Extended and dynamic linker histone-DNA Interactions control chromatosome compaction.

Chromatosomes play a fundamental role in chromatin regulation, but a detailed understanding of their structure is lacking, partially due to their complex dynamics. Using single-molecule DNA unzipping with optical tweezers, we reveal that linker histone interactions with DNA are remarkably extended, with the C-terminal domain binding both DNA linkers as far as approximately ±140 bp from the dyad. In addition to a symmetrical compaction of the nucleosome core governed by globular domain contacts at the dyad, the C-terminal domain compacts the nucleosome's entry and exit. These interactions are dynamic, exhibit rapid binding and dissociation, are sensitive to phosphorylation of a specific residue, and are crucial to determining the symmetry of the chromatosome's core. Extensive unzipping of the linker DNA, which mimics its invasion by motor proteins, shifts H1 into an asymmetric, off-dyad configuration and triggers nucleosome decompaction, highlighting the plasticity of the chromatosome structure and its potential regulatory role.

Single-Macromolecule Studies of Eukaryotic Genomic Maintenance.

Genomes are self-organized and self-maintained as long, complex macromolecules of chromatin. The inherent heterogeneity, stochasticity, phase separation, and chromatin dynamics of genome operation make it challenging to study genomes using ensemble methods. Various single-molecule force-, fluorescent-, and sequencing-based techniques rooted in different disciplines have been developed to fill critical gaps in the capabilities of bulk measurements, each providing unique, otherwise inaccessible, insights into the structure and maintenance of the genome. Capable of capturing molecular-level details about the organization, conformational changes, and packaging of genetic material, as well as processive and stochastic movements of maintenance factors, a single-molecule toolbox provides an excellent opportunity for collaborative research to understand how genetic material functions in health and malfunctions in disease. In this review, we discuss novel insights brought to genomic sciences by single-molecule techniques and their potential to continue to revolutionize the field-one molecule at a time.

Single molecule characterization of the binding kinetics of a transcription factor and its modulation by DNA sequence and methylation.

The interaction of transcription factors with their response elements in DNA is emerging as a highly complex process, whose characterization requires measuring the full distribution of binding and dissociation times in a well-controlled assay. Here, we present a single-molecule assay that exploits the thermal fluctuations of a DNA hairpin to detect the association and dissociation of individual, unlabeled transcription factors. We demonstrate this new approach by following the binding of Egr1 to its consensus motif and the three binding sites found in the promoter of the Lhb gene, and find that both association and dissociation are modulated by the 9 bp core motif and the sequences around it. In addition, CpG methylation modulates the dissociation kinetics in a sequence and position-dependent manner, which can both stabilize or destabilize the complex. Together, our findings show how variations in sequence and methylation patterns synergistically extend the spectrum of a protein's binding properties, and demonstrate how the proposed approach can provide new insights on the function of transcription factors.

The base pair-scale diffusion of nucleosomes modulates binding of transcription factors.

The structure of promoter chromatin determines the ability of transcription factors (TFs) to bind to DNA and therefore has a profound effect on the expression levels of genes. However, the role of spontaneous nucleosome movements in this process is not fully understood. Here, we developed a single-molecule optical tweezers assay capable of simultaneously characterizing the base pair-scale diffusion of a nucleosome on DNA and the binding of a TF, using the luteinizing hormone ß subunit gene (Lhb) promoter and Egr-1 as a model system. Our results demonstrate that nucleosomes undergo confined diffusion, and that the incorporation of the histone variant H2A.Z serves to partially relieve this confinement, inducing a different type of nucleosome repositioning. The increase in diffusion leads to exposure of a TF's binding site and facilitates its association with the DNA, which, in turn, biases the subsequent movement of the nucleosome. Our findings suggest the use of mobile nucleosomes as a general transcriptional regulatory mechanism.

Single-molecule DNA unzipping reveals asymmetric modulation of a transcription factor by its binding site sequence and context.

Most functional transcription factor (TF) binding sites deviate from their 'consensus' recognition motif, although their sites and flanking sequences are often conserved across species. Here, we used single-molecule DNA unzipping with optical tweezers to study how Egr-1, a TF harboring three zinc fingers (ZF1, ZF2 and ZF3), is modulated by the sequence and context of its functional sites in the Lhb gene promoter. We find that both the core 9 bp bound to Egr-1 in each of the sites, and the base pairs flanking them, modulate the affinity and structure of the protein-DNA complex. The effect of the flanking sequences is asymmetric, with a stronger effect for the sequence flanking ZF3. Characterization of the dissociation time of Egr-1 revealed that a local, mechanical perturbation of the interactions of ZF3 destabilizes the complex more effectively than a perturbation of the ZF1 interactions. Our results reveal a novel role for ZF3 in the interaction of Egr-1 with other proteins and the DNA, providing insight on the regulation of Lhb and other genes by Egr-1. Moreover, our findings reveal the potential of small changes in DNA sequence to alter transcriptional regulation, and may shed light on the organization of regulatory elements at promoters.

Pausing kinetics dominates strand-displacement polymerization by reverse transcriptase.

Reverse transcriptase (RT) catalyzes the conversion of the viral RNA into an integration-competent double-stranded DNA, with a variety of enzymatic activities that include the ability to displace a non-template strand concomitantly with polymerization. Here, using high-resolution optical tweezers to follow the activity of the murine leukemia Virus RT, we show that strand-displacement polymerization is frequently interrupted. Abundant pauses are modulated by the strength of the DNA duplex ∼8 bp ahead, indicating the existence of uncharacterized RT/DNA interactions, and correspond to backtracking of the enzyme, whose recovery is also modulated by the duplex strength. Dissociation and reinitiation events, which induce long periods of inactivity and are likely the rate-limiting step in the synthesis of the genome in vivo, are modulated by the template structure and the viral nucleocapsid protein. Our results emphasize the potential regulatory role of conserved structural motifs, and may provide useful information for the development of potent and specific inhibitors.

The mechano-chemistry of a monomeric reverse transcriptase.

Retroviral reverse transcriptase catalyses the synthesis of an integration-competent dsDNA molecule, using as a substrate the viral RNA. Using optical tweezers, we follow the Murine Leukemia Virus reverse transcriptase as it performs strand-displacement polymerization on a template under mechanical force. Our results indicate that reverse transcriptase functions as a Brownian ratchet, with dNTP binding as the rectifying reaction of the ratchet. We also found that reverse transcriptase is a relatively passive enzyme, able to polymerize on structured templates by exploiting their thermal breathing. Finally, our results indicate that the enzyme enters the recently characterized backtracking state from the pre-translocation complex.

Mitogen- and stress-activated protein kinase 1 is required for gonadotropin-releasing hormone-mediated activation of gonadotropin α-subunit expression.

Pituitary gonadotropin hormones are regulated by gonadotropin-releasing hormone (GnRH) via MAPK signaling pathways that stimulate gene transcription of the common α-subunit (Cga) and the hormone-specific ß-subunits of gonadotropin. We have reported previously that GnRH-induced activities at these genes include various histone modifications, but we did not examine histone phosphorylation. This modification adds a negative charge to residues of the histone tails that interact with the negatively charged DNA, is associated with closed chromatin during mitosis, but is increased at certain genes for transcriptional activation. Thus, the functions of this modification are unclear. We initially hypothesized that GnRH might induce phosphorylation of Ser-10 in histone 3 (H3S10p) as part of its regulation of gonadotropin gene expression, possibly involving cross-talk with H3K9 acetylation. We found that GnRH increases the levels of both modifications around the Cga gene transcriptional start site and that JNK inhibition dramatically reduces H3S10p levels. However, this modification had only a minor effect on Cga expression and no effect on H3K9ac. GnRH also increased H3S28p and H3K27ac levels and also those of activated mitogen- and stress-activated protein kinase 1 (MSK1). MSK1 inhibition dramatically reduced H3S28p levels in untreated and GnRH-treated cells and also affected H3K27ac levels. Although not affecting basal Cga expression, MSK1/2 inhibition repressed GnRH activation of Cga expression. Moreover, ChIP analysis revealed that GnRH-activated MSK1 targets the first nucleosome just downstream from the TSS. Given that the elongating RNA polymerase II (RNAPII) stalls at this well positioned nucleosome, GnRH-induced H3S28p, possibly in association with H3K27ac, would facilitate the progression of RNAPII.

RNA transcribed from a distal enhancer is required for activating the chromatin at the promoter of the gonadotropin α-subunit gene.

Since the discovery that many transcriptional enhancers are transcribed into long noncoding RNAs termed "enhancer RNAs" (eRNAs), their putative role in enhancer function has been debated. Very recent evidence has indicted that some eRNAs play a role in initiating or activating transcription, possibly by helping recruit and/or stabilize binding of the general transcription machinery to the proximal promoter of their target genes. The distal enhancer of the gonadotropin hormone α-subunit gene, chorionic gonadotropin alpha (Cga), is responsible for Cga cell-specific expression in gonadotropes and thyrotropes, and we show here that it encodes two bidirectional nonpolyadenylated RNAs whose levels are increased somewhat by exposure to gonadotropin-releasing hormone but are not necessarily linked to Cga transcriptional activity. Knockdown of the more distal eRNA led to a drop in Cga mRNA levels, initially without effect on the forward eRNA levels. With time, however, the repression on the Cga increased, and the forward eRNA levels were suppressed also. We demonstrate that the interaction of the enhancer with the promoter is lost after eRNA knockdown. Dramatic changes also were seen in the chromatin, with an increase in total histone H3 occupancy throughout this region and a virtual loss of histone H3 Lys 4 trimethylation at the promoter following the eRNA knockdown. Moreover, histone H3 Lys 27 (H3K27) acetylation, which was found at both enhancer and promoter in wild-type cells, appeared to have been replaced by H3K27 trimethylation at the enhancer. Thus, the Cga eRNA mediates the physical interaction between these genomic regions and determines the chromatin structure of the proximal promoter to allow gene expression.

Auxiliary ATP binding sites support DNA unwinding by RecBCD.

The RecBCD helicase initiates double-stranded break repair in bacteria by processively unwinding DNA with a rate approaching ∼1,600 bp·s-1, but the mechanism enabling such a fast rate is unknown. Employing a wide range of methodologies - including equilibrium and time-resolved binding experiments, ensemble and single-molecule unwinding assays, and crosslinking followed by mass spectrometry - we reveal the existence of auxiliary binding sites in the RecC subunit, where ATP binds with lower affinity and distinct chemical interactions as compared to the known catalytic sites. The essentiality and functionality of these sites are demonstrated by their impact on the survival of E.coli after exposure to damage-inducing radiation. We propose a model by which RecBCD achieves its optimized unwinding rate, even when ATP is scarce, by using the auxiliary binding sites to increase the flux of ATP to its catalytic sites.

Unravelling the role of epigenetics in reproductive adaptations to early-life environment.

Reproductive function adjusts in response to environmental conditions in order to optimize success. In humans, this plasticity includes age of pubertal onset, hormone levels and age at menopause. These reproductive characteristics vary across populations with distinct lifestyles and following specific childhood events, and point to a role for the early-life environment in shaping adult reproductive trajectories. Epigenetic mechanisms respond to external signals, exert long-term effects on gene expression and have been shown in animal and cellular studies to regulate normal reproductive function, strongly implicating their role in these adaptations. Moreover, human cohort data have revealed differential DNA methylation signatures in proxy tissues that are associated with reproductive phenotypic variation, although the cause-effect relationships are difficult to discern, calling for additional complementary approaches to establish functionality. In this Review, we summarize how adult reproductive function can be shaped by childhood events. We discuss why the influence of the childhood environment on adult reproductive function is an important consideration in understanding how reproduction is regulated and necessitates consideration by clinicians treating women with diverse life histories. The resolution of the molecular mechanisms responsible for human reproductive plasticity could also lead to new approaches for intervention by targeting these epigenetic modifications.

Synergy between RecBCD subunits is essential for efficient DNA unwinding.

The subunits of the bacterial RecBCD act in coordination, rapidly and processively unwinding DNA at the site of a double strand break. RecBCD is able to displace DNA-binding proteins, suggesting that it generates high forces, but the specific role of each subunit in the force generation is unclear. Here, we present a novel optical tweezers assay that allows monitoring the activity of RecBCD's individual subunits, when they are part of an intact full complex. We show that RecBCD and its subunits are able to generate forces up to 25-40 pN without a significant effect on their velocity. Moreover, the isolated RecD translocates fast but is a weak helicase with limited processivity. Experiments at a broad range of [ATP] and forces suggest that RecD unwinds DNA as a Brownian ratchet, rectified by ATP binding, and that the presence of the other subunits shifts the ratchet equilibrium towards the post-translocation state.

Multifaceted Targeting of the Chromatin Mediates Gonadotropin-Releasing Hormone Effects on Gene Expression in the Gonadotrope.

Gonadotropin-releasing hormone (GnRH) stimulates the expression of multiple genes in the pituitary gonadotropes, most notably to induce synthesis of the gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), but also to ensure the appropriate functioning of these cells at the center of the mammalian reproductive endocrine axis. Aside from the activation of gene-specific transcription factors, GnRH stimulates through its membrane-bound receptor, alterations in the chromatin that facilitate transcription of its target genes. These include changes in the histone and DNA modifications, nucleosome positioning, and chromatin packaging at the regulatory regions of each gene. The requirements for each of these events vary according to the DNA sequence which determines the basal chromatin packaging at the regulatory regions. Despite considerable progress in this field in recent years, we are only beginning to understand some of the complexities involved in the role and regulation of this chromatin structure, including new modifications, extensive cross talk, histone variants, and the actions of distal enhancers and non-coding RNAs. This short review aims to integrate the latest findings on GnRH-induced alterations in the chromatin of its target genes, which indicate multiple and diverse actions. Understanding these processes is illuminating not only in the context of the activation of these hormones during the reproductive life span but may also reveal how aberrant epigenetic regulation of these genes leads to sub-fertility.

Nucleosome mobility and the regulation of gene expression: Insights from single-molecule studies.

Nucleosomes at the promoters of genes regulate the accessibility of the transcription machinery to DNA, and function as a basic layer in the complex regulation of gene expression. Our understanding of the role of the nucleosome's spontaneous, thermally driven position changes in modulating expression is lacking. This is the result of the paucity of experimental data on these dynamics, at high-resolution, and for DNA sequences that belong to real, transcribed genes. We have developed an assay that uses partial, reversible unzipping of nucleosomes with optical tweezers to repeatedly probe a nucleosome's position over time. Using the nucleosomes at the promoters of two model genes, Cga and Lhb, we show that the mobility of nucleosomes is modulated by the sequence of DNA and by the use of alternative histone variants, and describe how the mobility can affect transcription, at the initiation and elongation phases.

Transcriptional enhancers: Transcription, function and flexibility.

Active transcriptional enhancers are often transcribed to eRNAs, whose changing levels mirror those of the target gene mRNA. We discuss some of the reported functions of these eRNAs and their likely diversity to allow utilization of distinct cis regulatory regions to enhance transcription in diverse developmental and cellular contexts.

H2A.Z controls the stability and mobility of nucleosomes to regulate expression of the LH genes.

The structure and dynamics of promoter chromatin have a profound effect on the expression levels of genes. Yet, the contribution of DNA sequence, histone post-translational modifications, histone variant usage and other factors in shaping the architecture of chromatin, and the mechanisms by which this architecture modulates expression of specific genes are not yet completely understood. Here we use optical tweezers to study the roles that DNA sequence and the histone variant H2A.Z have in shaping the chromatin landscape at the promoters of two model genes, Cga and Lhb. Guided by MNase mapping of the promoters of these genes, we reconstitute nucleosomes that mimic those located near the transcriptional start site and immediately downstream (+1), and measure the forces required to disrupt these nucleosomes, and their mobility along the DNA sequence. Our results indicate that these genes are basally regulated by two distinct strategies, making use of H2A.Z to modulate separate phases of transcription, and highlight how DNA sequence, alternative histone variants and remodelling machinery act synergistically to modulate gene expression.