Genome-scale chromatin binding dynamics of RNA Polymerase II general transcription machinery components.

A great deal of work has revealed, in structural detail, the components of the preinitiation complex (PIC) machinery required for initiation of mRNA gene transcription by RNA polymerase II (Pol II). However, less-well understood are the in vivo PIC assembly pathways and their kinetics, an understanding of which is vital for determining how rates of in vivo RNA synthesis are established. We used competition ChIP in budding yeast to obtain genome-scale estimates of the residence times for five general transcription factors (GTFs): TBP, TFIIA, TFIIB, TFIIE and TFIIF. While many GTF-chromatin interactions were short-lived ( < 1 min), there were numerous interactions with residence times in the range of several minutes. Sets of genes with a shared function also shared similar patterns of GTF kinetic behavior. TFIIE, a GTF that enters the PIC late in the assembly process, had residence times correlated with RNA synthesis rates. The datasets and results reported here provide kinetic information for most of the Pol II-driven genes in this organism, offering a rich resource for exploring the mechanistic relationships between PIC assembly, gene regulation, and transcription.

Transcriptional Profile of Exercise-Induced Protection Against Relapse to Cocaine Seeking in a Rat Model.

Background: Exercise has shown promise as a treatment for cocaine use disorder; however, the mechanism underlying its efficacy has remained elusive. Methods: We used a rat model of relapse (cue-induced reinstatement) and exercise (wheel running, 2 hours/day) coupled with RNA sequencing to establish transcriptional profiles associated with the protective effects of exercise (during early withdrawal [days 1-7] or throughout withdrawal [days 1-14]) versus noneffective exercise (during late withdrawal [days 8-14]) against cocaine-seeking and sedentary conditions. Results: As expected, cue-induced cocaine seeking was highest in the sedentary and late-withdrawal exercise groups; both groups also showed upregulation of a Grin1-associated transcript and enrichment of Drd1-Nmdar1 complex and glutamate receptor complex terms. Surprisingly, these glutamate markers were also enriched in the early- and throughout-withdrawal exercise groups, despite lower levels of cocaine seeking. However, a closer examination of the Grin1-associated transcript revealed a robust loss of transcripts spanning exons 9 and 10 in the sedentary condition relative to saline controls that was normalized by early- and throughout-withdrawal exercise, but not late-withdrawal exercise, indicating that these exercise conditions may normalize RNA mis-splicing induced by cocaine seeking. Our findings also revealed novel mechanisms by which exercise initiated during early withdrawal may modulate glutamatergic signaling in dorsomedial prefrontal cortex (e.g., via transcripts associated with non-NMDA glutamate receptors or those affecting signaling downstream of NMDA receptors), along with mechanisms outside of glutamatergic signaling such as circadian rhythm regulation and neuronal survival. Conclusions: These findings provide a rich resource for future studies aimed at manipulating these molecular networks to better understand how exercise decreases cocaine seeking.

Genome-scale chromatin interaction dynamic measurements for key components of the RNA Pol II general transcription machinery.

Background: A great deal of work has revealed in structural detail the components of the machinery responsible for mRNA gene transcription initiation. These include the general transcription factors (GTFs), which assemble at promoters along with RNA Polymerase II (Pol II) to form a preinitiation complex (PIC) aided by the activities of cofactors and site-specific transcription factors (TFs). However, less well understood are the in vivo PIC assembly pathways and their kinetics, an understanding of which is vital for determining on a mechanistic level how rates of in vivo RNA synthesis are established and how cofactors and TFs impact them. Results: We used competition ChIP to obtain genome-scale estimates of the residence times for five GTFs: TBP, TFIIA, TFIIB, TFIIE and TFIIF in budding yeast. While many GTF-chromatin interactions were short-lived (< 1 min), there were numerous interactions with residence times in the several minutes range. Sets of genes with a shared function also shared similar patterns of GTF kinetic behavior. TFIIE, a GTF that enters the PIC late in the assembly process, had residence times correlated with RNA synthesis rates. Conclusions: The datasets and results reported here provide kinetic information for most of the Pol II-driven genes in this organism and therefore offer a rich resource for exploring the mechanistic relationships between PIC assembly, gene regulation, and transcription. The relationships between gene function and GTF dynamics suggest that shared sets of TFs tune PIC assembly kinetics to ensure appropriate levels of expression.

Globally elevated levels of histone H3 lysine 9 trimethylation in early infancy are associated with poor growth trajectory in Bangladeshi children.

BACKGROUND: Stunting is a global health problem affecting hundreds of millions of children worldwide and contributing to 45% of deaths in children under the age of five. Current therapeutic interventions have limited efficacy. Understanding the epigenetic changes underlying stunting will elucidate molecular mechanisms and likely lead to new therapies. RESULTS: We profiled the repressive mark histone H3 lysine 9 trimethylation (H3K9me3) genome-wide in peripheral blood mononuclear cells (PBMCs) from 18-week-old infants (n = 15) and mothers (n = 14) enrolled in the PROVIDE study established in an urban slum in Bangladesh. We associated H3K9me3 levels within individual loci as well as genome-wide with anthropometric measurements and other biomarkers of stunting and performed functional annotation of differentially affected regions. Despite the relatively small number of samples from this vulnerable population, we observed globally elevated H3K9me3 levels were associated with poor linear growth between birth and one year of age. A large proportion of the differentially methylated genes code for proteins targeting viral mRNA and highly significant regions were enriched in transposon elements with potential regulatory roles in immune system activation and cytokine production. Maternal data show a similar trend with child's anthropometry; however, these trends lack statistical significance to infer an intergenerational relationship. CONCLUSIONS: We speculate that high H3K9me3 levels may result in poor linear growth by repressing genes involved in immune system activation. Importantly, changes to H3K9me3 were detectable before the overt manifestation of stunting and therefore may be valuable as new biomarkers of stunting.

Histone H3 lysine 27 acetylation profile undergoes two global shifts in undernourished children and suggests altered one-carbon metabolism.

BACKGROUND: Stunting is a condition in which a child does not reach their full growth potential due to chronic undernutrition. It arises during the first 2 years of a child's life and is associated with developmental deficiencies and life-long health problems. Current interventions provide some benefit, but new approaches to prevention and treatment grounded in a molecular understanding of stunting are needed. Epigenetic analyses are critical as they can provide insight into how signals from a poor environment lead to changes in cell function. RESULTS: Here we profiled histone H3 acetylation on lysine 27 (H3K27ac) in peripheral blood mononuclear cells (PBMCs) of 18-week-old (n = 14) and 1-year-old children (n = 22) living in an urban slum in Dhaka, Bangladesh. We show that 18-week-old children destined to become stunted have elevated levels of H3K27ac overall, functional analysis of which indicates activation of the immune system and stress response pathways as a primary response to a poor environment with high pathogen load. Conversely, overt stunting at 1-year-of age is associated with globally reduced H3K27ac that is indicative of metabolic rewiring and downregulation of the immune system and DNA repair pathways that are likely secondary responses to chronic exposure to a poor environment with limited nutrients. Among processes altered in 1-year-old children, we identified one-carbon metabolism, the significance of which is supported by integrative analysis with results from histone H3 trimethylation on lysine 4 (H3K4me3). Together, these results suggest altered one-carbon metabolism in this population of stunted children. CONCLUSIONS: The epigenomes of stunted children undergo two global changes in H3K27ac within their first year of life, which are associated with probable initial hyperactive immune responses followed by reduced metabolic capacity. Limitation of one-carbon metabolites may play a key role in the development of stunting. Trial registration ClinicalTrials.gov NCT01375647. Registered 17 June 2011, retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT01375647 .

Gut microbiome communication with bone marrow regulates susceptibility to amebiasis.

The microbiome provides resistance to infection. However, the underlying mechanisms are poorly understood. We demonstrate that colonization with the intestinal bacterium Clostridium scindens protects from Entamoeba histolytica colitis via innate immunity. Introduction of C. scindens into the gut microbiota epigenetically altered and expanded bone marrow granulocyte-monocyte progenitors (GMPs) and resulted in increased intestinal neutrophils with subsequent challenge with E. histolytica. Introduction of C. scindens alone was sufficient to expand GMPs in gnotobiotic mice. Adoptive transfer of bone marrow from C. scindens-colonized mice into naive mice protected against amebic colitis and increased intestinal neutrophils. Children without E. histolytica diarrhea also had a higher abundance of Lachnoclostridia. Lachnoclostridia C. scindens can metabolize the bile salt cholate, so we measured deoxycholate and discovered that it was increased in the sera of C. scindens-colonized specific pathogen-free and gnotobiotic mice, as well as in children protected from amebiasis. Administration of deoxycholate alone increased GMPs and provided protection from amebiasis. We elucidated a mechanism by which C. scindens and the microbially metabolized bile salt deoxycholic acid alter hematopoietic precursors and provide innate protection from later infection with E. histolytica.

MYBL2-Driven Transcriptional Programs Link Replication Stress and Error-prone DNA Repair With Genomic Instability in Lung Adenocarcinoma.

It has long been recognized that defects in cell cycle checkpoint and DNA repair pathways give rise to genomic instability, tumor heterogeneity, and metastasis. Despite this knowledge, the transcription factor-mediated gene expression programs that enable survival and proliferation in the face of enormous replication stress and DNA damage have remained elusive. Using robust omics data from two independent studies, we provide evidence that a large cohort of lung adenocarcinomas exhibit significant genome instability and overexpress the DNA damage responsive transcription factor MYB proto-oncogene like 2 (MYBL2). Across two studies, elevated MYBL2 expression was a robust marker of poor overall survival and disease-free survival outcomes, regardless of disease stage. Clinically, elevated MYBL2 expression identified patients with aggressive early onset disease, increased lymph node involvement, and increased incidence of distant metastases. Analysis of genomic sequencing data demonstrated that MYBL2 High lung adenocarcinomas had elevated somatic mutation burden, widespread chromosomal alterations, and alterations in single-strand DNA break repair pathways. In this study, we provide evidence that impaired single-strand break repair, combined with a loss of cell cycle regulators TP53 and RB1, give rise to MYBL2-mediated transcriptional programs. Omics data supports a model wherein tumors with significant genomic instability upregulate MYBL2 to drive genes that control replication stress responses, promote error-prone DNA repair, and antagonize faithful homologous recombination repair. Our study supports the use of checkpoint kinase 1 (CHK1) pharmacological inhibitors, in targeted MYBL2 High patient cohorts, as a future therapy to improve lung adenocarcinoma patient outcomes.

Conformational changes and catalytic inefficiency associated with Mot1-mediated TBP-DNA dissociation.

The TATA-box Binding Protein (TBP) plays a central role in regulating gene expression and is the first step in the process of pre-initiation complex (PIC) formation on promoter DNA. The lifetime of TBP at the promoter site is controlled by several cofactors including the Modifier of transcription 1 (Mot1), an essential TBP-associated ATPase. Based on ensemble measurements, Mot1 can use adenosine triphosphate (ATP) hydrolysis to displace TBP from DNA and various models for how this activity is coupled to transcriptional regulation have been proposed. However, the underlying molecular mechanism of Mot1 action is not well understood. In this work, the interaction of Mot1 with the DNA/TBP complex was investigated by single-pair Förster resonance energy transfer (spFRET). Upon Mot1 binding to the DNA/TBP complex, a transition in the DNA/TBP conformation was observed. Hydrolysis of ATP by Mot1 led to a conformational change but was not sufficient to efficiently disrupt the complex. SpFRET measurements of dual-labeled DNA suggest that Mot1's ATPase activity primes incorrectly oriented TBP for dissociation from DNA and additional Mot1 in solution is necessary for TBP unbinding. These findings provide a framework for understanding how the efficiency of Mot1's catalytic activity is tuned to establish a dynamic pool of TBP without interfering with stable and functional TBP-containing complexes.

Histone H3 lysine 4 methylation signature associated with human undernutrition.

Chronically undernourished children become stunted during their first 2 years and thereafter bear burdens of ill health for the rest of their lives. Contributors to stunting include poor nutrition and exposure to pathogens, and parental history may also play a role. However, the epigenetic impact of a poor environment on young children is largely unknown. Here we show the unfolding pattern of histone H3 lysine 4 trimethylation (H3K4me3) in children and mothers living in an urban slum in Dhaka, Bangladesh. A pattern of chromatin modification in blood cells of stunted children emerges over time and involves a global decrease in methylation at canonical locations near gene start sites and increased methylation at ectopic sites throughout the genome. This redistribution occurs at metabolic and immune genes and was specific for H3K4me3, as it was not observed for histone H3 lysine 27 acetylation in the same samples. Methylation changes in stunting globally resemble changes that occur in vitro in response to altered methylation capacity, suggesting that reduced levels of one-carbon nutrients in the diet play a key role in stunting in this population. A network of differentially expressed genes in stunted children reveals effects on chromatin modification machinery, including turnover of H3K4me3, as well as posttranscriptional gene regulation affecting immune response pathways and lipid metabolism. Consistent with these changes, reduced expression of the endocytic receptor gene LDL receptor 1 (LRP1) is a driver of stunting in a mouse model, suggesting a target for intervention.

Crystal structure of the full Swi2/Snf2 remodeler Mot1 in the resting state.

Swi2/Snf2 ATPases remodel protein:DNA complexes in all of the fundamental chromosome-associated processes. The single-subunit remodeler Mot1 dissociates TATA box-binding protein (TBP):DNA complexes and provides a simple model for obtaining structural insights into the action of Swi2/Snf2 ATPases. Previously we reported how the N-terminal domain of Mot1 binds TBP, NC2 and DNA, but the location of the C-terminal ATPase domain remained unclear (Butryn et al., 2015). Here, we report the crystal structure of the near full-length Mot1 from Chaetomium thermophilum. Our data show that Mot1 adopts a ring like structure with a catalytically inactive resting state of the ATPase. Biochemical analysis suggests that TBP binding switches Mot1 into an ATP hydrolysis-competent conformation. Combined with our previous results, these data significantly improve the structural model for the complete Mot1:TBP:DNA complex and suggest a general mechanism for Mot1 action.

An Improved Method for Measuring Chromatin-binding Dynamics Using Time-dependent Formaldehyde Crosslinking.

Formaldehyde crosslinking is widely used in combination with chromatin immunoprecipitation (ChIP) to measure the locations along DNA and relative levels of transcription factor (TF)-DNA interactions in vivo. However, the measurements that are typically made do not provide unambiguous information about the dynamic properties of these interactions. We have developed a method to estimate binding kinetic parameters from time-dependent formaldehyde crosslinking data, called crosslinking kinetics (CLK) analysis. Cultures of yeast cells are crosslinked with formaldehyde for various periods of time, yielding the relative ChIP signal at particular loci. We fit the data using the mass-action CLK model to extract kinetic parameters of the TF-chromatin interaction, including the on- and off-rates and crosslinking rate. From the on- and off-rate we obtain the occupancy and residence time. The following protocol is the second iteration of this method, CLKv2, updated with improved crosslinking and quenching conditions, more information about crosslinking rates, and systematic procedures for modeling the observed kinetic regimes. CLKv2 analysis has been applied to investigate the binding behavior of the TATA-binding protein (TBP), and a selected subset of other TFs. The protocol was developed using yeast cells, but may be applicable to cells from other organisms as well.

Second-generation method for analysis of chromatin binding with formaldehyde-cross-linking kinetics.

Formaldehyde-cross-linking underpins many of the most commonly used experimental approaches in the chromatin field, especially in capturing site-specific protein-DNA interactions. Extending such assays to assess the stability and binding kinetics of protein-DNA interactions is more challenging, requiring absolute measurements with a relatively high degree of physical precision. We previously described an experimental framework called the cross-linking kinetics (CLK) assay, which uses time-dependent formaldehyde-cross-linking data to extract kinetic parameters of chromatin binding. Many aspects of formaldehyde behavior in cells are unknown or undocumented, however, and could potentially affect CLK data analyses. Here, we report biochemical results that better define the properties of formaldehyde-cross-linking in budding yeast cells. These results have the potential to inform interpretations of "standard" chromatin assays, including chromatin immunoprecipitation. Moreover, the chemical complexity we uncovered resulted in the development of an improved method for measuring binding kinetics with the CLK approach. Optimum conditions included an increased formaldehyde concentration and more robust glycine-quench conditions. Notably, we observed that formaldehyde-cross-linking rates can vary dramatically for different protein-DNA interactions in vivo Some interactions were cross-linked much faster than the in vivo macromolecular interactions, making them suitable for kinetic analysis. For other interactions, we found the cross-linking reaction occurred on the same time scale or slower than binding dynamics; for these interactions, it was sometimes possible to compute the in vivo equilibrium-binding constant but not binding on- and off-rates. This improved method yields more accurate in vivo binding kinetics estimates on the minute time scale.

Transcriptomes of six mutants in the Sen1 pathway reveal combinatorial control of transcription termination across the Saccharomyces cerevisiae genome.

Transcriptome studies on eukaryotic cells have revealed an unexpected abundance and diversity of noncoding RNAs synthesized by RNA polymerase II (Pol II), some of which influence the expression of protein-coding genes. Yet, much less is known about biogenesis of Pol II non-coding RNA than mRNAs. In the budding yeast Saccharomyces cerevisiae, initiation of non-coding transcripts by Pol II appears to be similar to that of mRNAs, but a distinct pathway is utilized for termination of most non-coding RNAs: the Sen1-dependent or "NNS" pathway. Here, we examine the effect on the S. cerevisiae transcriptome of conditional mutations in the genes encoding six different essential proteins that influence Sen1-dependent termination: Sen1, Nrd1, Nab3, Ssu72, Rpb11, and Hrp1. We observe surprisingly diverse effects on transcript abundance for the different proteins that cannot be explained simply by differing severity of the mutations. Rather, we infer from our results that termination of Pol II transcription of non-coding RNA genes is subject to complex combinatorial control that likely involves proteins beyond those studied here. Furthermore, we identify new targets and functions of Sen1-dependent termination, including a role in repression of meiotic genes in vegetative cells. In combination with other recent whole-genome studies on termination of non-coding RNAs, our results provide promising directions for further investigation.

RNA synthesis is associated with multiple TBP-chromatin binding events.

Competition ChIP is an experimental method that allows transcription factor (TF) chromatin turnover dynamics to be measured across a genome. We develop and apply a physical model of TF-chromatin competitive binding using chemical reaction rate theory and are able to derive the physical half-life or residence time for TATA-binding protein (TBP) across the yeast genome from competition ChIP data. Using our physical modeling approach where we explicitly include the induction profile of the competitor in the model, we are able to estimate yeast TBP-chromatin residence times as short as 1.3 minutes, demonstrating that competition ChIP is a relatively high temporal-resolution approach. Strikingly, we find a median value of ~5 TBP-chromatin binding events associated with the synthesis of one RNA molecule across Pol II genes, suggesting multiple rounds of pre-initiation complex assembly and disassembly before productive elongation of Pol II is achieved at most genes in the yeast genome.

Molecular Mechanism of Mot1, a TATA-binding Protein (TBP)-DNA Dissociating Enzyme.

The essential Saccharomyces cerevisiae ATPase Mot1 globally regulates transcription by impacting the genomic distribution and activity of the TATA-binding protein (TBP). In vitro, Mot1 forms a ternary complex with TBP and DNA and can use ATP hydrolysis to dissociate the TBP-DNA complex. Prior work suggested an interaction between the ATPase domain and a functionally important segment of DNA flanking the TATA sequence. However, how ATP hydrolysis facilitates removal of TBP from DNA is not well understood, and several models have been proposed. To gain insight into the Mot1 mechanism, we dissected the role of the flanking DNA segment by biochemical analysis of complexes formed using DNAs with short single-stranded gaps. In parallel, we used a DNA tethered cleavage approach to map regions of Mot1 in proximity to the DNA under different conditions. Our results define non-equivalent roles for bases within a broad segment of flanking DNA required for Mot1 action. Moreover, we present biochemical evidence for two distinct conformations of the Mot1 ATPase, the detection of which can be modulated by ATP analogs as well as DNA sequence flanking the TATA sequence. We also show using purified complexes that Mot1 dissociation of a stable, high affinity TBP-DNA interaction is surprisingly inefficient, suggesting how other transcription factors that bind to TBP may compete with Mot1. Taken together, these results suggest that TBP-DNA affinity as well as other aspects of promoter sequence influence Mot1 function in vivo.

The Modifier of Transcription 1 (Mot1) ATPase and Spt16 Histone Chaperone Co-regulate Transcription through Preinitiation Complex Assembly and Nucleosome Organization.

Modifier of transcription 1 (Mot1) is a conserved and essential Swi2/Snf2 ATPase that can remove TATA-binding protein (TBP) from DNA using ATP hydrolysis and in so doing exerts global effects on transcription. Spt16 is also essential and functions globally in transcriptional regulation as a component of the facilitates chromatin transcription (FACT) histone chaperone complex. Here we demonstrate that Mot1 and Spt16 regulate a largely overlapping set of genes in Saccharomyces cerevisiae. As expected, Mot1 was found to control TBP levels at co-regulated promoters. In contrast, Spt16 did not affect TBP recruitment. On a global scale, Spt16 was required for Mot1 promoter localization, and Mot1 also affected Spt16 localization to genes. Interestingly, we found that Mot1 has an unanticipated role in establishing or maintaining the occupancy and positioning of nucleosomes at the 5' ends of genes. Spt16 has a broad role in regulating chromatin organization in gene bodies, including those nucleosomes affected by Mot1. These results suggest that the large scale overlap in Mot1 and Spt16 function arises from a combination of both their unique and shared functions in transcription complex assembly and chromatin structure regulation.

Formaldehyde crosslinking: a tool for the study of chromatin complexes.

Formaldehyde has been used for decades to probe macromolecular structure and function and to trap complexes, cells, and tissues for further analysis. Formaldehyde crosslinking is routinely employed for detection and quantification of protein-DNA interactions, interactions between chromatin proteins, and interactions between distal segments of the chromatin fiber. Despite widespread use and a rich biochemical literature, important aspects of formaldehyde behavior in cells have not been well described. Here, we highlight features of formaldehyde chemistry relevant to its use in analyses of chromatin complexes, focusing on how its properties may influence studies of chromatin structure and function.

Structural basis for recognition and remodeling of the TBP:DNA:NC2 complex by Mot1.

Swi2/Snf2 ATPases remodel substrates such as nucleosomes and transcription complexes to control a wide range of DNA-associated processes, but detailed structural information on the ATP-dependent remodeling reactions is largely absent. The single subunit remodeler Mot1 (modifier of transcription 1) dissociates TATA box-binding protein (TBP):DNA complexes, offering a useful system to address the structural mechanisms of Swi2/Snf2 ATPases. Here, we report the crystal structure of the N-terminal domain of Mot1 in complex with TBP, DNA, and the transcription regulator negative cofactor 2 (NC2). Our data show that Mot1 reduces DNA:NC2 interactions and unbends DNA as compared to the TBP:DNA:NC2 state, suggesting that Mot1 primes TBP:NC2 displacement in an ATP-independent manner. Electron microscopy and cross-linking data suggest that the Swi2/Snf2 domain of Mot1 associates with the upstream DNA and the histone fold of NC2, thereby revealing parallels to some nucleosome remodelers. This study provides a structural framework for how a Swi2/Snf2 ATPase interacts with its substrate DNA:protein complex.

Analysis of chromatin binding dynamics using the crosslinking kinetics (CLK) method.

Transcription factor binding sites in chromatin are routinely inventoried by the chromatin immunoprecipitation assay, and these binding patterns can provide precise and detailed information about cell state. However, some fundamental molecular questions regarding transcription factor function require an understanding of in vivo binding dynamics as well as location information. Here we describe the crosslinking kinetics (CLK) assay, in which the time-dependence of formaldehyde crosslinking is used to extract on- and off-rates for chromatin binding in vivo.

Measuring chromatin interaction dynamics on the second time scale at single-copy genes.

The chromatin immunoprecipitation (ChIP) assay is widely used to capture interactions between chromatin and regulatory proteins, but it is unknown how stable most native interactions are. Although live-cell imaging suggests short-lived interactions at tandem gene arrays, current methods cannot measure rapid binding dynamics at single-copy genes. We show, by using a modified ChIP assay with subsecond temporal resolution, that the time dependence of formaldehyde cross-linking can be used to extract in vivo on and off rates for site-specific chromatin interactions varying over a ~100-fold dynamic range. By using the method, we show that a regulatory process can shift weakly bound TATA-binding protein to stable promoter interactions, thereby facilitating transcription complex formation. This assay provides an approach for systematic, quantitative analyses of chromatin binding dynamics in vivo.