1. Volume 16, Issue 3, Pages 453-463 (November 2004)
Recruitment of Histone Modifications by USF Proteins at a Vertebrate Barrier Element 
Adam G. West, Suming Huang, Miklos Gaszner, Michael D. Litt, Gary Felsenfeld 
Molecular Cell 
Volume 16, Issue 3, Pages (November 2004)
DOI: /j.molcel

2. Figure 1
Footprint IV Sequences Are Required for the Recruitment of Histone Modifications to the 5′HS4 Insulator
(A) Schematic representation of the chicken β-globin cluster and surrounding loci. Boxes representing genes are not to scale. Arrows indicate DNaseI hypersensitive sites. The core of the 5′HS4 element is expanded to show the positions of the five footprinted sequences.
(B) Schematic representation of IL2R transgenes indicating the position of Taqman PCR primer sets.
(C and D) ChIP analysis of integrated IL2R transgenes in 6C2 cells. Fold enrichment of transgenic sequences following immunoprecipitation of nucleosomes with antibodies specific to dimethylated histone H3K4 (C) or diacetylated histone H3 K9/K14 (D). Enrichments were normalized to those observed at the endogenous HSA element of the folate receptor locus to adjust for IP efficiency. Black bars indicate endogenous reference sequences. ChIP analyses were completed after 45 days (post selection) of culture. No (−), variegated (+/−), and maximal (+) transgenic IL2R expression at the point of analysis is indicated (refer to Supplemental Figure S2).
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2 Purification of Footprint IV Binding Proteins
(A) Gel mobility shift assay with chicken erythrocyte nuclear extract using 32P-labeled footprint IV (FIV) oligonucleotide duplexes. Unlabeled competitor duplexes (indicated above each lane) were added at 50-fold molar excess. Arrows indicate footprint sequence-specific complexes.
(B) The sequences of wild-type and mutant FIV oligonucleotides. Footprinted bases are indicated by shading.
(C) Schematic representation of the steps used to purify FIV DNA binding proteins from chicken erythrocyte (RBC) nuclear pellet. Numbers represent the NaCl concentration (mM) at which DNA binding factors eluted from each column. Fractions were assayed for FIV binding activity, with sequence specificity checked following each purification stage (data not shown).
(D) Coomassie blue staining of proteins in the 400 mM eluate fraction following two rounds of FIV DNA affinity chromatography.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3 Analysis of Chicken USF1 and USF2
(A) 35S-labeled in vitro translated USF proteins analyzed by SDS-PAGE. Individually translated chicken USF1, USF2b, and human TFEB are in lanes 1, 2, and 4, respectively. Cotranslated chicken USF1 and USF2b (molar ratio of 1.4:1), human TFEB, and chicken USF1 (molar ratio of 1:3.2) and human TFEB and chicken USF2b (molar ratio of 1:4.5) are in lanes 3, 5, and 6, respectively.
(B) Western blot analyses of proteins in human K562 and chicken 6C2 nuclear extracts, and recombinant chicken USF1 detected by anti-USF1 antibodies.
(C) Western blot analysis of proteins purified by FIV DNA affinity chromatography detected by anti-USF1 antibodies. A control purification including free specific FIV competitor DNA (comp) is shown.
(D) Western blot analyses of proteins in human K562 and chicken 6C2 nuclear extracts detected by anti-USF2 antibodies.
(E) Western blot analysis of proteins purified by FIV DNA affinity chromatography detected by anti-USF2 antibodies.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4 USF1 and USF2 Interact with 5′HS4 through a Divergent E Box
(A) Gel mobility shift analysis of human K562 nuclear extract and recombinant human USF protein binding to labeled footprint IV duplexes. Proteins and antibodies used are indicated above each lane. Arrows indicate FIV sequence-specific (“A” and “B”) and nonspecific (ns; data not shown) complexes.
(B) Gel mobility shift analysis of recombinant protein binding to footprint IV duplexes. Proteins are indicated above each lane. Arrows indicate complexes formed with either human TFEB homodimers (a), chicken USF1 homodimers (b), chicken USF1/USF2b heterodimers (c), or chicken USF2b homodimers (d).
(C) Gel mobility shift analysis of chicken USF protein DNA binding specificity. Unlabeled competitor duplexes (indicated above each lane) were added at 50-fold molar excess. Chicken nuclear extract or recombinant chicken USF proteins used are indicated above each lane. Arrows indicate footprint sequence-specific complexes formed by chicken USF1 homodimers (b) and USF1/USF2b heterodimers (c).
(D) The sequences of the wild-type FIV and USF consensus oligonucleotides. Footprinted FIV bases are indicated by shading. Consensus and degenerate E box motifs are boxed.
(E) USF1 and USF2 specifically interact with the endogenous 5′HS4 insulator in vivo. ChIP analysis in 10 day chick embryo erythrocytes. A schematic representation of the chicken β-globin cluster is shown above. Open arrows indicate locations of TaqMan primer sets used in ChIP analysis. Fold enrichment of sequences following immunoprecipitation of crosslinked chromatin with anti-USF1 or anti-USF2 antibodies are shown as gray and black bars, respectively.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5
USF1 Recruits Histone Modifying Enzymes to the 5′HS4 Insulator
(A) USF1 associates with the H3-specific acetyltransferase PCAF and the H3K4-specific methyltransferase Set7/9. Western blot analyses of proteins immunoprecipitated from chicken erythrocyte nuclear extracts are shown.
(B) The HATs PCAF, CBP, and p300 specifically associate with the endogenous 5′HS4 insulator element. ChIP analysis in 10 day embryo erythrocytes. Fold enrichments of sequences following immunoprecipitation of crosslinked chromatin with anti-PCAF, anti-CBP, or anti-p300 antibodies are shown as white, gray, and black bars, respectively. Enrichments are normalized to the 16 kb condensed sequence, at which there are no enrichments with any of the antibodies used (not shown).
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
USF1 Is Required for Normal Histone Modification Patterns at the 5′HS4 Element
(A) Short hairpin-mediated knockdown of USF1 expression in 6C2 cells. Western blot analysis of whole-cell extracts from hairpin-expressing (kdUSF1) and parent (WT) 6C2 cell lines. USF1 expression is specifically knocked down to ∼10% of wild-type levels. The expression levels of GAPDH and the histone modifiers, SET7/9, PCAF, and CBP are unchanged.
(B) Histone modification patterns at the 5′HS4 insulator element are disrupted following USF1 knockdown. ChIP analyses of histone modifications at endogenous sequences in wild-type (black bars) or USF1 knockdown (gray bars) cells. Fold enrichments were normalized to those observed at either the HSA element or 16 kb condensed chromatin sequences to adjust for IP efficiency.
Molecular Cell  , DOI: ( /j.molcel )

8. Figure 7
Evidence Supports a Chain Terminator Model for the Barrier Function of a Vertebrate Insulator Element
Ubiquitous DNA binding proteins, including USF, interact with the constitutive 5′HS4 insulator element. USF mediates the recruitment of histone modifying enzymes, including those identified in this study. Nucleosomes in the immediate vicinity of the 5′HS4 element become constitutively enriched in H3K4-Me and histone acetylation at multiple sites (Ac) as a result. Immediately upstream of the 5′HS4 insulator is a 16 kb region of uninterrupted condensed chromatin that is deacetylated and enriched in H3K9-Me. Histone hyperacetylation and H3K4-Me at the 5′HS4 element act as a specific barrier to the propagation of H3K9-Me by HMTs. We do not exclude the possibility that the 5′HS4 element recruits as yet uncharacterized histone modifications that also oppose chromatin condensation pathways.
Molecular Cell  , DOI: ( /j.molcel )