Nucleosome-free DNA regions differentially affect distant communication in chromatin.

Communication between distantly spaced genomic regions is one of the key features of gene regulation in eukaryotes. Chromatin per se can stimulate efficient enhancer-promoter communication (EPC); however, the role of chromatin structure and dynamics in this process remains poorly understood. Here we show that nucleosome spacing and the presence of nucleosome-free DNA regions can modulate chromatin structure/dynamics and, in turn, affect the rate of EPC in vitro and in silico. Increasing the length of internucleosomal linker DNA from 25 to 60 bp results in more efficient EPC. The presence of longer nucleosome-free DNA regions can positively or negatively affect the rate of EPC, depending upon the length and location of the DNA region within the chromatin fiber. Thus the presence of histone-free DNA regions can differentially affect the efficiency of EPC, suggesting that gene regulation over a distance could be modulated by changes in the length of internucleosomal DNA spacers.

Structural analysis of nucleosomal barrier to transcription.

Thousands of human and Drosophila genes are regulated at the level of transcript elongation and nucleosomes are likely targets for this regulation. However, the molecular mechanisms of formation of the nucleosomal barrier to transcribing RNA polymerase II (Pol II) and nucleosome survival during/after transcription remain unknown. Here we show that both DNA-histone interactions and Pol II backtracking contribute to formation of the barrier and that nucleosome survival during transcription likely occurs through allosterically stabilized histone-histone interactions. Structural analysis indicates that after Pol II encounters the barrier, the enzyme backtracks and nucleosomal DNA recoils on the octamer, locking Pol II in the arrested state. DNA is displaced from one of the H2A/H2B dimers that remains associated with the octamer. The data reveal the importance of intranucleosomal DNA-protein and protein-protein interactions during conformational changes in the nucleosome structure on transcription. Mechanisms of nucleosomal barrier formation and nucleosome survival during transcription are proposed.

Analysis of the mechanism of nucleosome survival during transcription.

Maintenance of nucleosomal structure in the cell nuclei is essential for cell viability, regulation of gene expression and normal aging. Our previous data identified a key intermediate (a small intranucleosomal DNA loop, Ø-loop) that is likely required for nucleosome survival during transcription by RNA polymerase II (Pol II) through chromatin, and suggested that strong nucleosomal pausing guarantees efficient nucleosome survival. To evaluate these predictions, we analysed transcription through a nucleosome by different, structurally related RNA polymerases and mutant yeast Pol II having different histone-interacting surfaces that presumably stabilize the Ø-loop. The height of the nucleosomal barrier to transcription and efficiency of nucleosome survival correlate with the net negative charges of the histone-interacting surfaces. Molecular modeling and analysis of Pol II-nucleosome intermediates by DNase I footprinting suggest that efficient Ø-loop formation and nucleosome survival are mediated by electrostatic interactions between the largest subunit of Pol II and core histones.

Histone chaperone FACT action during transcription through chromatin by RNA polymerase II.

FACT (facilitates chromatin transcription) is a histone chaperone that promotes chromatin recovery during transcription, with additional roles in cell differentiation. Although several models of the action of FACT during transcription have been proposed, they remain to be experimentally evaluated. Here we show that human FACT (hFACT) facilitates transcription through chromatin and promotes nucleosome recovery in vitro. FACT action depends on the presence of histone H2A/H2B dimers in the nucleosome. Kinetic analysis suggests that hFACT decreases the lifetime of nonproductive RNA polymerase II (Pol II)-nucleosome complexes and facilitates the formation of productive complexes containing nucleosomal DNA partially uncoiled from the octamer. Taken together, our data suggest that hFACT interacts with DNA-binding surfaces of H2A/H2B dimers, facilitating uncoiling of DNA from the histone octamer. Thus, hFACT-H2A/H2B interactions play a key role in overcoming the nucleosomal barrier by Pol II and promoting nucleosome survival during transcription.

Mechanism of transcription through a nucleosome by RNA polymerase II.

Efficient maintenance of chromatin structure during passage of RNA polymerase II (Pol II) is critical for cell survival and functioning. Moderate-level transcription of eukaryotic genes by Pol II is accompanied by nucleosome survival, extensive exchange of histones H2A/H2B and minimal exchange of histones H3/H4. Complementary in vitro studies have shown that transcription through chromatin by single Pol II complexes is uniquely coupled with nucleosome survival via formation of a small intranucleosomal DNA loop (Ø-loop) containing the transcribing enzyme. In contrast, transient displacement and exchange of all core histones are observed during intense transcription. Indeed, multiple transcribing Pol II complexes can efficiently overcome the high nucleosomal barrier and displace the entire histone octamer in vitro. Thus, various Pol II complexes can remodel chromatin to different extents. The mechanisms of nucleosome survival and displacement during transcription and the role of DNA-histone interactions and various factors during this process are discussed. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.

Internucleosomal interactions mediated by histone tails allow distant communication in chromatin.

Action across long distances on chromatin is a hallmark of eukaryotic transcriptional regulation. Although chromatin structure per se can support long-range interactions, the mechanisms of efficient communication between widely spaced DNA modules in chromatin remain a mystery. The molecular simulations described herein suggest that transient binary internucleosomal interactions can mediate distant communication in chromatin. Electrostatic interactions between the N-terminal tails of the core histones and DNA enhance the computed probability of juxtaposition of sites that lie far apart along the DNA sequence. Experimental analysis of the rates of communication in chromatin constructs confirms that long-distance communication occurs efficiently and independently of distance on tail-containing, but not on tailless, chromatin. Taken together, our data suggest that internucleosomal interactions involving the histone tails are essential for highly efficient, long-range communication between regulatory elements and their targets in eukaryotic genomes.

RNA polymerase complexes cooperate to relieve the nucleosomal barrier and evict histones.

Maintenance of the chromatin states and histone modification patterns during transcription is essential for proper gene regulation and cell survival. Histone octamer survives moderate transcription, but is evicted during intense transcription in vivo by RNA polymerase II (Pol II). Previously we have shown that nucleosomes can survive transcription by single Pol II complexes in vitro. To study the mechanism of histone displacement from DNA, the encounter between multiple complexes of RNA polymerase and a nucleosome was analyzed in vitro. Multiple transcribing Pol II complexes can efficiently overcome the high nucleosomal barrier and displace the entire histone octamer, matching the observations in vivo. DNA-bound histone hexamer left behind the first complex of transcribing enzyme is evicted by the next Pol II complex. Thus transcription by single Pol II complexes allows survival of the original H3/H4 histones, while multiple, closely spaced complexes of transcribing Pol II can induce displacement of all core histones along the gene.

Interferon regulatory factors IRF5 and IRF7 inhibit growth and induce senescence in immortal Li-Fraumeni fibroblasts.

Cellular immortalization is one of the prerequisite steps in carcinogenesis. By gene expression profiling, we have found that genes in the interferon (IFN) pathway were dysregulated during the spontaneous cellular immortalization of fibroblasts from Li-Fraumeni syndrome (LFS) patients with germ-line mutations in p53. IFN signaling pathway genes were down-regulated by epigenetic silencing during immortalization, and some of these same IFN-regulated genes were activated during replicative senescence. Bisulfite sequencing of the promoter regions of two IFN regulatory transcription factors (IRF5 and IRF7) revealed that IRF7, but not IRF5, was epigenetically silenced by methylation of CpG islands in immortal LFS cells. The induction of IRF7 gene by IFNalpha in immortal LFS cells was potentiated by pretreatment with the demethylation agent 5-aza-2'-deoxycytidine. Overexpression of IRF5 and IRF7 revealed that they can act either alone or in tandem to activate other IFN-regulated genes. In addition, they serve to inhibit the proliferation rate and induce a senescence-related phenotype in immortal LFS cells. Furthermore, polyinosinic:polycytidylic acid treatment of the IRF-overexpressing cells showed a more rapid induction of several IFN-regulated genes. We conclude that the epigenetic inactivation of the IFN pathway plays a critical role in cellular immortalization, and the reactivation of IFN-regulated genes by transcription factors IRF5 and/or IRF7 is sufficient to induce cellular senescence. The IFN pathway may provide valuable molecular targets for therapeutic interventions at early stages of cancer development.

Preparation and analysis of uniquely positioned mononucleosomes.

Short DNA fragments containing single, uniquely positioned nucleosome cores have been extensively employed as simple model experimental systems for analysis of many intranuclear processes, including binding of proteins to nucleosomes, transcription, DNA repair and ATP-dependent chromatin remodeling. In many cases such simple model templates faithfully recapitulate numerous important aspects of these processes. Here we describe several recently developed procedures for obtaining and analysis of mononucleosomes that are uniquely positioned on 150-600 bp DNA fragments.

Mechanism of FACT removal from transcribed genes by anticancer drugs curaxins.

Human FACT (facilitates chromatin transcription) is a multifunctional protein complex that has histone chaperone activity and facilitates nucleosome survival and transcription through chromatin. Anticancer drugs curaxins induce FACT trapping on chromatin of cancer cells (c-trapping), but the mechanism of c-trapping is not fully understood. Here, we show that in cancer cells, FACT is highly enriched within the bodies of actively transcribed genes. Curaxin-dependent c-trapping results in redistribution of FACT from the transcribed chromatin regions to other genomic loci. Using a combination of biochemical and biophysical approaches, we have demonstrated that FACT is bound to and unfolds nucleosomes in the presence of curaxins. This tight binding to the nucleosome results in inhibition of FACT-dependent transcription in vitro in the presence of both curaxins and competitor chromatin, suggesting a mechanism of FACT trapping on bulk nucleosomes (n-trapping).

Topological analysis of plasmid chromatin from yeast and mammalian cells.

Yeast has proven to be a powerful system for investigation of chromatin structure. However, the extent to which yeast chromatin can serve as a model for mammalian chromatin is limited by the significant number of differences that have been reported. To further investigate the structural relationship between the two chromatins, we have performed a DNA topological analysis of pRSSVO, a 5889 base-pair plasmid that can replicate in either yeast or mammalian cells. When grown in mammalian cells, pRSSVO contains an average of 33 negative supercoils, consistent with one nucleosome per 181 bp. This is close to the measured nucleosome repeat length of 190 bp. However, when grown in yeast cells, pRSSVO contains an average of only 23 negative supercoils, which is indicative of only one nucleosome per 256 bp. This is dramatically different from the measured nucleosome repeat length of 165 bp. To account for these observations, we suggest that yeast chromatin is composed of relatively short ordered arrays of nucleosomes with a repeat of 165 bp, separated by substantial gaps, possibly corresponding to regulatory regions.

Transcription through chromatin by RNA polymerase II: histone displacement and exchange.

The process of transcript elongation by RNA polymerase II (Pol II) involves transcription-dependent exchange and displacement of all core histones and is tightly controlled by numerous protein complexes modifying chromatin structure. These processes can contribute to regulation of transcription initiation and elongation, as well as the chromatin state. Recent data suggest that the histone octamer is displaced from DNA at a high rate of transcription, but can survive less frequent transcription that is accompanied only by partial loss of H2A/H2B histones. Here we propose that critical density of Pol II molecules could be required for displacement of the histone octamer and discuss mechanisms that are most likely involved in the processes of histone exchange.

Expression profiling identifies three pathways altered in cellular immortalization: interferon, cell cycle, and cytoskeleton.

Abrogation of cellular senescence, resulting in immortalization, is a necessary step in the tumorigenic transformation of a cell. Four independent, spontaneously immortalized Li-Fraumeni syndrome (LFS) cell lines were used to analyze the gene expression changes that may have given these cell lines the growth advantage required to become immortal. A cellular senescence-like phenotype can be induced in immortal LFS cells by treating them with the DNA methyltransferase (DNMT) inhibitor 5-aza-deoxycytidine. We hypothesized, therefore, that genes epigenetically silenced by promoter methylation are potentially key regulators of senescence. We used microarrays to compare the epigenetic gene expression profiles of precrisis LFS cells with immortal LFS cells. Gene ontology analysis of the expression data revealed a statistically significant contribution of interferon pathway, cell cycle, and cytoskeletal genes in the process of immortalization. The identification of the genes and pathways regulating immortalization will lead to a better understanding of cellular immortalization and molecular targets in cancer and aging.

Overcoming a nucleosomal barrier to replication.

Efficient overcoming and accurate maintenance of chromatin structure and associated histone marks during DNA replication are essential for normal functioning of the daughter cells. However, the molecular mechanisms of replication through chromatin are unknown. We have studied traversal of uniquely positioned mononucleosomes by T7 replisome in vitro. Nucleosomes present a strong, sequence-dependent barrier for replication, with particularly strong pausing of DNA polymerase at the +(31-40) and +(41-65) regions of the nucleosomal DNA. The exonuclease activity of T7 DNA polymerase increases the overall rate of progression of the replisome through a nucleosome, likely by resolving nonproductive complexes. The presence of nucleosome-free DNA upstream of the replication fork facilitates the progression of DNA polymerase through the nucleosome. After replication, at least 50% of the nucleosomes assume an alternative conformation, maintaining their original positions on the DNA. Our data suggest a previously unpublished mechanism for nucleosome maintenance during replication, likely involving transient formation of an intranucleosomal DNA loop.

Large-scale ATP-independent nucleosome unfolding by a histone chaperone.

DNA accessibility to regulatory proteins is substantially influenced by nucleosome structure and dynamics. The facilitates chromatin transcription (FACT) complex increases the accessibility of nucleosomal DNA, but the mechanism and extent of its nucleosome reorganization activity are unknown. Here we determined the effects of FACT from the yeast Saccharomyces cerevisiae on single nucleosomes by using single-particle Förster resonance energy transfer (spFRET) microscopy. FACT binding results in dramatic ATP-independent, symmetrical and reversible DNA uncoiling that affects at least 70% of the DNA within a nucleosome, occurs without apparent loss of histones and proceeds via an 'all-or-none' mechanism. A mutated version of FACT is defective in uncoiling, and a histone mutation that suppresses phenotypes caused by this FACT mutation in vivo restores the uncoiling activity in vitro. Thus, FACT-dependent nucleosome unfolding modulates the accessibility of nucleosomal DNA, and this activity is an important function of FACT in vivo.

Epigenetic silencing of multiple interferon pathway genes after cellular immortalization.

Abrogating cellular senescence is a necessary step in the formation of a cancer cell. Promoter hypermethylation is an epigenetic mechanism of gene regulation known to silence gene expression in carcinogenesis. Treatment of spontaneously immortal Li-Fraumeni fibroblasts with 5-aza-2'-deoxycytidine (5AZA-dC), an inhibitor of DNA methyltransferase (DNMT), induces a senescence-like state. We used microarrays containing 12 558 genes to determine the gene expression profile associated with cellular immortalization and also regulated by 5AZA-dC. Remarkably, among 85 genes with methylation-dependent downregulation (silencing) after immortalization, 39 (46%) are known to be regulated during interferon signaling, a known growth-suppressive pathway. This work indicates that gene silencing may be associated with an early event in carcinogenesis, cellular immortalization.

Preparation and analysis of positioned mononucleosomes.

Short DNA fragments containing single nucleosomes have been extensively employed as simple model experimental systems for analysis of many intranuclear processes, including binding of proteins to nucleosomes, covalent histone modifications, transcription, DNA repair, and ATP-dependent chromatin remodeling. Here we describe several recently developed procedures for obtaining and analysis of mononucleosomes assembled on 200-350-bp DNA fragments.

Experimental analysis of hFACT action during Pol II transcription in vitro.

FACT (facilitates chromatin transcription) is a histone chaperone that facilitates transcription through chromatin and promotes histone recovery during transcription. Here, we describe a highly purified experimental system that recapitulates many important properties of transcribed chromatin and the key aspects of hFACT action during this process in vitro. We present the protocols describing how to prepare different forms of nucleosomes, including intact nucleosome, covalently conjugated nucleosome, nucleosome missing one of the two H2A/2B dimers (hexasome) and tetrasome (a nucleosome missing both H2A/2B dimers). These complexes allow analysis of various aspects of FACT's function. These approaches and other methods described below can also be applied to the study of other chromatin remodelers and chromatin-targeted factors.

Structure of transcribed chromatin is a sensor of DNA damage.

Early detection and repair of damaged DNA is essential for cell functioning and survival. Although multiple cellular systems are involved in the repair of single-strand DNA breaks (SSBs), it remains unknown how SSBs present in the nontemplate strand (NT-SSBs) of DNA organized in chromatin are detected. The effect of NT-SSBs on transcription through chromatin by RNA polymerase II was studied. NT-SSBs localized in the promoter-proximal region of nucleosomal DNA and hidden in the nucleosome structure can induce a nearly quantitative arrest of RNA polymerase downstream of the break, whereas more promoter-distal SSBs moderately facilitate transcription. The location of the arrest sites on nucleosomal DNA suggests that formation of small intranucleosomal DNA loops causes the arrest. This mechanism likely involves relief of unconstrained DNA supercoiling accumulated during transcription through chromatin by NT-SSBs. These data suggest the existence of a novel chromatin-specific mechanism that allows the detection of NT-SSBs by the transcribing enzyme.

Preparation of mononucleosomal templates for analysis of transcription with RNA polymerase using spFRET.

Single positioned nucleosomes have been extensively employed as simple model experimental systems for analysis of various intranuclear processes. Here we describe an experimental system containing positioned mononucleosomes allowing transcription by various RNA polymerases. Each DNA template contains a pair of fluorescent labels (Cy3 and Cy5) allowing measuring relative distances between the neighboring coils of nucleosomal DNA using Forster resonance energy transfer (FRET). The single-particle FRET (spFRET) approach for analysis of DNA uncoiling from the histone octamer during transcription through chromatin is described in detail.