Multidomain Transcription Factor Research Articles

Simulations reveal unique roles for the FXR hinge in the FXR-RXR nuclear receptor heterodimer.

Nuclear receptors are multidomain transcription factors whose full-length quaternary architecture is poorly described and understood. Most nuclear receptors bind DNA as heterodimers or homodimers, which could encompass a variety of arrangements of the individual domains. Only a handful of experimental structures currently exist describing these architectures. Given that domain interactions and protein-DNA interactions within transcriptional complexes are tightly linked to function, understanding the arrangement of nuclear receptor domains on DNA is of utmost importance. Here, we employ modeling and molecular dynamics (MD) simulations to describe the structure of the full-length farnesoid X receptor (FXR) and retinoid X receptor alpha (RXR) heterodimer bound to DNA. Combining over 100 microseconds of atomistic MD simulations with enhanced sampling simulations, we characterize the dynamic behavior of eight FXR-RXR-DNA complexes, showing that these complexes support a range of quaternary architectures. Our simulations reveal critical roles for the hinge in the DNA-bound dimer in facilitating interdomain allostery, mediating DNA binding and driving the dynamic flexibility of the complex. These roles have been hard to appreciate previously due to experimental limitations in studying the flexible hinge. These studies provide a much-needed framework that will enable the field to obtain a complete understanding of nuclear receptor quaternary architectures.

Oct4 interprets and enhances nucleosome dynamics.

Transcription factors are proteins that bind to DNA to regulate gene expression. Historically, DNA accessibility has been considered a prerequisite indispensable for transcription factor function. Nevertheless, in recent years certain transcription factors have been shown to have “pioneering activity”, meaning that they can bind their specific binding sites in packed regions of the genome, where the DNA is wrapped around a nucleoprotein complex called the nucleosome. Oct4 is a pioneer transcription factor, involved in pluripotency maintenance and that can be used for reprogramming of adult cells into stem-cell like cells. Using experimental data, we built models of Oct4 bound to two different native nucleosomes. We then performed over 50 µs of atomistic simulations of the Oct4-nucleosome complexes, which we compared to our already existing library of 25 µs of nucleosome alone simulations. We found that nucleosome flexibility is a requirement for a multidomain transcription factor like Oct4 to bind. Furthermore, we described two different mechanisms in which Oct4 can alter nucleosome dynamics, either by stabilizing naturally occurring nucleosome opening events or inducing large opening events not seen in the nucleosome alone simulations. The atomistic resolution allowed us to observe an interplay between the histone tails and the two DNA binding domains of Oct4 for the opening events to occur. Overall, our findings provide a mechanistic description of the effects of a pioneer transcription factor bound to a nucleosome, at an unprecedented resolution. This work allows us to begin understanding the changes of genome architecture that happen upon expression and binding of certain transcription factors, and ultimately gives us the tools to understand cell-fate transitions.

Zeb2 regulates the balance between retinal interneurons and Müller glia by inhibition of BMP–Smad signaling

The interplay between signaling molecules and transcription factors during retinal development is key to controlling the correct number of retinal cell types. Zeb2 (Sip1) is a zinc-finger multidomain transcription factor that plays multiple roles in central and peripheral nervous system development. Haploinsufficiency of ZEB2 causes Mowat-Wilson syndrome, a congenital disease characterized by intellectual disability, epilepsy and Hirschsprung disease. In the developing retina, Zeb2 is required for generation of horizontal cells and the correct number of interneurons; however, its potential function in controlling gliogenic versus neurogenic decisions remains unresolved. Here we present cellular and molecular evidence of the inhibition of Müller glia cell fate by Zeb2 in late stages of retinogenesis. Unbiased transcriptomic profiling of control and Zeb2-deficient early-postnatal retina revealed that Zeb2 functions in inhibiting Id1/2/4 and Hes1 gene expression. These neural progenitor factors normally inhibit neural differentiation and promote Müller glia cell fate. Chromatin immunoprecipitation (ChIP) supported direct regulation of Id1 by Zeb2 in the postnatal retina. Reporter assays and ChIP analyses in differentiating neural progenitors provided further evidence that Zeb2 inhibits Id1 through inhibition of Smad-mediated activation of Id1 transcription. Together, the results suggest that Zeb2 promotes the timely differentiation of retinal interneurons at least in part by repressing BMP–Smad/Notch target genes that inhibit neurogenesis. These findings show that Zeb2 integrates extrinsic cues to regulate the balance between neuronal and glial cell types in the developing murine retina.

Assessing Allostery in Intrinsically Disordered Proteins With Ensemble Allosteric Model.

Intrinsically disordered (ID) proteins have been shown to play a major role in signaling in a broad range of proteins, using a process known as allostery, wherein the protein can integrate one or a number of inputs to regulate its function. The disorder-mediated allostery can be understood energetically with ensemble allosteric model (EAM). In this model, the molecule without effectors is considered as an ensemble of preexisting conformations, and effector binding is treated as an energetic perturbation of the ensemble to redistribute the microstates that are favorable or unfavorable to the second binding partner. As it only considers the intrinsic energetics of the system and does not depend on a crystallographic structure, it can be applied to both structured proteins, ID proteins, and mixed proteins with both structured and ID domains. Simulation with EAM on the basis of experimental data can help quantitatively explain experimental observations, as well as to make predictions to direct future research. This has recently been illustrated with the case of human glucocorticoid receptor, a multidomain transcription factor that contains both structured and disordered regions. In this chapter, we describe the assays for measuring the transcriptional activity, binding affinity to cognate DNA, conformational stability, either on single domain or tandem coupled domains in the GR two-domain isoforms. We then explain how these data are utilized as input parameters or constraints in the EAM for quantitative estimates of stabilities and coupling energies for each domain through global minimization method.

The Disordered Linker in p53 Participates in Nonspecific Binding to and One-Dimensional Sliding along DNA Revealed by Single-Molecule Fluorescence Measurements.

The tumor suppressor p53 is a multidomain transcription factor that can quickly bind to its target DNA by sliding along the DNA strand. We hypothesized that the intrinsically disordered and positively charged linker of p53 regulates its search dynamics first by directly interacting with DNA and second by modulating hopping of the core domain. To test the two hypotheses, we prepared five variants of p53 in which the length and charge of the linker were modulated. The affinity for and sliding along nonspecific DNA of p53 were altered by the charge of the linker, but not by the linker length. In particular, charge neutralization significantly reduced the affinity, suggesting that the linker directly contacts the DNA. Charge neutralization eliminated the slow mode of sliding, in which the core domain was assumed to contact nonspecific DNA. In contrast, the affinity of p53 for the target DNA was not affected by linker mutations. These results demonstrate that the linker participates in a target search of p53 by contacting nonspecific DNA and recruiting the core domain to contact DNA.

Cooperative DNA Recognition Modulated by an Interplay between Protein-Protein Interactions and DNA-Mediated Allostery.

Highly specific transcriptional regulation depends on the cooperative association of transcription factors into enhanceosomes. Usually, their DNA-binding cooperativity originates from either direct interactions or DNA-mediated allostery. Here, we performed unbiased molecular simulations followed by simulations of protein-DNA unbinding and free energy profiling to study the cooperative DNA recognition by OCT4 and SOX2, key components of enhanceosomes in pluripotent cells. We found that SOX2 influences the orientation and dynamics of the DNA-bound configuration of OCT4. In addition SOX2 modifies the unbinding free energy profiles of both DNA-binding domains of OCT4, the POU specific and POU homeodomain, despite interacting directly only with the first. Thus, we demonstrate that the OCT4-SOX2 cooperativity is modulated by an interplay between protein-protein interactions and DNA-mediated allostery. Further, we estimated the change in OCT4-DNA binding free energy due to the cooperativity with SOX2, observed a good agreement with experimental measurements, and found that SOX2 affects the relative DNA-binding strength of the two OCT4 domains. Based on these findings, we propose that available interaction partners in different biological contexts modulate the DNA exploration routes of multi-domain transcription factors such as OCT4. We consider the OCT4-SOX2 cooperativity as a paradigm of how specificity of transcriptional regulation is achieved through concerted modulation of protein-DNA recognition by different types of interactions.

Search by proteins for their DNA target site: 2. The effect of DNA conformation on the dynamics of multidomain proteins.

Multidomain transcription factors, which are especially abundant in eukaryotic genomes, are advantageous to accelerate the search kinetics for target site because they can follow the intersegment transfer via the monkey-bar mechanism in which the protein forms a bridged intermediate between two distant DNA regions. Monkey-bar dynamics highly depends on the properties of the multidomain protein (the affinity of each of the constituent domains to the DNA and the length of the linker) and the DNA molecules (their inter-distance and inter-angle). In this study, we investigate using coarse-grained molecular dynamics simulations how the local conformation of the DNA may affect the DNA search performed by a multidomain protein Pax6 in comparison to that of the isolated domains. Our results suggest that in addition to the common rotation-coupled translation along the DNA major groove, for curved DNA the tethered domains may slide in a rotation-decoupled sliding mode. Furthermore, the multidomain proteins move by longer jumps on curved DNA compared with those performed by the single domain protein. The long jumps originate from the DNA curvature bringing two sequentially distant DNA sites into close proximity with each other and they suggest that multidomain proteins may move on highly curved DNA faster than linear DNA.

Please, Get in My Way So That I Can Be More Efficient!

Please, Get in My Way So That I Can Be More Efficient!

Transient expression, purification and characterisation of human full-length PPARγ2 in HEK293 cells

Effective anti-diabetic drugs known as thiazolidinediones (e.g. rosiglitazone, pioglitazone) exert their therapeutic effects through their agonistic activity at the peroxisome proliferator-activated receptor gamma (PPARγ). As a multidomain transcription factor, PPARγ forms heterodimers with different retinoid X receptors (RXRs) to modulate target gene expression at the transcriptional level in response to natural or synthetic ligands. Difficulties in producing either of the two major human PPARγ isoforms (PPARγ1 and PPARγ2) as pure full-length proteins in adequate quantity has hindered detailed mechanistic studies of PPARγ and its ancillary protein partners. Here we report an efficient transient expression system to produce recombinant human full-length PPARγ2 protein. The DNA encoding the human full-length PPARγ2 was cloned into a mammalian episomal vector and transiently expressed in human embryonic kidney 293 (HEK293-6E) cells with an expression level of 10mg/L culture. Identity of the purified recombinant PPARγ2 protein was confirmed by mass spectrometry analysis. The purified PPARγ2 protein was active in ligand binding and could be phosphorylated in vitro by Cdk5/p25 at Ser 273. Further studies showed that selected PPARγ modulators inhibited Cdk5-mediated PPARγ2 Ser 273 phosphorylation in vitro. Our results demonstrate the feasibility of producing large quantities of pure and functional human full-length PPARγ2 suitable for drug discovery applications.

Facilitated DNA Search by Multidomain Transcription Factors: Cross Talk via a Flexible Linker

More than 70% of eukaryotic proteins are composed of multiple domains. However, most studies of the search for DNA focus on individual protein domains and do not consider potential cross talk within a multidomain transcription factor. In this study, the molecular features of the DNA search mechanism were explored for two multidomain transcription factors: human Pax6 and Oct-1. Using a simple computational model, we compared a DNA search of multidomain proteins with a search of isolated domains. Furthermore, we studied how manipulating the binding affinity of a single domain to DNA can affect the overall DNA search of the multidomain protein. Tethering the two domains via a flexible linker increases their affinity to the DNA, resulting in a higher propensity for sliding along the DNA, which is more significant for the domain with the weaker DNA-binding affinity. In this case, the domain that binds DNA more tightly anchors the multidomain protein to the DNA and, via the linker, increases the local concentration of the weak DNA-binding domain (DBD). The tethered domains directly exchange between two parallel DNA molecules via a bridged intermediate, where intersegmental transfer is promoted by the weaker DBD. We found that, in general, the relative affinity of the two domains can significantly affect the cross talk between them and thus their overall capability to search DNA efficiently. The results we obtained by examining various multidomain DNA-binding proteins support the necessity of discrepancies between the DNA-binding affinities of the constituent domains.

Structure of the intact PPAR-γ–RXR-α nuclear receptor complex on DNA

Nuclear receptors are multi-domain transcription factors that bind to DNA elements from which they regulate gene expression. The peroxisome proliferator-activated receptors (PPARs) form heterodimers with the retinoid X receptor (RXR), and PPAR-gamma has been intensively studied as a drug target because of its link to insulin sensitization. Previous structural studies have focused on isolated DNA or ligand-binding segments, with no demonstration of how multiple domains cooperate to modulate receptor properties. Here we present structures of intact PPAR-gamma and RXR-alpha as a heterodimer bound to DNA, ligands and coactivator peptides. PPAR-gamma and RXR-alpha form a non-symmetric complex, allowing the ligand-binding domain (LBD) of PPAR-gamma to contact multiple domains in both proteins. Three interfaces link PPAR-gamma and RXR-alpha, including some that are DNA dependent. The PPAR-gamma LBD cooperates with both DNA-binding domains (DBDs) to enhance response-element binding. The A/B segments are highly dynamic, lacking folded substructures despite their gene-activation properties.

Global jumping and domain-specific intersegment transfer between DNA cognate sites of the multidomain transcription factor Oct-1

At high DNA concentration, as found in the nucleus, DNA-binding proteins search for specific binding sites by hopping between separate DNA strands. Here, we use (15)N(z)-exchange transverse relaxation optimized NMR spectroscopy to characterize the mechanistic details of intermolecular hopping for the multidomain transcription factor, human Oct-1. Oct-1 is a member of the POU family of transcription factors and contains two helix-turn-helix DNA-binding domains, POU(HD) and POU(S), connected by a relatively short flexible linker. The two domains were found to exchange between specific sites at significantly different rates. The cotranscription factor, Sox2, decreases the exchange rate and equilibrium dissociation constant for Oct-1 > or = 5-fold and approximately 20-fold, respectively, by slowing the exchange rate for the POU(S) domain. DNA-dependent exchange rates measured at physiological ionic strength indicate that the two domains use both an intersegmental transfer mechanism, which does not involve the intermediary of free protein, and a fully dissociative or jumping mechanism to translocate between cognate sites. These data represent an example of dissecting domain-specific kinetics for protein-DNA association involving a multidomain protein and provide evidence that intersegmental transfer involves a ternary intermediate, or transition state in which the DNA-binding domains bridge two different DNA fragments simultaneously.

Regulation of DNA Binding of p53 by its C-terminal Domain

The tumor suppressor p53 is a tetrameric multi-domain transcription factor. Its C-terminal domain is thought to regulate the binding of its core domain to specific recognition sequences in promoters. The mechanism of regulation by the C-terminal domain and the role of its post-translational modification are controversial. We have examined the binding of DNA in solution to a series of unmodified p53 constructs that lack various domains. The specific DNA sequences bind tightly to the core domain, irrespective of whether or not the C-terminal domain is part of the construct. Unmodified p53 is accordingly an active DNA binding protein. Non-specific DNA sequences do not inhibit directly the binding of the specific sequences to the core but bind to the C terminus and inhibit p53 via that binding mode. Using NMR, we identified the residues of the C terminus that interact with the non-specific DNA. They include residues that are known to be modified post-translationally. Our data provide direct support for the regulatory role of the C terminus in the activity of p53 and show that p53 containing the unmodified C terminus actively binds to short double-stranded DNA.

Cupins: the most functionally diverse protein superfamily?

The cupin superfamily of proteins, named on the basis of a conserved β-barrel fold (‘cupa’ is the Latin term for a small barrel), was originally discovered using a conserved motif found within germin and germin-like proteins from higher plants. Previous analysis of cupins had identified some 18 different functional classes that range from single-domain bacterial enzymes such as isomerases and epimerases involved in the modification of cell wall carbohydrates, through to two-domain bicupins such as the desiccation-tolerant seed storage globulins, and multidomain transcription factors including one linked to the nodulation response in legumes. Recent advances in comparative genomics, and the resolution of many more 3-D structures have now revealed that the largest subset of the cupin superfamily is the 2-oxyglutarate-Fe2+ dependent dioxygenases. The substrates for this subclass of enzyme are many and varied and in total amount to probably 50–100 different biochemical reactions, including several involved in plant growth and development. Although the majority of enzymatic cupins contain iron as an active site metal, other members contain either copper, zinc, cobalt, nickel or manganese ions as a cofactor, with each cofactor allowing a different type of chemistry to occur within the conserved tertiary structure. This review discusses the range of structures and functions found in this most diverse of superfamilies.

Retinoid metabolizing enzymes in development.

It has long been known that retinoids are necessary for normal embryogenesis. To understand the role of retinoids in this process, it is important to elucidate the retinoid signalling pathway. The retinoid signal transduction pathway consists of three major components: (i) the metabolism of biologically active retinoids, (ii) nuclear receptors which bind to and are activated by retinoids, and (iii) the genes whose expression is regulated by the receptor/ligand complex. The initiation of the retinoid signal transduction pathway critically depends of the presence of biologically active retinoids. The production and degradation of these biologically active retinoids will be the main focus this brief review. In addition, we will discuss the developmental aspects of retinoid metabolism. Several retinoids including all-trans-retinoic acid, 9-cis-retinoic acid, 4-oxo-all-trans-retinoic acid, and 3,4-didehydro-all-trans-retinoic acid have been shown to be biologically active in the developing embryo [7–10,13,17,18,32,33,35]. The all-trans isomers of these biologically active retinoids bind to retinoic acid receptors (RARs) while 9-cis-retinoic acid binds to retinoid X receptors (RXRs) as well as to RARs. RARs and RXRs belong to the family of nuclear hormone receptors. These are multidomain transcription factors that bind to specific enhancer elements (retinoic acid response elements or RAREs) in a ligand dependent fashion [23,24]. Typically, RAR’s and RXR’s bind to enhancer elements as heterodimers. In the absence of ligand, a co-repressor is bound to this RXR/RAR heterodimer causing repression of transcription from the associated promoter. Upon binding of ligand, the co-repressor is released and transcription is activated through binding of a co-activator. Ultimately, co-activators and co-repressors affect the chromatin structure of the ligand-regulated gene [16,37].

The Prevalence of Foot Defects Among Wartime Recruits

<h3>Abstract</h3> <i>POGZ</i>, which encodes a multi-domain transcription factor, has been found frequently mutated in neurodevelopmental disorders, particularly autism spectrum disorder (ASD) and intellectual disability (ID). However, little is known about its functions in embryonic stem cells (ESCs) and in transcriptional regulation. Here, we show that POGZ plays key roles in the maintenance of ESCs by association with the SWI-SNF (BAF) chromatin remodeler complex and heterochromatin protein 1 (HP1) proteins. Loss of POGZ induces differentiation of ESCs, likely by up-regulation of primitive endoderm and mesoderm lineage genes and by down-regulation of pluripotency-related and cell cycle genes. Genome-wide binding analysis shows that POGZ is primarily localized to gene promoter and enhancer regions where POGZ is required to maintain an open chromatin. Regulation of chromatin under control of POGZ depends on esBAF complex. Furthermore, there is an extensive overlap of POGZ and OCT4 peaks genome-wide, and both factors interact with each other. We propose that POGZ is an important pluripotency-associated factor, and its absence causes failure to maintain a proper ESC-specific chromatin state and transcriptional circuitry, which eventually leads to loss of ESC phenotype. Our work provides important insights into the roles of POGZ in the maintenance of ESC identity as well as regulation of transcription, which will be useful for understanding the etiology of neurodevelopmental disorders by <i>POGZ</i> mutation.