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Technologies such as ChIP-seq, MNase-seq, FAIRE-seq, DNase-seq, and ATAC-seqcombine next generation sequencing (NGS) with new biochemical techniques, or modifications of established ones, to enable genome-wide investigations of a broad spectrum of chromatin phenomena (Figure 1).

Figure 1. Overview of ChIP-seq, DNase-seq, ATAC-seq and MNase-seq experiments.

A genomic locus analyzed by complementary chromatin profiling experiments reveals different facets of chromatin structure; ChIP-seq reveals binding sites of specific transcription factors, DNase-seq and ATAC-seq reveal regions of open chromatin while MNase-seq identifies well-positioned nucleosomes. In ChIP-seq chromatin immunoprecipitation (ChIP) is used to extract DNA fragments that are bound to the target protein, either directly or via other proteins in a complex containing the target factor. In DNase-seq, chromatin is lightly digested by the DNase I endonuclease. Size selection is used to enrich for fragments that are produced in regions of chromatin where the DNA is highly sensitive to DNase I attack. ATAC-seq is an alternative to DNase-seq that uses an engineered Tn5 transposase to cleave DNA and to integrate primer DNA sequences into the cleaved genomic DNA. Micrococcal nuclease (MNase) is an endo-exo- nuclease that processively digests DNA until an obstruction such as a nucleosome is reached.

Reference:

Mayer, C.A., Liu, X.S. Identifying and Mitigating Bias in Next-Generation Sequencing Methods for Chromatin Biology. Nat Rev Genet. 2014. 15(11): 709–721.

(Elisa Bono)

ChIP-Seq (Samuele Irudal):

ChIP-Seq combines chromatin immunoprecipitation with DNA sequencing, to obtain the DNA sequences which are able to interact with the immunoprecipitated proteins. This is very useful to identify transcription factors and nucleosome-associated sequences. Workflow is very simple: first, the cells need to be treated with formaldehyde, to create cross-link between the proteins and the DNA. Then, cells have to be lysed, DNA is extracted and fragmented with sonication; an antibody against the protein of interest is added. Precipitation could be achieved via beads associated to A/G proteins and centrifugation. Subsequently the pellet needs to be eluted and treated with high ionic force or high temperature, to resolve the cross-links and dissociate DNA fragments from the protein of interest. DNA has to be purified from contaminants (RNA and proteins). Finally, adapters have to be added at the ends of the fragments: ends must be blunted to allow ligase reaction between the fragments and the adapters. Specific primers for PCR phase (able to recognize these adapters) will be used to amplify and sequence each fragments. Also, a library can be created.

ATAC-Seq

ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) is a technique used in molecular biology to study chromatin accessibility genome-wide. The technique was first described in 2013, as an alternative or complementary method to MNase-seq (sequencing of micrococcal nuclease sensitive sites), FAIRE-seq and DNAse-seq. It aims to identify accessible DNA regions, equivalent to DNase I hypersensitive sites.

The key part of the ATAC-seq procedure is the action of the transposase Tn5 (transposase of prokaryotic origin) on the genomic DNA of the sample. Transposases are enzymes that bind to the end of a transposon and catalyze the movement of transposons to other parts of the genome. While naturally occurring transposases have a low level of activity (necessary to reduce the risk of fatal mutations in the host), ATAC-seq employs a mutated hyperactive transposase.

Hyperactive Tn5 transposase endogenously functions through the “cut and paste” mechanism requiring sequence-specific excision of a locus containing 19 base-pair inverted repeats.

The Tn5 transposase reagent is loaded with sequencing adapters creating an active dimeric transposome complex. The enzyme so can provide the cut of exposed DNA and the simultaneous ligation of specific sequences, called adapters.

This transposase preferentially inserts sequencing adapters into unprotected regions of DNA, therefore acting as a probe for measuring chromatin accessibility genome-wide.

Adapter-ligated DNA fragments are then isolated, amplified by PCR and used for next generation.

An ATAC-seq experiment will typically produce millions of next generation sequencing reads that can be successfully mapped on the reference genome. After elimination of duplicates, each sequencing read points to a position on the genome where one transposition (or cutting) event took place during the experiment. One can then assign a cut count for each genomic position and create a signal with base-pair resolution.

Regions of the genome where DNA was accessible during the experiment will contain significantly more sequencing reads (since that is where the transposase preferentially acts), and form peaks in the ATAC-seq signal that are detectable with peak calling tools. These regions can be further categorized into the various regulatory element types - promoters, enhancers, insulators, etc.- by integrating further genomic and epigenomic data such as information about histone modifications or evidence for active transcription. Inside the regions where the ATAC-seq signal is enriched, one can also observe sub-regions with depleted signal. These subregions, typically only a few base pairs long, are considered to be “footprints” of DNA-binding proteins. These proteins will protect the DNA strand from transposase cleavage and will consequently cause a depletion in the signal.

An ATAC-seq experiment can also be used to infer nucleosome positions.

References:

-       Buenrostro, J., Wu, B., Chang, H., Greenleaf, W., (2015) ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide, Curr Protoc Mol Biol. ; 109: 21.29.1–21.29.9

-       Wikipedia

(Elisa Bono)

DNase-seq

DNase-seq (DNase I hypersensitive sites sequencing) is a technique used to identify the location of cis-regulatory regions (eg promoters, enhancers, silencers) in the genome. These regulatory areas are nucleosome-free and, therefore, are accessible and sensitive to DNase I. DNase I is an E. coli’s endonuclease which only shows specificity for DNA regions not wrapped on histones, independently from the nucleotidic composition.

Briefly, cells are lysed, chromatin in extracted and DNase I digestion occur. Then the solution is deproteinized so only DNA fragments are cleaved by restriction enzymes. The remaining fragments are now separated on electrophoresis gel. In the classical DNase hypersensitivity assay the following step is Southern blot with very ends probes; on the contrary, in DNase-seq it is necessary to amplify cleavage products by PCR and prepare a library for high-throughput next generation sequencing. NGS will give the precise nucleotidic sequence of nucleosome-depleted DNA regions, which are the regulatory ones.

FAIRE-seq is a DNA-seq successor.

(Giada Cipollina)

FAIRE

FAIRE-Seq (Formaldehyde-Assisted Isolation of Regulatory Elements Sequencing) is a technique used for isolation and sequencing of nucleosome-depleted regions of the genome. To perform FAIRE, chromatin is crosslinked with formaldehyde in vivo, sheared by sonication, and phenol-chloroform extracted. The DNA recovered in the aqueous phase is fluorescently labelled and sequenced with NGS techniques or hybridized to a DNA microarray. FAIRE performed in human cells strongly enriches DNA coincident with the location of DNaseI hypersensitive sites, transcriptional start sites, and active promoters. Evidence for cell-type–specific patterns of FAIRE enrichment is also presented. FAIRE has utility as a positive selection for genomic regions associated with regulatory activity, including regions traditionally detected by nuclease hypersensitivity assays.

 

FAIRE was first proved in Saccharomyces cerevisiae, where was demonstrated that following phenol-chloroform extraction of formaldehyde-crosslinked yeast chromatin, the genomic regions immediately upstream of genes were preferentially segregated into the aqueous phase. The enrichment of regulatory regions in the aqueous phase was interpreted to indicate relatively inefficient crosslinking between proteins and DNA at these regions, symptom of a general lack of proteins (e.g. histones) at these sites. More recent experiments, in fact, confirmed that FAIRE enrichment has a strong negative correlation with nucleosome occupancy.

 

Brief example of FAIRE procedure:

Formaldehyde is added directly to plates of living cells (e.g. fibroblasts) at room temperature (22–25°C) to a final concentration of 1% and plates are then incubated for 1-7 minutes. Glycine is then added at room temperature to quench the formaldehyde. Afterward, cells are rinsed with phosphate buffered saline, spun and then snap frozen. Cell lysis can be achieved through glass beads disruption in a lysis buffer solution and, afterward, the whole sample is sonicated to obtain DNA fragments.

DNA is isolated by adding an equal volume of phenol-chloroform, vortexing and spinning at 4°C. The aqueous phase is isolated and stored in a separate tube. The organic phase undergoes again the process and the new aqueous phase is added to the previously isolated one. Only at this point the final phenol-chloroform extraction is performed, to be sure that the whole protein content has been eliminated.

DNA is precipitated with Sodium acetate and resuspended in a solution containing RNase A. Crosslinked samples are incubated at 65°C overnight to ensure that any DNA–DNA crosslinks do not interfere with downstream enzymatic steps.

 

FAIRE-extracted DNA fragments can be analysed in a high-throughput way using next-generation sequencing techniques (NGS). In general, libraries are made by ligating specific adapters to the DNA fragments that allow them to cluster on a platform and be amplified resulting in the DNA sequences being read/determined, and this in parallel for millions of the DNA fragments.

Depending on the size of the genome FAIRE-seq is performed on, a minimum of reads is required to create an appropriate coverage of the data, ensuring a proper signal can be determined. In addition, a reference or input genome, which has not been cross-linked, is often sequenced alongside to determine the level of background noise.

Extracted FAIRE-fragments can be quantified in an alternative method by using quantitative PCR. However, this method does not allow a genome wide / high-throughput quantification of the extracted fragments.

Giresi, P.G. et al. (2007) FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) isolates active regulatory elements from human chromatin, Genome Res 17(6), 877–885

https://en.wikipedia.org/wiki/FAIRE-Seq

(Cecilia Castelli)

Nucleosome positioning (Samuele Irudal)

Different assay can bu used to assess nucleosoe positioning, but two can be the more relevant: one is an in tube assay, the other is an in vivo one. Both are single locus analysis, so the focus will be only one one gene.

• In the first, DNA associated to nucleosomes have to be extracted; then, nucleosomes are dissociated from the main sequence. After a fixed time, a specific RE is used to cut a restriction site in our gene of reference; the samples are then separeted on gel-electrophoresis and cut is spotted via Southern-blot, using a probe against our gene. Different output can be observed, according to the new position taken by the nucleosomes: if the nucleosomes covered the RE site, one high band will be observed; if nuclesomes moved on the sequence, different bands with different MW will be observed. Nucleosomes positioning can be forced to change adding Tfs to the tube, causing (e.g.) the coverage of the RE site. Fig. 1 shows the point of the assay.

• The second assay involves the use of Micrococcus Nuclease (MNase 1). First, nuclei are extracted from the cell colture and then treatment with MNase 1 occurs. This enzyme is an endonuclease, able to cut in the middle of the sequence and to digest DNA until an obstacle is encountered (e.g.: nucleosome associated to DNA). After this passage, the samples are deproteinized and RE treatment is followed: this will grant a point of reference for MW misuration in the next passage. Subsequently, separation on gel-electrophoresis is executed with a following Southern blot analysis. Being a single locus analysis, a probe against a specific gene is used. Different output can be observed: if in a sample nucleosome positioning is random, many bands will be shown according to the different length of the fragments (Fig.2a). If nucleosomes positioning in the sample is ordered, bands number will be indeed fixed and and lower (Fig.2b).

other