin, preparing semisynthetic tubulin dimers. Extending the method to native PTMs will open the door for an in-depth study of tubulin modification using a chemically defined system.LCB
Heterochromatin protein 1 recruitment and chromatin conformational dynamics at the single-molecule level
Get PDFChromatin is the template on which DNA-associated transactions take place in eukaryotic organisms. Nucleosomes consisting of the four histones H2A, H2B, H3 and H4 each organize ~150bp of DNA and constitute a first layer of chromatin. The three-dimensional organization of chromatin as well as histone post-translational modifications (PTMs) regulate recruitment of chromatin-associated effector proteins (effectors). Heterochromatin protein 1 (HP1) is an effector associated with silenced genome regions. HP1 recognizes histone H3 trimethylated at lysine 9 (H3 K9me3) and can dimerize. This results in a protein with two binding domains allowing multivalent engagement of target chromatin. HP1 can further promote chromatin condensation and inter-fiber contacts. The effector p53 binding protein (53BP1) is a key regulator in the DNA damage repair pathway. It is known to target a trio of PTMs; H4 dimethylated at K20 (H4 K20me2), H2A(.X) ubiquitylated at K15 (H2A.X K15ub) and H2A.X phosphorylated at S139 (H2AX S139ph). Although details about the function of the individual domains of these proteins have been uncovered, little is known about the binding mechanism of the holoproteins and how this affects chromatin conformation. The aim of this thesis was to develop chromatin engineering and single-molecule fluorescence based methods to; i) Interrogate recruitment kinetics of HP1 to post-translationally modified chromatin. ii) Understand how HP1 binding alters the conformational dynamics of chromatin secondary structure. iii) Prepare histones carrying the PTM signature recognized by 53BP1. We established an assay to monitor binding kinetics of HP1α to modified chromatin using co-localization single-molecule microscopy (CoSM). H3 K9me3 octamers, labeled HP1α, dimerized HP1α and labeled array DNA formed the basis for this. With this, we found that HP1a multivalency induced by dimerization functions as a platform to enhance HP1a binding to target chromatin up to 9-fold by both accelerating association and prolonging retention. This was further corroborated by FRAP measurements using specific mutants in live mouse fibroblasts. Chromatin conformational dynamics were investigated by ensemble and single-molecule FRET (smFRET). Multiple combinations of FRET positions allowed us to obtain multi-perspective information on the conformational changes in chromatin upon compaction. This showed distinct steps in the local folding of chromatin. Histone acetylation of histone H4 prevented the last steps of this folding pathway. HP1-mediated compaction promotes earlier steps of compaction while maintaining conformational dynamics. Finally, towards similar studies with 53BP1, we devised schemes for synthesis of histones containing the target PTMs. Histones with the individual PTMs were prepared by ligation and desulfurization. H2A.X with both an N-terminal ubiquitin and C-terminal phosphorylation was prepared through a convergent route using recombinant SUMO and a split intein from Nostoc Punctiforme as orthogonal recombinant protection groups. Together the work described in this thesis combines advanced protein and chromatin engineering with CoSM and smFRET. This resulted in mechanistic insight into the spatio-temporal regulation of HP1 recruitment, chromatin conformational dynamics, templates for similar investigations with 53BP1, and tools for further investigation of nucleosome function in the context of chromatin.LCB
Deciphering the chromatin binding mechanisms of human HP1 variants at the single-molecule level
No full textIn eukaryotic cells, DNA is tightly packed in the form chromatin. The basic structure of chromatin is a nucleosome composed of 147 bp DNA wrapped around eight histone proteins; two copies of H2A, H2B, H3 and H4. These histone proteins are decorated with patterns of post-translational modifications leading to a direct change in chromatin structure or recruitment of effector proteins.
Heterochromatin protein 1 (HP1) is an effector protein, that binds with low micromolar affinity to trimethylated lysine 9 on histone H3 (H3K9me3). It has been shown that HP1 localizes to specific domains where its binding is highly dynamic, however it is not well understood how HP1 is efficiently recruited to heterochromatin sites enacting stable gene silencing. The aim of this project was therefore to develop a single molecule microscopy method in total internal fluorescence (TIRF) to study the dynamic binding of HP1 towards chemically synthesized chromatin fibers. This would enable us to understand how HP1, which has low affinity towards H3K9me3, is able to increase its affinity and localization to chromatin. Initially, three models were studied; 1) The co-existence of many low-affinity binding sites in chromatin fibers allows rapid re-binding of HP1a after dissociation. 2) Stable and long-lived complexes are the result of HP1a oligomerization on the chromatin fiber. 3) Multivalent binding interactions of dimeric HP1a increases binding affinity towards chromatin. We found that HP1α residence time on chromatin depends on the density of H3K9me3, where the dissociated protein can rapidly rebind to a neighbouring site. Multivalency by HP1a dimerization leads to longer retention and accelerates the association rate and HP1a does not form oligomers but competes for free H3K9me3 sites.
Following on from these results we aimed to improve our understanding of how HP1 dynamically samples the chromatin landscape, by studying the influence of chromatin states over HP1a binding, as well as finding out the binding differences between the HP1 isoforms (HP1a, b and g) and the influence of a phosphorylation mark on HP1a. We found that HP1a exhibits the longest residence times and fastest binding rates due to DNA interaction as well as H3K9me3 binding, confirmed by in vivo fluorescence recovery after photobleaching (FRAP) experiments. Interestingly, phosphorylated HP1a increases retention through strengthening of multivalency while reducing DNA binding. From the smTIRF results, a kinetic model was developed to dissect the detailed mechanism of HP1-chromatin binding, revealing a multivalent interaction network provided by multiple weak protein and DNA interactions.
The final part of this thesis was focused towards improving our single molecule TIRF set up by developing a multiplexed read-out of nucleosomes. This will provide a way to measure the interaction of a chromatin binding protein towards a library of nucleosomes in one single molecule experiment. The technique relies on the nucleosomal DNA containing a short ssDNA site where the complementary labeled DNA piece can hybridize dynamically.
The work presented in this thesis, was based on using a single molecule microscopy technique to shed light on the recruitment of an effector protein to chromatin and on the development of a new technique to further investigate more complicated chromatin-binding proteins.LCB
Dynamic Chromatin Invasion and Remodeling by the Transcription Factor Rap1
No full textPioneer transcription factors (PTF) are a subset of transcription factors with nucleosome-binding properties allowing them to bind specific sequences in condensed chromatin. Although the biological functions of PTFs including target genes and epigenetic changes have been well characterized in vivo, their mechanism of action remains unknown.
Here, we investigate dynamic chromatin binding of Rap1 (an essential Yeast PTF) in highly defined in-vitro single-molecule experiments. Rap1 is an essential regulatory protein whose DNA-binding sites are found in three types of chromosomal elements: promoters, silencers and telomeres. Moreover, its DNA targets are well characterized on a genome wide scale. Further, Rap1 binding sites have been found to co-localize with fragile, i.e. unstable and partially accessible nucleosomes. Due to its role as a PTF, Rap1 might be able to directly interact with compact chromatin and open chromatin structure in the process. Alternatively, it might capture transiently open states, stabilizing fragile nucleosomes.
To discriminate between these possibilities, we will investigate dynamic chromatin binding of Rap1 in highly defined in vitro single-molecule experiments. Our experiments include single-molecule FRET and single-molecule TIRF to probe the search and binding kinetics of Rap1 in different environments (i.e free DNA, mono-nucleosomes and chromatin) but also study the effect of Rap1 binding on chromatin. For this, we have designed a library of nucleosome positioning sequences containing Rap1 binding sites based on a well characterized promoter region found in vivo (Ribosomal Protein L30 promoter). Subsequently, these modular DNA sequences can be fluorescently labelled and inserted into large arrays of DNA allowing too later form defined chromatin fibres.
We have so far shown that Rap1 is indeed capable of binding nucleosomal DNA and that its binding is specific and modulated by the local chromatin environment. Furthermore, the sequence composition and position of its recognition site within the nucleosome and chromatin structure alters binding kinetics. Importantly, we also find that Rap1 opens local chromatin structure upon binding, thereby potentially providing access to the gene expression machinery.LCB
Investigating G-quadruplex dynamics in nucleosomes
No full textSCGCGR-53 1006; EPFL SB ISIC LCBM. Consultable sur demande à la Bibliothèque de l'EPFL / Offered in consultation at the EPFL library
Dynamic protein-chromatin interactions in heterochromatin and during the DNA damage response on the single molecule level
No full textDynamic regulation of chromatin, a structure consisting of DNA and histone proteins, is mediated through histone post-translational modifications (PTMs) and effector proteins. HP1a and PRC2 are recruited to repressive PTMs (H3K9me3, H3K27me3) and enable heterochromatin formation, which leads to gene silencing. During the DNA damage response, tight spatial and temporal control of the chromatin compaction state is required and novel PTMs are installed to guide proteins through dynamic signalling pathways. In the RNF168-mediated signalling cascade, appropriate DNA repair mechanisms are selected through ubiquitination (H2AK15ub) of chromatin. Improper regulation of these processes leads to cancer and neurodegenerative diseases. Here, we present a method to study chromatin-protein interactions employing total internal reflection fluorescence microscopy (TIRFM) and in vitro chromatin assembly of chemically defined, modified histones. Colocalization experiments of fluorescently labelled chromatin fibres and proteins allowed us to observe dynamic chromatin-protein interactions on the single-molecule level. We tested different binding models of HP1a, and we measured a highly dynamic behaviour of HP1a towards chromatin in dependence of the available binding sites or HP1a concentration. HP1a dimerization allows multivalent binding, increased chromatin association and decreased dissociation and thus leads to a more stable mode of chromatin binding. Further, we studied PRC2 recruitment to chromatin and DNA in dependence of the accessory factor PHF1. We measured increased residence times of PRC2 when PHF1 was included. Structure prediction and mutagenesis studies identified a novel winged-helix (WH) motif of PHF1, which was responsible for the enhanced binding effect in PRC2-PHF1 measurements. Moreover, binding of PRC2 towards chromatin was prolonged in the presence of H3K27me3, H3K27M or H3K36me3 histone PTMs. Next, we characterized the chromatin writer RNF168. We show altered ubiquitination activities dependent on accessibility of the nucleosome acidic patch, which was tested by addition of a competitor (RAPTA-C) or by introducing H4K16 acetylation, which leads to an open chromatin conformation. RNF168 is a multivalent reader of ubiquitin marks and we show binding specificity of ubiquitin binding module 1 (UDM1) towards native and synthetic K63-linked ubiquitin chains, while no interaction with K48-linked ubiquitin chains was detected. In contrast, we could not determine a clear contribution of the second binding module (UDM2), which recognizes H2A.XK15ub. In summary, we gained insight into different binding modes of HP1a, PRC2 and RNF168. Understanding basal protein binding mechanisms and chromatin regulation is essential to find new drug targets.LCB
Development and in vitro characterization of a genetically encoded split fluorescent probe targeting bivalent chromatin
No full textSCGCGR-53 1049; EPFL SB ISIC LCBM. Consultable sur demande à la Bibliothèque de l'EPFL / Offered in consultation at the EPFL library. Prix BAS
Development of multiplexed single-molecule imaging techniques to study chromatin invasion dynamics of chromatin effector proteins
No full textDNA-binding proteins physically interact with the DNA and directly affect genomic functions. The eukaryotic genome is compacted into chromatin, limiting the DNA access to nuclear factors. In this Ph.D. thesis, I explored the dynamic mechanisms, that allow the target recognition and access to genomic regions occupied by nucleosomes for the genome editor Cas9, the DNA-sensor protein cGAS, and the transcription factor MYC-MAX.
For mammalian genome editing applications, the bacterial Cas9 nuclease must target chromatinized DNA. Previous enzymatic studies showed that Cas9 nuclease activity is reduced by the presence of nucleosomes but there is still no systematic understanding of the mechanisms governing Cas9 targeting and nuclease activity in the chromatin context. To investigate if this loss of activity in chromatinized DNA is due to impaired DNA access or due to reduced residence time, I set out to systematically image chromatin invasion by Cas9 in real-time on the single-molecule level. Single-molecule measurements provide detailed mechanistic insight into intricate molecular processes, and are thus suitable to reveal how Cas9 dynamically engages nucleosomes. However, single-molecule experiments, in particular of complex chromatin samples, are difficult to perform and the reproducible and quantitative determination of parameters can be challenging.
I addressed this challenge and developed a new method - XSCAN (multiplexed single-molecule detection of chromatin association) - to parallelize dynamic single-molecule observations, where the interactions of dCas9 to many different types of nucleosomes are observed simultaneously in one single-molecule experiment. I provided each nucleosome type with an identifying DNA sequence, called â barcodeâ , within its nucleosomal DNA. Parallel experiments were subsequently spatially decoded, via the detection of specific binding of dye labelled DNA probes.
Using XSCAN, I then revealed that the time required for stable dCas9 binding is greatly increased for target sequences located further within nucleosome structure, as it is coupled to transient DNA unwrapping events at the nucleosome periphery. Moreover, nucleosomes decrease the association rate constant and suppress non-specific binding in vitro by shortening the residence time of dCas9 for sgRNA containing mismatches by up to 3-fold at more internal sites. This study provided the kinetics of nucleosome inhibition of Cas9 nuclease activity which is critical to the success of genome or epigenome editing applications.
In the following chapters of this thesis, I further optimized XSCAN and adopted the single-molecule colocalization microscopy techniques to study two other biological questions. First, I explored on the single-molecule level the inhibitory mechanism of the cGAS DNA sensor's self-activation by Barrier-to-autointegration factor. Secondly, to clarify the cryo-EM structure of transcription factor MYC-MAX bound to a nucleosome, I probed the MYC-MAX nucleosome binding on the single-molecule level.
Together, the work presented in this Ph.D. thesis shed light on the complex mechanisms of chromatin effectors â DNA/chromatin interactions. Furthermore, the newly established XSCAN is a robust, fast and semi-automated single-molecule technique, which I expect to be used by the scientific community in order to address new questions in chromatin biology.LCB
Mechanism of Chromatin Ubiquitination by PRC1 on the Single-Molecule Level
No full textMulticellular eukaryotic organisms contain identical genetic information within each cell, stored
within the nucleus as chromatin fibers. The regulation of gene expression allows cell-type-specific
temporal and spatial gene activation or repression despite the identical DNA sequence that determines
differentiation and cell identity. Chromatin 'writer' enzymes can alter chromatin by installing
post-translational modifications (PTMs) on histone proteins, which are then recognized by 'reader'
proteins. PTMs are epigenetic marks that regulate a dynamic yet balanced gene expression. Polycomb
group (PcG) proteins form a regulatory system of 'readers' and 'writers', essential for
transcriptional repression during development and differentiation.
Polycomb repressive complexes 1 (PRC1), a key member of the PcG family, function as the primary
E3 ligase 'writers' of ubiquitination on histone H2A at lysine 119 (H2AK119ub). The H2AK119ub
modification recruits Polycomb repressive complexes 2 (PRC2) to install H3K27me1-3 and form
repressive chromatin domains. Thus, variant PRC1 (vPRC1) is the primary catalyst of PcG-initialized
gene repression, with its activity dependent on its multisubunit composition, among others, the
Polycomb group of ring finger (PCGF) subunit. vPRC1 complexes dynamically engage chromatin,
but how these transient interactions are coupled to ubiquitin writing is unknown.
Here, I present a single-molecule fluorescence approach to directly observe ubiquitination dynamics
by vPRC1 in real-time on defined chromatin. Initially, I dissected the recruitment dynamics of
PRC1 complexes on immobilized synthetic chromatin. Subsequently, I introduced vPRC1 with a
preloaded E2~Ub to initiate H2AK119 ubiquitination. The observed binding and enzymatic kinetics
were used to construct a mechanistic model of real-time chromatin 'reading' and 'writing'.
vPRC1 transiently recruited chromatin fibers with non-specific and specific binding modes characterized
by short and long residence times. The presence of E2 increased the specific
residence time and the association rate, while a pre-installed H2AK119ub rescued the dissociation
and association times observed on wild-type chromatin. vPRC1 complexes dynamically searched
chromatin until they bound nucleosomes in a catalytically competent state, followed by E2~Ub
recruitment and ubiquitin transfer. The ubiquitination rate increased over time on wild-type H2A
chromatin fibers, while the ubiquitination efficiency on H2AK119R/K120R was minimal. vPRC1
showed a small processivity with up to two ubiquitin being transferred per single binding event.
The reactive binding and catalytic times of isolated events were independent of vPRC1 subunit
composition, while PCGF1-containing enzymes showed faster global ubiquitination due to more
frequent catalytically competent states. The efficiency of E2~Ub recruitment and the formation
of the catalytically active state determined the global activity differences between PRC1 subtypes
containing either PCGF1 or PCGF4. The findings demonstrate that the key factor influencing vPRC1
ubiquitination activity is the dynamic assembly of a ternary complex between chromatin, vPRC1
and E2.
The methodology may be expanded to study the enzymatic kinetics of other E3 ligase enzymes
and deubiquitinases on chromatin or other substrates. In particular, single-molecule enzymology
can be applied to characterize drugs' effects on the binding and enzymatic kinetics of E3 ligasLCB
A chemical biology approach to decipher chromatin ubiquitylation by RNF168
No full textDNA damage signaling following DNA double-strand breaks (DSBs) involves numerous regulating proteins, which dynamically recognize ('read') and alter ('write' or 'erase') histone post-translational modifications (PTMs). Among these PTMs, the ubiquitin system plays a key role in the two major pathways of DSB repair, homologous recombination (HR) and non-homologous end-joining (NHEJ), which are deregulated in many diseases, especially in cancer. Ubiquitylation of histone H2A at lysines 13 and 15 by the E3 ligase RNF168 plays a key role in orchestrating DSB repair, which is often deregulated in cancer. RNF168 activity is triggered by DSB signaling cascades, reportedly through K63-linked poly-ubiquitylation of linker histone H1. However, mechanistic insights into how ubiquitin recognition by RNF168 affects H2AK15Ub deposition have remained elusive.
This work aimed to examine the role of the E3 ubiquitin ligase RNF168 as a reader and writer of ubiquitylation marks in DNA repair from a chemical biology perspective. Here, I developed a method to chemically site-specifically di-ubiquitylate H1 on four different DNA repair-associated ubiquitylation hotspots on H1 (H1KxUb2, x = 17, 46, 64, 97). Incorporated H1KxUb2 variants in 'designer' chromatin fibers via in vitro reconstitution revealed the simulation of the E3 ligase activity of RNF168. Strikingly, the presence of H1KxUb2 enhanced the ubiquitylation activity of RNF168 compared to H1-containing and unmodified chromatin arrays, suggesting an effect beyond chromatin fiber opening. The stimulation of RNF168's ubiquitylation activity depended on its ubiquitin-binding capacity, confirming that ubiquitin recognition of di-ubiquitin on H1 was the driving force underlying the observed increase in ubiquitylation activity. Furthermore, the stimulatory effect of RNF168 ubiquitylation activity depended on the attachment site of ubiquitin moieties on H1, in particular, position 17 (H1K17Ub2). Finally, I studied the behavior of RNF168 in the presence of H1K17Ub2 in cellulo to gain insights into its binding behavior in a more complex environment. As the cellular context precludes selective control over PTMs, the previously synthesized, non-hydrolyzable triazole-linked H1K17Ub2 was introduced via bead-loading into U-2 OS cells, which displayed a dispersed nuclear distribution. With increasing bead-loading efficiencies of H1K17Ub2 (but not H1), RNF168 nuclear foci formation was disrupted, even in the presence of DNA-damaging treatment. Thus, H1K17Ub2 likely delocalized RNF168, thereby impeding its interaction with endogenous ubiquitin marks, such as poly-ubiquitylated H1, necessary for proper foci recruitment. Together, these observations indicate that K63-linked ubiquitin installed on H1 directs RNF168 recruitment to chromatin in cells.
Overall, this work provides mechanistic insights into the crosstalk of H1 and H2A ubiquitylation via RNF168. In particular, the streamlined synthesis of H1KxUb2 variants enables mechanistic studies into RNF168 regulation, with potential implications for its inhibition in susceptible cancers. In summary, the results strengthen the hypothesis that the direct binding of poly-ubiquitylated H1 by the RNF168 UDM1 domain is a key driving force underlying RNF168 recruitment to DNA damage sites.LCB
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