1. Dynamics of Enhancer-Promoter Communication during Differentiation-Induced Gene Activation 
Pantelis Hatzis, Iannis Talianidis 
Molecular Cell 
Volume 10, Issue 6, Pages (December 2002)
DOI: /S (02)

2. Figure 1
Activation of the HNF-4α Gene during CaCo-2 Cell Differentiation and Mapping of the Upstream Regulatory Region
(A) Total RNAs prepared from CaCo-2 cells at the indicated hours after reaching confluence were analyzed by RT-PCR using specific primers HNF-4α, Enh-3′, Int.1, and ARP PO as control. Quantitation of HNF-4α mRNA levels were performed by phosphoimage analysis and verified by real-time PCR. Values at the bottom represent normalized HNF-4α reaction products obtained by real-time PCR from the same cDNA samples.
(B) DNase-I hypersensitive analysis. Nuclei from the indicated time postconfluent CaCo-2 cells were digested with 0 to 20 units of DNase-I, and genomic DNA was prepared and digested with either HindIII or EcoRI. Digestion products obtained with 10 units of DNase-I from each time point were separated on 1% agarose gels and subjected to Southern blot hybridization with the indicated probes. The scheme below shows the positions of the major hypersensitive sites relative to the transcription start site.
Molecular Cell  , DOI: ( /S (02) )

3. Figure 2
Nucleosome Structure Analysis of the HNF-4α Regulatory Regions in Differentiating CaCo-2 Cells
(A and B) Nuclei from the indicated times postconfluent CaCo-2 cells and A2780 cells were digested with 0 to 170 units of micrococcal nuclease. Total DNA was prepared and digested either with AccI (A) or MscI (B). Digestion products obtained with 50 units of micrococcal nuclease were separated on 1.5% agarose gels, stained with ethidium bromide, and, after photography (shown in the panel EtBr), blotted to nitrocellulose filters and hybridized with the indicated probes (left panels).
(C) Schematic presentation of the HNF-4α proximal promoter and enhancer relative to nucleosome positions.
(D) Aliquots of nuclei preparations of (A) were digested with 50 units of BglII, and genomic DNA was prepared, which was fully digested with AccI and analyzed in Southern blots with Probe 1.
Molecular Cell  , DOI: ( /S (02) )

4. Figure 3
Order of Recruitment of Transcription Factors at the HNF-4α Enhancer and Promoter in Differentiating CaCo-2 Cells
(A) Schematic presentation of the position of PCR primers used in the chromatin immunoprecipitation analysis. Numbers indicate the 5′ nucleotide positions of the primers relative to the transcription start site.
(B) Chromatin immunoprecipitation (ChIP) assays. Soluble chromatin from crosslinked cells was immunoprecipitated with the indicated antibodies, and the DNAs in the immunoprecipitates were amplified (19–25 cycles) with the indicated oligonucleotides. Autoradiographic images of the products separated on 5% polyacrylamide gels are shown.
Molecular Cell  , DOI: ( /S (02) )

5. Figure 4
Order of Recruitment of General Transcription Factors and RNA Pol-II to the HNF-4α Promoter in Differentiating CaCo-2 Cells
Chromatin immunoprecipitation assays were performed with the indicated antibodies as detailed in Figure 3B. Note that the antibody labeled RNA pol-II (Santa Cruz Biotechnologies, sc-9001) was raised against the N terminus of the protein and recognizes both unphosphorylated and hyperphosphorylated forms of the molecule, while CTD-Ser5P and CTD-Ser2P (Covance H14 and H5) specifically recognize the carboxy-terminal domain of pol-II phosphorylated at Ser5 and Ser2, respectively.
Molecular Cell  , DOI: ( /S (02) )

6. Figure 5
Stable Enhancer-Promoter Complex Formation in 80 Hr Postconfluent Cells
Complexes immunoprecipitated with the indicated first antibodies were eluted from the protein-G-Sepharose beads and, after dilution, were reimmunoprecipitated with the indicated second antibodies. PCR reactions were performed with primer sets amplifying the HNF-4a enhancer (Enh), proximal promoter (Prom), and coding region (Cod).
Molecular Cell  , DOI: ( /S (02) )

7. Figure 6
Histone Modifications and Recruitment of Acetyltransferases and the Brg-1 Chromatin Remodeling Factor to the HNF-4α Regulatory Regions in Differentiating CaCo-2 Cells
Chromatin immunoprecipitation experiments were performed as in Figure 3B, with antibodies raised against histone tail peptides bearing the indicated modifications (A) or antibodies recognizing CBP, P/CAF, and Brg-1 (B).
Molecular Cell  , DOI: ( /S (02) )

8. Figure 7
Model Depicting the Sequential Steps Involved in the Formation of an Active Preinitiation Complex on the HNF-4α Regulatory Region
1. Poised or committed state of the HNF-4α gene.
2. Recruitment of CBP, P/CAF, and Brg-1 to the enhancer region and assembly of the RNA pol-II holoenzyme at the proximal promoter region.
3. Unidirectional movement of the DNA-protein complex formed on the HNF-4α enhancer along the intervening sequences and spreading of histone hyperacetylation.
4. Formation of a stable enhancer-promoter complex, hyperacetylation of nucleosomes located at the promoter, remodeling of the nucleosome located at the transcription start site, and release of RNA pol-II from the promoter. See Discussion for details.
Molecular Cell  , DOI: ( /S (02) )