Formaldehyde Cross-linking Research Articles

Direct recruitment of N-myc to target gene promoters

The N-myc gene is amplified in 20-25% of human neuroblastomas, and this amplification serves as a poor prognostic factor. However, few genes have been determined to be direct targets of N-myc. Our current studies focused on identifying N-myc target genes, especially those affected in cells such as neuroblastomas that have high levels of N-myc protein. To pursue this goal, we performed differential expression screens with cell-culture systems containing high versus low levels of N-myc. The design of our experiments was such that we should identify genes both upregulated and downregulated by N-myc. Accordingly, we identified 22 genes upregulated by N-myc and one gene downregulated by N-myc. However, only five of these genes responded to increased N-myc levels in more than one system. Further analysis of the regulation of these genes required determining whether they were direct or indirect targets of N-myc. Therefore, we used a formaldehyde crosslinking and immunoprecipitation procedure to determine whether N-myc was bound to the promoters of these putative target genes in living cells. We found that low levels of N-myc were bound to the promoters of the telomerase and prothymosin genes in neuroblastoma cells having low amounts of N-myc but that the amounts of N-myc bound to these promoters greatly increased with overexpression of N-myc. However, the amount of max bound to the promoters was high before and after induction of N-myc. Therefore, our studies suggest that N-myc competes with other max partners for binding to target promoters. Our use of the chromatin immunoprecipitation assay suggests a molecular explanation for the consequences of amplification of the N-myc gene in neuroblastomas.

Genome-wide mapping of meiosis-induced DNA double-strand breaks

The major applications of DNA microarray technology to date have been genome-wide analysis of gene expression and analysis of DNA variation. Our goal is to develop an approach using DNA microarray technology to quantitatively study the distribution of DNA-associated proteins at the whole-genome level. An initial approach will determine the genomic distribution of DNA double-strand breaks (DSBs) that initiate meiotic recombination in Saccharomyces cerevisiae. DSB formation involves an intermediate stage where DNA ends are covalently linked to Spo11p. In a rad50S mutant background these intermediates accumulate throughout meiosis. Our approach is to purify Spo11p::His6/DNA intermediates by immobilized metal-affinity chromatography. The DNA purified from these intermediates is then hybridized to DNA microarrays to map the distribution and frequency of Spo11p-linked double-strand breaks throughout the genome. With the addition of a formaldehyde cross-linking step, this approach could be used to map the distribution of any protein that interacts with the genome, either independently or as part of a protein complex. The affinity chromatography approach allows large amounts of material to be purified, and should enable the detection of minor or transient protein/DNA intermediates.

In Vivo Cross-Linking and Immunoprecipitation for Studying Dynamic Protein:DNA Associations in a Chromatin Environment

Chromatin structure plays important roles in regulating many DNA-templated processes, such as transcription, replication, and recombination. Considerable progress has recently been made in the identification of large, multisubunit complexes dedicated to these nuclear processes, all of which occur on nucleosomal templates. Mapping specific genomic loci relative to the position of selectively modified or unique histone variants or nonhistone components provides valuable insights into how these proteins (and their modifications) function in their normal chromatin context. Here we describe a versatile and high-resolution method which involves two basic steps: (1) in vivo formaldehyde cross-linking of intact cells followed by (2) selective immunoprecipitation of protein–DNA complexes with specific antibodies. This method allows for detailed analyses of protein–DNA interactions in a native chromatin environment. Recently, this technique has been successfully employed to map the boundaries of specifically modified (e.g., acetylated) histones along target genes, to define the cell cycle-regulated assembly of origin-dependent replication and centromere-specific complexes with remarkable precision, and to map the in vivo position of reasonably rare transcription factors on cognate DNA sites. Thus, the basic chromatin immunoprecipitation technique is remarkably versatile and has now been used in a wide range of cell types, including budding yeast, fly, and human cells. As such, it seems likely that many more studies, centered around chromatin structure and protein–DNA interactions in its native setting, will benefit from this technique. In this article, a brief review of the history of this powerful approach and a discussion of the basic method are provided. Procedures for protein recovery as well as limitations and extensions of the method are also presented.

Site-directed disulfide bonding reveals an interaction site between energy-coupling protein TonB and BtuB, the outer membrane cobalamin transporter.

Transport of vitamin B(12) across the outer membrane of Escherichia coli, like that of iron-siderophore complexes, is an active transport process requiring a specific outer membrane transporter BtuB, the proton motive force, and the trans-periplasmic energy coupling protein TonB. Interaction between TonB and two of the TonB-dependent siderophore transporters has been detected previously by formaldehyde crosslinking. Here, site-directed disulfide crosslinking demonstrates contact between a conserved region of BtuB, called the TonB-box, and a portion of TonB, previously implicated as the site of suppressors of TonB-box mutations. The specific pattern of disulfide bonding to alternating residues in the TonB-box allowed deduction of the conformation and parallel orientation of the contact region between these two protein segments. Crosslinking at several positions was increased when BtuB was loaded with substrate, and the crosslinking pattern was altered by the presence of substitutions in BtuB that cause a TonB-uncoupled phenotype. This crosslinking process thus reflects protein interactions that are involved in coupling to active transport.

Transcription factor TFIIH is required for promoter melting in vivo.

The Rad25 protein in yeast is a DNA helicase and a subunit of the general transcription factor TFIIH. While in vitro studies have led to the hypothesis that TFIIH helicase activity plays a role in promoter melting, in vivo tests are lacking. Using potassium permanganate, which preferentially modifies single-stranded DNA, we show that a temperature-sensitive rad25(ts) mutant severely reduces the normally extensive promoter melting observed in vivo on the highly expressed genes TDH2 and PDC1 and on the induced heat shock gene HSP82. Loss of promoter melting can be observed in as little as 30 s after a shift to the nonpermissive temperature and is accompanied by a dramatic reduction in transcription. These effects on the promoter are specific, since the mutation does not affect TATA box occupancy or, in the case of HSP82, recruitment of TATA-binding protein to the TATA element or that of heat shock factor to heat shock elements. Additionally, using the technique of formaldehyde cross-linking coupled with restriction endonuclease cleavage and ligation-mediated PCR, we were able to map the polymerase density on the promoter of HSP82. This high-resolution mapping allowed us to determine that the polymerase II (Pol II) density on the promoter is also dramatically reduced after inactivation of TFIIH. These data provide strong support for the hypothesis that TFIIH functions with Pol II in the transcriptionally required step of promoter melting and show, surprisingly, that the extent of TFIIH-dependent promoter melting observed in vivo is several times larger than that seen in vitro.

Formaldehyde Cross-Linking for Studying Nucleosomal Dynamics

Methods are described for the utilization of formaldehyde as a reversible cross-linking agent for the characterization of protein–protein and protein–DNA interactions. The methods include a description of procedures to: (1) isolate and characterize transcriptionally active chromatin from cells cross-linked with formaldehyde; (2) study histone mobility during replication and transcription by the characterization of the formaldehyde-cross-linked histone octamer that is isolated from cells labeled with density-labeled amino acids; and (3) cross-link thein vitroreconstituted histone–DNA complex in order to maintain its structural state during subsequent characterization. Included in these methods are procedures for a second dimensional analysis of protein–protein cross-links in which the monomer components are electrophoretically resolved in the second dimension. The methods also include procedures to selectively reverse protein–DNA cross-links while maintaining the protein–protein cross-links. Potential artifacts are also discussed; i.e., data are presented which indicate that the helical pitch of DNA can be altered if the ionic strength is not properly controlled. The stability of the cross-linked nucleosome in the presence of altered pH or salt/urea concentrations is described in order to indicate that there are limitations to procedures that can be used for the subsequent characterization of the cross-linked complex.

Mapping DNA target sites of chromatin proteins in vivo by formaldehyde crosslinking.

Mapping DNA target sites of chromatin proteins in vivo by formaldehyde crosslinking.

Mapping DNA interaction sites of chromosomal proteins. Crosslinking studies in yeast.

Eucaryotes use a common theme for packaging genomic DNA: 146 bp of DNA are wrapped around an octameric protein core consisting of two molecules each of the histones H2A, H2B, H3, and H4. Although this nucleosomal arrangement is ubiquitous throughout the genome, different chromosomal portions are nonetheless functionally and structurally distinct. This is owing to regional interactions between regulatory chromosomal proteins (i.e., transcription factors, structural components, histone-modifying enzymes) and specific portions of the chromosome. Therefore it is important to determine the natural sites of action of these chromatin (re-)modeling factors and to gain insight into the parameters governing their association with chromatin in vivo. To address this problem, Orlando and Paro () have used formaldehyde crosslinking to prevent redistribution of chromatin components during chromatin preparation, immunoprecipitation to isolate the chromosomal factor(s) to be investigated and hybridization to enable the detection of genomic sequences contacted by the factor(s) (see Chapter 33). A similar approach has been used by Braunstein et al. (), employing antibodies against sites of histone acetylation, to determine the acetylation state of histones in chromatin sites. We have streamlined and further developed this protocol and included a polymerase chain reaction (PCR) step to more sensitively and rapidly map the chromosomal distribution of regulatory proteins in Saccharomyces cerevisiae (Fig. 1). Open image in new window Fig. 1. Strategy for the mapping of chromosomal interaction sites of nonhistone chromatin associated factors. Yeast cells are crosslinked with formaldehyde in vivo (A) and subsequently disrupted mechanically. Chromatin fragments released from the cells are sheared by sonication (B) to an average size suitable for the desired accuracy and resolution of the mapping experiment. Antibodies recognizing a chromosomal non-histone protein are added and protein-DNA complexes are recovered by adsorption to proteinA sepharose beads (C). Samples from the INPUT and PRECIPITATE are heat-treated to reverse the crosslinks and DNA is isolated (D). The purified DNA is then used as template in multiplex PCR reactions with primer pairs derived from different chromosomal regions. Control PCR reactions with decreasing amounts of template are analyzed in parallel to assure that the PCR was still in the exponential phase to allow for a quantitative interpretation of the results (E). (F) Polyacrylamide gel electrophores’s is used to separate and visualize different sized PCR products. Bound as well as unbound sequences are equally represented in the genome and will be amplified accordingly from the INPUT sample. Only sequences specifically bound by the regulatory protein in vivo coimmunoprecipitate with this factor (the shaded area in the P1/P2 region symbolizes an interaction site) and will give rise to PCR products from the PRECIPITATE (F).

Interactions in the TonB-dependent energy transduction complex: ExbB and ExbD form homomultimers.

The cytoplasmic membrane proteins ExbB and ExbD support TonB-dependent active transport of iron siderophores and vitamin B12 across the essentially unenergized outer membrane of Escherichia coli. In this study, in vivo formaldehyde cross-linking analysis was used to investigate the interactions of T7 epitope-tagged ExbB or ExbD proteins. ExbB and ExbD each formed two unique cross-linked complexes which were not dependent on the presence of TonB, the outer membrane receptor protein FepA, or the other Exb protein. Cross-linking analysis of ExbB- and ExbD-derived size variants demonstrated instead that these ExbB and ExbD complexes were homodimers and homotrimers and suggested that ExbB also interacted with an unidentified protein(s). Cross-linking analysis of epitope-tagged ExbB and ExbD proteins with TonB antisera afforded detection of a previously unrecognized TonB-ExbD cross-linked complex and confirmed the composition of the TonB-ExbB cross-linked complex. The implications of these findings for the mechanism of TonB-dependent energy transduction are discussed.

C-Myc target gene specificity is determined by a post-DNAbinding mechanism.

Uncertainty as to which member of a family of DNA-binding transcription factors regulates a specific promoter in intact cells is a problem common to many investigators. Determining target gene specificity requires both an analysis of protein binding to the endogenous promoter as well as a characterization of the functional consequences of transcription factor binding. By using a formaldehyde crosslinking procedure and Gal4 fusion proteins, we have analyzed the timing and functional consequences of binding of Myc and upstream stimulatory factor (USF)1 to endogenous cellular genes. We demonstrate that the endogenous cad promoter can be immunoprecipitated with antibodies against Myc and USF1. We further demonstrate that although both Myc and USF1 can bind to cad, the cad promoter can respond only to the Myc transactivation domain. We also show that the amount of Myc bound to the cad promoter fluctuates in a growth-dependent manner. Thus, our data analyzing both DNA binding and promoter activity in intact cells suggest that cad is a Myc target gene. In addition, we show that Myc binding can occur at many sites in vivo but that the position of the binding site determines the functional consequences of this binding. Our data indicate that a post-DNA-binding mechanism determines Myc target gene specificity. Importantly, we have demonstrated the feasibility of analyzing the binding of site-specific transcription factors in vivo to single copy mammalian genes.

Enzymatic degradation of formaldehyde-crosslinked gelatin

A general method is proposed for characterizing the enzymatic degradability of formaldehyde-crosslinked gelatin which is simple and uses subtilisin as a readily available, commercial alkaline proteinase. Solubilization of gelatin involved dissolution of species not chemically bonded to the crosslinked network, as well as the soluble fractions resulting to the enzymatic degradation. There was a linear relationship between the complete solubilization time of the gelatin and its exposure time to the formaldehyde crosslinking procedure. © Rapid Science Ltd. 1998

Loading of an Mcm Protein onto DNA Replication Origins Is Regulated by Cdc6p and CDKs

In eukaryotic cells, firing of DNA replication origins normally does not recur until after M phase. This characteristic is thought to be due to the properties of “initiation” proteins like Orc, Cdc6, and Mcms. Using formaldehyde cross-linking, we show that Cdc6p and Mcm7p associate specifically with replication origins during G1 but not during G2 in S. cerevisiae. Mcm7p's association with origins depends on Cdc6p. Ectopic expression of Cdc6p enables it to associate with origins during G2, but this fails to recruit Mcm7p. Our data suggest that the loading of Mcm proteins onto origins is regulated by two mechanisms: first, by Cdc6p occupancy, and second, by S- and M-CDKs, whose activity during S, G2, and M phases prevents Mcm loading.

Co-localization of Polycomb protein and GAGA factor on regulatory elements responsible for the maintenance of homeotic gene expression.

The Polycomb group and trithorax group genes of Drosophila are required for maintaining the differential expression state of developmental regulators, such as the homeotic genes, in a stable and heritable manner throughout development. The Polycomb group genes have been suggested to act by regulating higher order chromatin and packaging repressed chromosomal domains in a heterochromatin-like structure. We have mapped, at high resolution, the distribution of Polycomb protein on the bithorax complex of Drosophila tissue culture cells, using an improved formaldehyde cross-linking and immunoprecipitation technique. Polycomb protein is not distributed homogeneously on the regulatory regions of the repressed Ultrabithorax and abdominal-A genes, but is highly enriched at discrete sequence elements, many of which coincide with previously mapped Polycomb group response elements (PREs). Our results further suggest that Polycomb protein spreads locally over a few kilobases of DNA surrounding PREs, perhaps to stabilize silencing complexes. GAGA factor/Trithorax-like, a member of the trithorax group, is also bound at those PREs which contain GAGA consensus-binding sites. Two modes of binding can be distinguished: a high level binding to elements in the regulatory domain of the expressed Abdominal-B gene, and a low level of binding to Polycomb-bound PREs in the inactive domains of the bithorax complex. We propose that GAGA factor binds constitutively to regulatory elements in the bithorax complex, which function both as PREs and as trithorax group response elements.

Localization of Sir2p: the nucleolus as a compartment for silent information regulators.

In wild-type budding yeast strains, the proteins encoded by SIR3, SIR4 and RAP1 co-localize with telomeric DNA in a limited number of foci in interphase nuclei. Immunostaining of Sir2p shows that in addition to a punctate staining that coincides with Rap1 foci, Sir2p localizes to a subdomain of the nucleolus. The presence of Sir2p at both the spacer of the rDNA repeat and at telomeres is confirmed by formaldehyde cross-linking and immunoprecipitation with anti-Sir2p antibodies. In strains lacking Sir4p, Sir3p becomes concentrated in the nucleolus, by a pathway requiring SIR2 and UTH4, a gene that regulates life span in yeast. The unexpected nucleolar localization of Sir2p and Sir3p correlates with observed effects of sir mutations on rDNA stability and yeast longevity, defining a new site of action for silent information regulatory factors.

Chromatin structure and methylation of rat rRNA genes studied by formaldehyde fixation and psoralen cross-linking.

By using formaldehyde cross-linking of histones to DNA and gel retardation assays we show that formaldehyde fixation, similar to previously established psoralen photocross-linking, discriminates between nucleosome- packed (inactive) and nucleosome-free (active) fractions of ribosomal RNA genes. By both cross-linking techniques we were able to purify fragments from agarose gels, corresponding to coding, enhancer and promoter sequences of rRNA genes, which were further investigated with respect to DNA methylation. This approach allows us to analyse independently and in detail methylation patterns of active and inactive rRNA gene copies by the combination of Hpa II and Msp I restriction enzymes. We found CpG methylation mainly present in enhancer and promoter regions of inactive rRNA gene copies. The methylation of one single Hpa II site, located in the promoter region, showed particularly strong correlation with the transcriptional activity.

SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast.

Yeast core telomeric heterochromatin can silence adjacent genes and requires RAP1, SIR2, SIR3, and SIR4 and histones H3 and H4 for this telomere position effect. SIR3 overproduction can extend the silenced domain. We examine here the nature of these multiprotein complexes. SIR2 and SIR4 were immunoprecipitated from whole-cell extracts. In addition, using formaldehyde cross-linking we have mapped SIR2, SIR4, and RAP1 along telomeric chromatin before and after SIR3 overexpression. Our data demonstrate that SIR2 and SIR4 interact in a protein complex and that SIR2, SIR3, SIR4, and RAP1 map to the same sites along telomeric heterochromatin in wild-type cells. However, when overexpressed, SIR3 spreads along the chromosome and its interactions are dominant to those of SIR4 and especially SIR2, whose detection is decreased in extended heterochromatin. RAP1 binding at the core region is unaffected by SIR3 overproduction and RAP1 shows no evidence of spreading. Thus, we propose that the structure of core telomeric heterochromatin differs from that extended by SIR3.

綿繊維の気相ホルムアルデヒド架橋に及ぼす前処理および触媒付与方法の効果

Effects of pre-treatments and catalysts-application methods on formaldehyde cross-linking density in cotton fibers finished with vapor-phase-formaldehyde were studied.Cotton fibers were pre-treated with aqueous sodium hydroxide solution, liquid ammonia and their combination.For vapor-phase-formaldehyde-finishing, two kinds of catalysts application methods were examined.One is MgC12 with aqueous solution and the other is SO2 gas.It was found that the degree of formaldehyde cross-linkig in vapor-phase-formaldehyde treated cotton fibers depends on the initial moisture regain for MgC12 solution system, while depends on both initial moisture regain and pre-treated method for SO2 gas system.

Tympanic Membrane Reconstruction using Formaldehyde‐Formed Autogenous Temporalis Fascia: Twenty Years' Experience

This study describes 20 years' experience with autogenous temporalis fascia that is formed and shaped by formaldehyde cross-linking with special Fasciaform (Hear America, Palo Alto, Calif.) molds in the repair of large tympanic membrane perforations. One hundred twenty operations in 113 patients were performed between 1973 and 1993 to close large perforations in patients with intact ossicular chains. All perforations were successfully closed by this technique. Audiometric studies indicated that the postoperative air-bone gap was closed to within 0 to 10 dB in 63% and to within 0 to 20 dB in 97%. One patient had a 15-dB sensorineural hearing impairment. Graft lateralization requiring revision occurred in three patients, two of whom had previous unsuccessful tympanoplasties. Comparisons of adult vs. pediatric groups and primary vs. revision groups were made. The technique ensures the removal of any ectopic epithelium on the medial surface of the tympanic membrane remnant and provides for easy graft placement and stability during healing without the use of middle ear Gelfoam (Upjohn Co., Kalamazoo, Mich.). The formaldehyde-formed fascia graft or Fasciaform graft technique provides an effective method of closing large perforations with excellent functional results and minimal complications.

Tympanic membrane reconstruction using formaldehyde-formed autogenous temporalis fascia: Twenty years' experience

This study describes 20 years' experience with autogenous temporalis fascia that is formed and shaped by formaldehyde cross-linking with special Fasciaform (Hear America, Palo Alto, Calif.) molds in the repair of large tympanic membrane perforations. One hundred twenty operations in 113 patients were performed between 1973 and 1993 to close large perforations in patients with intact ossicular chains. All perforations were successfully closed by this technique. Audiometric studies indicated that the postoperative air-bone gap was closed to within 0 to 10 dB in 63% and to within 0 to 20 dB in 97%. One patient had a 15-dB sensorineural hearing impairment. Graft lateralization requiring revision occurred in three patients, two of whom had previous unsuccessful tympanoplasties. Comparisons of adult vs. pediatric groups and primary vs. revision groups were made. The technique ensures the removal of any ectopic epithelium on the medial surface of the tympanic membrane remnant and provides for easy graft placement and stability during healing without the use of middle ear Gelfoam (Upjohn Co., Kalamazoo, Mich.). The formaldehyde-formed fascia graft or Fasciaform graft technique provides an effective method of closing large perforations with excellent functional results and minimal complications. (Otolaryngol Head Neck Surg 1996;114:366-79.)

Immunoprecipitation of chromatin

This chapter discusses the immunoprecipitation of chromatin. The chapter discusses three standard protocols, all of which are used to analyze the role of specific proteins within defined regions of the eukaryotic genome. It analyzes both the DNA and the protein after immunoprecipitation. This requires a careful examination of the protocol chosen as not all are amenable to protein analysis. The conformation of protein antigens is likely to vary depending on whether they are in their native state in solution, formaldehyde-fixed, bound to nitrocellulose filters, or fixed to plastic microtiter plates. Nonspecific binding of the antibody to chromatin should also be carefully controlled. Antibodies may be added in excess if complete depletion of the target protein is required. Affinity purification of antisera also minimizes background and, if possible, should be routinely carried out. Some studies requiring the precise positioning of DNA-binding proteins are hampered by the exchange, sliding, or artificial movements of proteins during chromatin isolation. Treatment with formaldehyde dimethyl sulfate/borohydride or UV irradiation efficiently cross-links proteins to DNA. Methods for formaldehyde cross-linking of chromatin in nuclei is described in detail.