Regulation of chromatin architecture by transcription factor binding.

Transcription factors (TF) bind to chromatin and regulate the expression of genes. The pair Myc:Max binds to E-box regulatory DNA elements throughout the genome to control the transcription of a large group of specific genes. We introduce an implicit modeling protocol for Myc:Max binding to mesoscale chromatin fibers at nucleosome resolution to determine TF effect on chromatin architecture and shed light into its mechanism of gene regulation. We first bind Myc:Max to different chromatin locations and show how it can direct fiber folding and formation of microdomains, and how this depends on the linker DNA length. Second, by simulating increasing concentrations of Myc:Max binding to fibers that differ in the DNA linker length, linker histone density, and acetylation levels, we assess the interplay between Myc:Max and other chromatin internal parameters. Third, we study the mechanism of gene silencing by Myc:Max binding to the Eed gene loci. Overall, our results show how chromatin architecture can be regulated by TF binding. The position of TF binding dictates the formation of microdomains that appear visible only at the ensemble level. At the same time, the level of linker histone and tail acetylation, or different linker DNA lengths, regulates the concentration-dependent effect of TF binding. Furthermore, we show how TF binding can repress gene expression by increasing fiber folding motifs that help compact and occlude the promoter region. Importantly, this effect can be reversed by increasing linker histone density. Overall, these results shed light on the epigenetic control of the genome dictated by TF binding.

Regulation of Chromatin Architecture by Transcription Factor Binding.

Transcription factors (TF) bind to chromatin and regulate the expression of genes. The pair Myc:Max binds to E-box regulatory DNA elements throughout the genome, controlling transcription of a large group of specific genes. We introduce an implicit modeling protocol for Myc:Max binding to mesoscale chromatin fibers to determine TF effect on chromatin architecture and shed light on its mechanism of gene regulation. We first bind Myc:Max to different chromatin locations and show how it can direct fiber folding and formation of microdomains, and how this depends on the linker DNA length. Second, by simulating increasing concentrations of Myc:Max binding to fibers that differ in the DNA linker length, linker histone density, and acetylation levels, we assess the interplay between Myc:Max and other chromatin internal parameters. Third, we study the mechanism of gene silencing by Myc:Max binding to the Eed gene loci. Overall, our results show how chromatin architecture can be regulated by TF binding. The position of TF binding dictates the formation of microdomains that appear visible only at the ensemble level. On the other hand, the presence of linker histone, acetylations, or different linker DNA lengths regulates the concentration-dependent effect of TF binding. Furthermore, we show how TF binding can repress gene expression by increasing fiber folding motifs that help compact and occlude the promoter region. Importantly, this effect can be reversed by increasing linker histone density. Overall, these results shed light on the epigenetic control of the genome dictated by TF binding.

Techniques for and challenges in reconstructing 3D genome structures from 2D chromosome conformation capture data.

Chromosome conformation capture technologies that provide frequency information for contacts between genomic regions have been crucial for increasing our understanding of genome folding and regulation. However, such data do not provide direct evidence of the spatial 3D organization of chromatin. In this opinion article, we discuss the development and application of computational methods to reconstruct chromatin 3D structures from experimental 2D contact data, highlighting how such modeling provides biological insights and can suggest mechanisms anchored to experimental data. By applying different reconstruction methods to the same contact data, we illustrate some state-of-the-art of these techniques and discuss our gene resolution approach based on Brownian dynamics and Monte Carlo sampling.

Effect of Single-Residue Mutations on CTCF Binding to DNA: Insights from Molecular Dynamics Simulations.

In humans and other eukaryotes, DNA is condensed into chromatin fibers that are further wound into chromosomes. This organization allows regulatory elements in the genome, often distant from each other in the linear DNA, to interact and facilitate gene expression through regions known as topologically associating domains (TADs). CCCTC-binding factor (CTCF) is one of the major components of TAD formation and is responsible for recruiting a partner protein, cohesin, to perform loop extrusion and facilitate proper gene expression within TADs. Because single-residue CTCF mutations have been linked to the development of a variety of cancers in humans, we aim to better understand how these mutations affect the CTCF structure and its interaction with DNA. To this end, we compare all-atom molecular dynamics simulations of a wildtype CTCF-DNA complex to those of eight different cancer-linked CTCF mutant sequences. We find that most mutants have lower binding energies compared to the wildtype protein, leading to the formation of less stable complexes. Depending on the type and position of the mutation, this loss of stability can be attributed to major changes in the electrostatic potential, loss of hydrogen bonds between the CTCF and DNA, and/or destabilization of specific zinc fingers. Interestingly, certain mutations in specific fingers can affect the interaction with the DNA of other fingers, explaining why mere single mutations can impair CTCF function. Overall, these results shed mechanistic insights into experimental observations and further underscore CTCF's importance in the regulation of chromatin architecture and gene expression.

Genome modeling: From chromatin fibers to genes.

The intricacies of the 3D hierarchical organization of the genome have been approached by many creative modeling studies. The specific model/simulation technique combination defines and restricts the system and phenomena that can be investigated. We present the latest modeling developments and studies of the genome, involving models ranging from nucleosome systems and small polynucleosome arrays to chromatin fibers in the kb-range, chromosomes, and whole genomes, while emphasizing gene folding from first principles. Clever combinations allow the exploration of many interesting phenomena involved in gene regulation, such as nucleosome structure and dynamics, nucleosome-nucleosome stacking, polynucleosome array folding, protein regulation of chromatin architecture, mechanisms of gene folding, loop formation, compartmentalization, and structural transitions at the chromosome and genome levels. Gene-level modeling with full details on nucleosome positions, epigenetic factors, and protein binding, in particular, can in principle be scaled up to model chromosomes and cells to study fundamental biological regulation.

Brownian dynamics simulations of mesoscale chromatin fibers.

The relationship between chromatin architecture and function defines a central problem in biology and medicine. Many computational chromatin models with atomic, coarse-grained, mesoscale, and polymer resolution have been used to shed light onto the mechanisms that dictate genome folding and regulation of gene expression. The associated simulation techniques range from Monte Carlo to molecular, Brownian, and Langevin dynamics. Here, we present an efficient Compute Unified Device Architecture (CUDA) implementation of Brownian dynamics (BD) to simulate chromatin fibers at the nucleosome resolution with our chromatin mesoscale model. With the CUDA implementation for computer architectures with graphic processing units (GPUs), we significantly accelerate compute-intensive hydrodynamic tensor calculations in the BD simulations by massive parallelization, boosting the performance a hundred-fold compared with central processing unit calculations. We validate our BD simulation approach by reproducing experimental trends on fiber diffusion and structure as a function of salt, linker histone binding, and histone-tail composition, as well as Monte Carlo equilibrium sampling results. Our approach proves to be physically accurate with performance that makes feasible the study of chromatin fibers in the range of kb or hundreds of nucleosomes (small gene). Such simulations are essential to advance the study of biological processes such as gene regulation and aberrant genome-structure related diseases.

Chromatin transitions triggered by LH density as epigenetic regulators of the genome.

Motivated by experiments connecting linker histone (LH) deficiency to lymphoma progression and retinal disorders, we study by mesoscale chromatin modeling how LH density (ρ) induces gradual, as well sudden, changes in chromatin architecture and how the process depends on DNA linker length, LH binding dynamics and binding mode, salt concentration, tail modifications, and combinations of ρ and linker DNA length. We show that ρ tightly regulates the overall shape and compaction of the fiber, triggering a transition from an irregular disordered state to a compact and ordered structure. Such a structural transition, resembling B to A compartment transition connected with lymphoma of B cells, appears to occur around ρ = 0.5. The associated mechanism is DNA stem formation by LH binding, which is optimal when the lengths of the DNA linker and LH C-terminal domain are similar. Chromatin internal and external parameters are key regulators, promoting or impeding the transition. The LH density thus emerges as a critical tunable variable in controlling cellular functions through structural transitions of the genome.

Local chromatin fiber folding represses transcription and loop extrusion in quiescent cells.

A longstanding hypothesis is that chromatin fiber folding mediated by interactions between nearby nucleosomes represses transcription. However, it has been difficult to determine the relationship between local chromatin fiber compaction and transcription in cells. Further, global changes in fiber diameters have not been observed, even between interphase and mitotic chromosomes. We show that an increase in the range of local inter-nucleosomal contacts in quiescent yeast drives the compaction of chromatin fibers genome-wide. Unlike actively dividing cells, inter-nucleosomal interactions in quiescent cells require a basic patch in the histone H4 tail. This quiescence-specific fiber folding globally represses transcription and inhibits chromatin loop extrusion by condensin. These results reveal that global changes in chromatin fiber compaction can occur during cell state transitions, and establish physiological roles for local chromatin fiber folding in regulating transcription and chromatin domain formation.

Biomolecular modeling thrives in the age of technology.

The biomolecular modeling field has flourished since its early days in the 1970s due to the rapid adaptation and tailoring of state-of-the-art technology. The resulting dramatic increase in size and timespan of biomolecular simulations has outpaced Moore's law. Here, we discuss the role of knowledge-based versus physics-based methods and hardware versus software advances in propelling the field forward. This rapid adaptation and outreach suggests a bright future for modeling, where theory, experimentation and simulation define three pillars needed to address future scientific and biomedical challenges.

Biomolecular Modeling and Simulation: A Prospering Multidisciplinary Field.

We reassess progress in the field of biomolecular modeling and simulation, following up on our perspective published in 2011. By reviewing metrics for the field's productivity and providing examples of success, we underscore the productive phase of the field, whose short-term expectations were overestimated and long-term effects underestimated. Such successes include prediction of structures and mechanisms; generation of new insights into biomolecular activity; and thriving collaborations between modeling and experimentation, including experiments driven by modeling. We also discuss the impact of field exercises and web games on the field's progress. Overall, we note tremendous success by the biomolecular modeling community in utilization of computer power; improvement in force fields; and development and application of new algorithms, notably machine learning and artificial intelligence. The combined advances are enhancing the accuracy andscope of modeling and simulation, establishing an exemplary discipline where experiment and theory or simulations are full partners.

Mesoscale Modeling and Single-Nucleosome Tracking Reveal Remodeling of Clutch Folding and Dynamics in Stem Cell Differentiation.

Nucleosomes form heterogeneous groups in vivo, named clutches. Clutches are smaller and less dense in mouse embryonic stem cells (ESCs) compared to neural progenitor cells (NPCs). Using coarse-grained modeling of the pluripotency Pou5f1 gene, we show that the genome-wide clutch differences between ESCs and NPCs can be reproduced at a single gene locus. Larger clutch formation in NPCs is associated with changes in the compaction and internucleosome contact probability of the Pou5f1 fiber. Using single-molecule tracking (SMT), we further show that the core histone protein H2B is dynamic, and its local mobility relates to the structural features of the chromatin fiber. H2B is less stable and explores larger areas in ESCs compared to NPCs. The amount of linker histone H1 critically affects local H2B dynamics. Our results have important implications for how nucleosome organization and H2B dynamics contribute to regulate gene activity and cell identity.

A MULTISCALE VISION-ILLUSTRATIVE APPLICATIONS FROM BIOLOGY TO ENGINEERING.

Modeling and simulation have quickly become equivalent pillars of research along with traditional theory and experimentation. The growing realization that most complex phenomena of interest span many orders of spatial and temporal scales has led to an exponential rise in the development and application of multiscale modeling and simulation over the past two decades. In this perspective, the associate editors of the International Journal for Multiscale Computational Engineering and their co-workers illustrate current applications in their respective fields spanning biomolecular structure and dynamics, civil engineering and materials science, computational mechanics, aerospace and mechanical engineering, and more. Such applications are highly tailored, exploit the latest and ever-evolving advances in both computer hardware and software, and contribute significantly to science, technology, and medical challenges in the 21st century.

Histone H1 loss drives lymphoma by disrupting 3D chromatin architecture.

Linker histone H1 proteins bind to nucleosomes and facilitate chromatin compaction1, although their biological functions are poorly understood. Mutations in the genes that encode H1 isoforms B-E (H1B, H1C, H1D and H1E; also known as H1-5, H1-2, H1-3 and H1-4, respectively) are highly recurrent in B cell lymphomas, but the pathogenic relevance of these mutations to cancer and the mechanisms that are involved are unknown. Here we show that lymphoma-associated H1 alleles are genetic driver mutations in lymphomas. Disruption of H1 function results in a profound architectural remodelling of the genome, which is characterized by large-scale yet focal shifts of chromatin from a compacted to a relaxed state. This decompaction drives distinct changes in epigenetic states, primarily owing to a gain of histone H3 dimethylation at lysine 36 (H3K36me2) and/or loss of repressive H3 trimethylation at lysine 27 (H3K27me3). These changes unlock the expression of stem cell genes that are normally silenced during early development. In mice, loss of H1c and H1e (also known as H1f2 and H1f4, respectively) conferred germinal centre B cells with enhanced fitness and self-renewal properties, ultimately leading to aggressive lymphomas with an increased repopulating potential. Collectively, our data indicate that H1 proteins are normally required to sequester early developmental genes into architecturally inaccessible genomic compartments. We also establish H1 as a bona fide tumour suppressor and show that mutations in H1 drive malignant transformation primarily through three-dimensional genome reorganization, which leads to epigenetic reprogramming and derepression of developmentally silenced genes.

Nucleosome Clutches are Regulated by Chromatin Internal Parameters.

Nucleosomes cluster together when chromatin folds in the cell to form heterogeneous groups termed "clutches". These structural units add another level of chromatin regulation, for example during cell differentiation. Yet, the mechanisms that regulate their size and compaction remain obscure. Here, using our chromatin mesoscale model, we dissect clutch patterns in fibers with different combinations of nucleosome positions, linker histone density, and acetylation levels to investigate their role in clutch regulation. First, we isolate the effect of each chromatin parameter by studying systems with regular nucleosome spacing; second, we design systems with naturally-occurring linker lengths that fold onto specific clutch patterns; third, we model gene-encoding fibers to understand how these combined factors contribute to gene structure. Our results show how these chromatin parameters act together to produce different-sized nucleosome clutches. The length of nucleosome free regions (NFRs) profoundly affects clutch size, while the length of linker DNA has a moderate effect. In general, higher linker histone densities produce larger clutches by a chromatin compaction mechanism, while higher acetylation levels produce smaller clutches by a chromatin unfolding mechanism. We also show that it is possible to design fibers with naturally-occurring DNA linkers and NFRs that fold onto specific clutch patterns. Finally, in gene-encoding systems, a complex combination of variables dictates a gene-specific clutch pattern. Together, these results shed light into the mechanisms that regulate nucleosome clutches and suggest a new epigenetic mechanism by which chromatin parameters regulate transcriptional activity via the three-dimensional folded state of the genome at a nucleosome level.

Bridging chromatin structure and function over a range of experimental spatial and temporal scales by molecular modeling.

Chromatin structure, dynamics, and function are being intensely investigated by a variety of methods, including microscopy, X-ray diffraction, nuclear magnetic resonance, biochemical crosslinking, chromosome conformation capture, and computation. A range of experimental techniques combined with modeling is clearly valuable to help interpret experimental data and, importantly, generate configurations and mechanisms related to the 3D organization and function of the genome. Contact maps, in particular, as obtained by a variety of chromosome conformation capture methods, are of increasing interest due to their implications on genome structure and regulation on many levels. In this perspective, using seven examples from our group's studies, we illustrate how molecular modeling can help interpret such experimental data. Specifically, we show how computed contact maps related to experimental systems can be used to explain structures of nucleosomes, chromatin higher-order folding, domain segregation mechanisms, gene organization, and the effect on chromatin structure of external and internal fiber parameters, such as nucleosome positioning, presence of nucleosome free regions, histone posttranslational modifications, and linker histone binding. We argue that such computations on multiple spatial and temporal scales will be increasingly important for the integration of genomic, epigenomic, and biophysical data on chromatin structure and related cellular processes.

Sensitive effect of linker histone binding mode and subtype on chromatin condensation.

The complex role of linker histone (LH) on chromatin compaction regulation has been highlighted by recent discoveries of the effect of LH binding variability and isoforms on genome structure and function. Here we examine the effect of two LH variants and variable binding modes on the structure of chromatin fibers. Our mesoscale modeling considers oligonucleosomes with H1C and H1E, bound in three different on and off-dyad modes, and spanning different LH densities (0.5-1.6 per nucleosome), over a wide range of physiologically relevant nucleosome repeat lengths (NRLs). Our studies reveal an LH-variant and binding-mode dependent heterogeneous ensemble of fiber structures with variable packing ratios, sedimentation coefficients, and persistence lengths. For maximal compaction, besides dominantly interacting with parental DNA, LHs must have strong interactions with nonparental DNA and promote tail/nonparental core interactions. An off-dyad binding of H1E enables both; others compromise compaction for bendability. We also find that an increase of LH density beyond 1 is best accommodated in chromatosomes with one on-dyad and one off-dyad LH. We suggest that variable LH binding modes and concentrations are advantageous, allowing tunable levels of chromatin condensation and DNA accessibility/interactions. Thus, LHs add another level of epigenetic regulation of chromatin.

Differential Kinetics of Two-Cysteine Peroxiredoxin Disulfide Formation Reveal a Novel Model for Peroxide Sensing.

Two-cysteine peroxiredoxins (Prx) have a three-step catalytic cycle consisting of (1) reduction of peroxide and formation of sulfenic acid on the enzyme, (2) condensation of the sulfenic acid with a thiol to form disulfide, also known as resolution, and (3) reduction of the disulfide by a reductant protein. By following changes in protein fluorescence, we have studied the pH dependence of reaction 2 in human peroxiredoxins 1, 2, and 5 and in Salmonella typhimurium AhpC and obtained rate constants for the reaction and p Ka values of the thiol and sulfenic acid involved for each system. The observed reaction 2 rate constant spans 2 orders of magnitude, but in all cases, reaction 2 appears to be slow compared to the same reaction in small-molecule systems, making clear the rates are limited by conformational features of the proteins. For each Prx, reaction 2 will become rate-limiting at some critical steady-state concentration of H2O2 producing the accumulation of Prx as sulfenic acid. When this happens, an alternative and faster-resolving Prx (or other peroxidase) may take over the antioxidant role. The accumulation of sulfenic acid Prx at distinct concentrations of H2O2 is embedded in the kinetic limitations of the catalytic cycle and may constitute the basis of a H2O2-mediated redox signal transduction pathway requiring neither inactivation nor posttranslational modification. The differences in the rate constants of resolution among Prx coexisting in the same compartment may partially explain their complementation in antioxidant function and stepwise sensing of H2O2 concentration.

Catalytic Thr or Ser Residue Modulates Structural Switches in 2-Cys Peroxiredoxin by Distinct Mechanisms.

Typical 2-Cys Peroxiredoxins (2-Cys Prxs) reduce hydroperoxides with extraordinary rates due to an active site composed of a catalytic triad, containing a peroxidatic cysteine (CP), an Arg, and a Thr (or Ser). 2-Cys Prx are involved in processes such as cancer; neurodegeneration and host-pathogen interactions. During catalysis, 2-Cys Prxs switch between decamers and dimers. Analysis of 2-Cys Prx structures in the fully folded (but not locally unfolded) form revealed a highly conserved, non-conventional hydrogen bond (CH-π) between the catalytic triad Thr of a dimer with an aromatic residue of an adjacent dimer. In contrast, structures of 2-Cys Prxs with a Ser in place of the Thr do not display this CH-π bond. Chromatographic and structural data indicate that the Thr (but not Ser) destabilizes the decamer structure in the oxidized state probably through steric hindrance. As a general trend, mutations in a yeast 2-Cys Prx (Tsa1) favoring the dimeric state also displayed a decreased catalytic activity. Remarkably, yeast naturally contains Thr-Ser variants (Tsa1 and Tsa2, respectively) with distinct oligomeric stabilities in their disulfide states.

Deconstructing the catalytic efficiency of peroxiredoxin-5 peroxidatic cysteine.

Human peroxiredoxin-5 (PRDX5) is a thiol peroxidase that reduces H2O2 10(5) times faster than free cysteine. To assess the influence of two conserved residues on the reactivity of the critical cysteine (C47), we determined the reaction rate constants of PRDX5, wild type (WT), T44V and R127Q with one substrate electrophile (H2O2) and a nonspecific electrophile (monobromobimane). We also studied the corresponding reactions of low molecular weight (LMW) thiolates in order to construct a framework against which we could compare our proteins. To obtain a detailed analysis of the structural and energetic changes involved in the reaction between WT PRDX5 and H2O2, we performed ONIOM quantum mechanics/molecular mechanics (QM/MM) calculations with a QM region including 60 atoms of substrate and active site described by the B3LYP density functional and the 6-31+G(d,p) basis set; the rest of the protein was included in the MM region. Brønsted correlations reveal that the absence of T44 can increase the general nucleophilicity of the C47 but decreases the specific reactivity toward H2O2 by a factor of 10(3). The R127Q mutation causes C47 to behave like a LMW thiolate in the two studied reactions. QM/MM results with WT PRDX5 showed that hydrogen bonds in the active site are the cornerstone of two effects that make catalysis possible: the enhancement of thiolate nucleophilicity upon substrate ingress and the stabilization of the transition state. In both effects, T44 has a central role. These effects occur in a precise temporal sequence that ensures that the selective nucleophilicity of C47 is available only for peroxide substrates.