Nucleosome Binding Affinities Research Articles

Chromatin analysis of adult pluripotent stem cells reveals a unique stemness maintenance strategy.

Many highly regenerative organisms maintain adult pluripotent stem cells throughout their life, but how the long-term maintenance of pluripotency is accomplished is unclear. To decipher the regulatory logic of adult pluripotent stem cells, we analyzed the chromatin organization of stem cell genes in the planarian Schmidtea mediterranea. We identify a special chromatin state of stem cell genes, which is distinct from that of tissue-specific genes and resembles constitutive genes. Where tissue-specific promoters have detectable transcription factor binding sites, the promoters of stem cell-specific genes instead have sequence features that broadly decrease nucleosome binding affinity. This genic organization makes pluripotency-related gene expression the default state in these cells, which is maintained by the activity of chromatin remodelers ISWI and SNF2 in the stem cells.

Structural Features of Transcription Factors Associating with Nucleosome Binding.

Fate-changing transcription factors (TFs) scan chromatin to initiate new genetic programs during cell differentiation and reprogramming. Yet the protein structure domains that allow TFs to target nucleosomal DNA remain unexplored. We screened diverse TFs for binding to nucleosomes containing motif-enriched sequences targeted by pioneer factors invivo. FOXA1, OCT4, ASCL1/E12α, PU1, CEBPα, and ZELDA display a range of nucleosome binding affinities that correlate with their cell reprogramming potential. We further screened 593 full-length human TFs on protein microarrays against different nucleosome sequences, followed by confirmation in solution, to distinguish among factors that bound nucleosomes, such as the neuronal AP-2α/β/γ, versus factors that only bound free DNA. Structural comparisons of DNA binding domains revealed that efficient nucleosome binders use short anchoring α helices to bind DNA, whereas weak nucleosome binders use unstructured regions and/or β sheets. Thus, specific modes of DNA interaction allow nucleosome scanning that confers pioneer activity to transcription factors.

A Robust Method for the Purification and Characterization of Recombinant Human Histone H1 Variants.

Higher order compaction of the eukaryotic genome is key to the regulation of all DNA-templated processes, including transcription. This tightly controlled process involves the formation of mononucleosomes, the fundamental unit of chromatin, packaged into higher order architectures in an H1 linker histone-dependent process. While much work has been done to delineate the precise mechanism of this event in vitro and in vivo, major gaps still exist, primarily due to a lack of molecular tools. Specifically, there has never been a successful purification and biochemical characterization of all human H1 variants. Here we present a robust method to purify H1 and illustrate its utility in the purification of all somatic variants and one germline variant. In addition, we performed a first ever side-by-side biochemical comparison, which revealed a gradient of nucleosome binding affinities and compaction capabilities. These data provide new insight into H1 redundancy and lay the groundwork for the mechanistic investigation of disease-driving mutations.

A bromodomain-DNA interaction facilitates acetylation-dependent bivalent nucleosome recognition by the BET protein BRDT.

Bromodomains are critical components of many chromatin modifying/remodelling proteins and are emerging therapeutic targets, yet how they interact with nucleosomes, rather than acetylated peptides, remains unclear. Using BRDT as a model, we characterized how the BET family of bromodomains interacts with site-specifically acetylated nucleosomes. Here we report that BRDT interacts with nucleosomes through its first (BD1), but not second (BD2) bromodomain, and that acetylated histone recognition by BD1 is complemented by a bromodomain–DNA interaction. Simultaneous DNA and histone recognition enhances BRDT's nucleosome binding affinity and specificity, and its ability to localize to acetylated chromatin in cells. Conservation of DNA binding in bromodomains of BRD2, BRD3 and BRD4, indicates that bivalent nucleosome recognition is a key feature of these bromodomains and possibly others. Our results elucidate the molecular mechanism of BRDT association with nucleosomes and identify structural features of the BET bromodomains that may be targeted for therapeutic inhibition.

Structural analyses of the chromatin remodelling enzymes INO80-C and SWR-C.

INO80-C and SWR-C are conserved members of a subfamily of ATP-dependent chromatin remodeling enzymes that function in transcription and genome-maintenance pathways. A crucial role for these enzymes is to control chromosomal distribution of the H2A.Z histone variant. Here we use electron microscopy (EM) and two-dimensional (2D) class averaging to demonstrate that these remodeling enzymes have similar overall architectures. Each enzyme is characterized by a dynamic ‘tail’ domain and a compact ‘head’ that contains Rvb1/Rvb2 subunits organized as hexameric rings. EM class averages and mass spectrometry support the existence of single heterohexameric rings in both SWR-C and INO80-C. EM studies define the position of the Arp8/Arp4/Act1 module within INO80-C, and we find that this module enhances nucleosome binding affinity but is largely dispensable for remodeling activities. In contrast, the Ies6/Arp5 module is essential for INO80-C remodeling, and furthermore this module controls conformational changes that may couple nucleosome binding to remodeling.

The Effect of Linker Histone's Nucleosome Binding Affinity on Chromatin Unfolding Mechanisms

Eukaryotic gene activation requires selective unfolding of the chromatin fiber to access the DNA for processes such as DNA transcription, replication, and repair. Mutation/modification experiments of linker histone (LH) H1 suggest the importance of dynamic mechanisms for LH binding/dissociation, but the effects on chromatin's unfolding pathway remain unclear. Here we investigate the stretching response of chromatin fibers by mesoscale modeling to complement single-molecule experiments, and present various unfolding mechanisms for fibers with different nucleosome repeat lengths (NRLs) with/without LH that are fixed to their cores or bind/unbind dynamically with different affinities. Fiber softening occurs for long compared to short NRL (due to facile stacking rearrangements), dynamic compared to static LH/core binding as well as slow rather than fast dynamic LH rebinding (due to DNA stem destabilization), and low compared to high LH concentration (due to DNA stem inhibition). Heterogeneous superbead constructs—nucleosome clusters interspersed with extended fiber regions—emerge during unfolding of medium-NRL fibers and may be related to those observed experimentally. Our work suggests that fast and slow LH binding pools, present simultaneously in vivo, might act cooperatively to yield controlled fiber unfolding at low forces. Medium-NRL fibers with multiple dynamic LH pools offer both flexibility and selective DNA exposure, and may be evolutionarily suitable to regulate chromatin architecture and gene expression.

Accessory subunit of ISW2, Itc1 senses extranucleosomal DNA for regulating nucleosome remodeling

AbstractATP‐dependent chromatin remodeling complexes utilize the energy from ATP hydrolysis to bring about changes in the chromatin structure and make DNA accessible to regulatory factors. One such ATP‐dependent chromatin remodeling enzyme from Saccharomyces cerevisiae is ISW2. ISW2, a four subunit complex, consists of Isw2, the catalytic subunit, Itc1, the largest accessory subunit and two histone fold subunits Dpb4 and Dls1. ISW2 complex is well characterized in terms of its function in vivo and in vitro, which makes it a model remodeling enzyme to study biological functions. This work aims at defining the role of Itc1 subunit in nucleosome remodeling by ISW2. Systematic C‐terminal truncations of Itc1 were created with FLAG epitope tag for affinity purification of native complexes. Nucleosome binding affinity was reduced with shortening of the C‐terminus indicating the role of Itc1 in stabilizing the interaction of ISW2 with nucleosomes. Remodeling activity of most of these mutants was unaffected except one which resulted in nucleosome positioning defect. This mutant ISW2 complex was named as DNP (Defect in Nucleosome Position) ISW2. The defect in nucleosome positioning is not due to the loss of complex integrity or change in stoichometry of subunits. Further characterization of DNP ISW2 by site‐directed mapping of nucleosome positions showed that the truncation of the C‐terminus of Itc1 resulted in loss of ability to sense extranucleosomal DNA to effectively remodel nucleosomes.This work was supported by Public Health Service grant GM 70864 from the National Institute of Health

Nucleosome-binding affinity as a primary determinant of the nuclear mobility of the pioneer transcription factor FoxA

FoxA proteins are pioneer transcription factors, among the first to bind chromatin domains in development and enable gene activity. The Fox DNA-binding domain structurally resembles linker histone and binds nucleosomes stably. Using fluorescence recovery after photobleaching, we found that FoxA1 and FoxA2 move much more slowly in nuclei than other transcription factor types, including c-Myc, GATA-4, NF-1, and HMGB1. We find that slower nuclear mobility correlates with high nonspecific nucleosome binding, and point mutations that disrupt nonspecific binding markedly increase nuclear mobility. FoxA's distinct nuclear mobility is consistent with its pioneer activity in chromatin.

Repetitive DNA elements, nucleosome binding and human gene expression

We evaluated the epigenetic contributions of repetitive DNA elements to human gene regulation. Human proximal promoter sequences show distinct distributions of transposable elements (TEs) and simple sequence repeats (SSRs). TEs are enriched distal from transcriptional start sites (TSSs) and their frequency decreases closer to TSSs, being largely absent from the core promoter region. SSRs, on the other hand, are found at low frequency distal to the TSS and then increase in frequency starting ∼ 150 bp upstream of the TSS. The peak of SSR density is centered around the − 35 bp position where the basal transcriptional machinery assembles. These trends in repetitive sequence distribution are strongly correlated, positively for TEs and negatively for SSRs, with relative nucleosome binding affinities along the promoters. Nucleosomes bind with highest probability distal from the TSS and the nucleosome binding affinity steadily decreases reaching its nadir just upstream of the TSS at the same point where SSR frequency is at its highest. Promoters that are enriched for TEs are more highly and broadly expressed, on average, than promoters that are devoid of TEs. In addition, promoters that have similar repetitive DNA profiles regulate genes that have more similar expression patterns and encode proteins with more similar functions than promoters that differ with respect to their repetitive DNA. Furthermore, distinct repetitive DNA promoter profiles are correlated with tissue-specific patterns of expression. These observations indicate that repetitive DNA elements mediate chromatin accessibility in proximal promoter regions and the repeat content of promoters is relevant to both gene expression and function.

Nucleosome positioning signals in genomic DNA

Although histones can form nucleosomes on virtually any genomic sequence, DNA sequences show considerable variability in their binding affinity. We have used DNA sequences of Saccharomyces cerevisiae whose nucleosome binding affinities have been experimentally determined (Yuan et al. 2005) to train a support vector machine to identify the nucleosome formation potential of any given sequence of DNA. The DNA sequences whose nucleosome formation potential are most accurately predicted are those that contain strong nucleosome forming or inhibiting signals and are found within nucleosome length stretches of genomic DNA with continuous nucleosome formation or inhibition signals. We have accurately predicted the experimentally determined nucleosome positions across a well-characterized promoter region of S. cerevisiae and identified strong periodicity within 199 center-aligned mononucleosomes studied recently (Segal et al. 2006) despite there being no periodicity information used to train the support vector machine. Our analysis suggests that only a subset of nucleosomes are likely to be positioned by intrinsic sequence signals. This observation is consistent with the available experimental data and is inconsistent with the proposal of a nucleosome positioning code. Finally, we show that intrinsic nucleosome positioning signals are both more inhibitory and more variable in promoter regions than in open reading frames in S. cerevisiae.