1. Structural and Dynamic Functions Establish Chromatin Domains
Kojiro Ishii, Ulrich K. Laemmli 
Molecular Cell 
Volume 11, Issue 1, Pages (January 2003)
DOI: /S (03)

2. Figure 1
Diagnostic Epigenetic State for Different Heterochromatin Protection Activities
This cartoon shows the three main epigenetic expression states of the HML reporter KIY54.
(A) If the UASg sites are empty or occupied by a neutral protein, ADE2 and URA3 are predominantly silenced (OFF/OFF, indicated by coils) by the E and I silencer elements.
(B) If the UASg sites are occupied by a chimeric, genuine boundary protein (BA), which blocks spreading of heterochromatin and lacks transcription activation potential, an ON/OFF epigenetic state is predominantly established. That is, ADE2 is stochastically protected (= ON, indicated by a line) from silencing, while URA3 remains silenced (coils) as it remains exposed to the I silencer.
(C) Transcription activators (TAs) establish an ON/ON state.
(D) Desilencing activities (DAs), in contrast to BAs, block heterochromatin with a bidirectional (arrows) chromatin modifying activity; they also establish in KIY54 an ON/ON expression state. Other HML reporters will be used to experimentally distinguish between the DA and TA functions.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2
The Drosophila BEAF Protein Harbors a Robust, Evolutionarily Conserved BA in Yeast
(A) The structure of the HML reporter KIY54 is shown. The different Gbd-fusion proteins (indicated at left) were expressed in KIY54 and then spotted with 10-fold serial dilution, onto four different agar plates. The epigenetic expression states assayed by this procedure are indicated at the bottom, and the plates are labeled at the top. All epigenetic states grow on the nonselective control plate which scores for the applied cell concentration. The plate called −Ade (lacks adenine) measures the fraction of cells that are either ADE2/URA3 = ON/ON or ON/OFF. The plate called +FOA measures the fraction of cells harboring a silenced URA3 gene; cells that are either ON/OFF or OFF/OFF grow on this plate, since ADE2 can be ON or OFF. The double-selection plate labeled −Ade/+FOA identifies cells expressing genuine BAs that protect ADE2 from silencing while maintaining URA3 silenced (ON/OFF). Quantitative values of those ON/OFF cell fractions are indicated on the right of each panel. Note that full-length BEAF (Gbd-32A/B) and its C-terminal domain (aa 203–283, Gbd-C) exhibit a strong BA function (−Ade/+FOA). Further note that constructs carrying the middle domain of BEAF (aa 82–202, Gbd-M and Gbd-MC) reduce the fraction of URA3-silenced cells (+FOA plate).
(B) These spot test series show that Gbd-fusion proteins of CTCF GAF and Gcn5p are not BAs in yeast. The experimental assay was carried out as described above. Note, however, that Gbd-CTCF, Gbd-GAF, and Gbd-Gcn5 (not the BEAF construct Gbd-C) reduce the fraction of URA3-silenced cells (+FOA plate); this reflects their desilencing activity (see Figure 3).
(C) Spot test series with reporter KIY195 are shown for a number of expressed proteins. Heterochromatin protection is observed by a reduction of the fraction of URA3-silenced cells (+FOA plate) relative to the control plate (control). CTCF and to a lesser extent GAF and Gcn5p work in this assay.
(D) This bar graph presents the β-galactosidase activity obtained with different ectopically expressed Gbd-fusion proteins that were targeted to the UASg sites of a nonsilenced lacZ reporter in KIY27 (except that Gal4-ON activity was in SFY526). Note that full-length BEAF (32A/B), its C-terminal domain (C), and all DAs (CTCF, GAF, and Gcn5p) are not acting as transcription activators.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3 CTCF, GAF, and Gcn5p Block Heterochromatin as DAs
Strains KIY83, KIY86, and KYIY181 serve to distinguish, together with KIY54, DAs from BAs and TAs. DAs, although inactive as TAs in KIY181, KIY86, and KIY27 and also inactive as BAs in KIY54, can still block heterochromatin in KIY83 (interposed binding).
(A) These spot test series with reporter KIY83 show that the indicated Gbd-fusion proteins of BEAF variants, CTCF, GAF, and Gcn5p protect URA3 from silencing if bound interposed between the E silencer and URA3. Sir3p is overproduced to obtain sufficient silencing of URA3 in this strain.
(B) These spot test series with reporter KIY86 show that BAs and DAs lack activity if not interposed between the I silencer and URA3.
(C) KIY181 is an HML reporter where both silencers were deleted and URA3 was replaced with minimal HIS3. The spot test series show that the Gbd-fusion proteins do not stimulate the basal level expression of the HIS3 gene (−His plate), which is required for growth on the plate containing 2 mM of 3-amino-1, 2, 4-triazole inhibitor (−His/+AT). Note that Gbd-VP16:DE reduces HIS3 expression (reduced growth) supposedly due to a squelching phenomenon (Gill and Ptashne, 1988; Sadowski et al., 1988).
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4 Sp1 Harbors a Robust BA in Yeast
Numerous mammalian TAs and other factors were expressed as Gbd-fusion proteins and tested in KIY54 as described in Figure 2. Fractional numbers of cells growing on −Ade/+FOA plate are indicated on the right. Note the robust BA function of the glutamine domains Q1 and Q2 of Sp1 and that many factors (not the BAs) reduce the fraction of URA3-silenced genes (FOA plate).
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5 Many Inactive TAs Block Heterochromatin as DAs
The mammalian and other factors tested in KIY54 (Figure 4) were also examined in KIY83 and KIY86 to identify those with DA functions as described in Figure 3. Most factors exhibit DA functions in KIY83; this is manifested by a reduction of growth on the +FOA plate. No activity is measured in KIY86 where the UASg sites are not interposed. The absence of growth on the control and +FOA plate observed with construct Gbd-VP16:DE is likely indirect, arising from a squelching rather than a desilencing activity.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6
Telomeric Silencing Is Blocked by Proteins Acting as DAs but Not BAs
A number of Gbd-fusion proteins were tested with three different constructs harboring a telomere-linked URA3 gene whose 3′ end is near the telomere. As depicted, YDS634 contains UASg sites interposed between the telomere and the downstream side of URA3. KIY40 contains UASg upstream of URA3. KIY41 has bracketing UASg sites at either side of URA3. Note the only DAs protect (desilence) telomere-linked genes.
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7
Genuine Boundary Proteins and Desilencers Block Heterochromatin by Different Mechanisms
This cartoon summarizes the results obtained.
(A) BAs separate heterochromatin (coils) from euchromatin (line) sharply and in a unidirectional manner. BAs only block heterochromatin if interposed. That is, a gene on the right is ON, while a gene on the left, toward the silencing source, remains OFF. This conclusion is independent of gene orientation.
(B) DAs act bidirectionally at the chromatin level possibly by histone acetylation (Ac?); they create a local euchromatic island. DAs work if interposed (= ON, right side), where gene orientation is inconsequential. Although the DAs lack TA function when tested with nonsilenced yeast genes, they can desilence a noninterposed, silenced gene (= ON, left side), if its promoter is proximal and supposedly within the reach of the bidirectional euchromatic island formed around the DA binding site. This same gene remains OFF if its promoter is distal.
(C) Structural bulwarks set up by BAs do not block silencing of a telomere-linked gene. The cartoon represents strain KIY41 (see Figure 6). Looping of the ends of chromosomes is proposed to lead to multidirectional silencing from the downstream and upstream directions (horizontal arrows) but also from pairing (vertical arrows) and possibly from telomere clustering. We propose that BAs do not block silencing mediated by the latter two processes, pairing and clustering.
(D) DAs acting from downstream (YDS634 is shown, see Figure 6) can desilence a promoter. One explanation is that the formation of loops brings a histone acetylation function bound upstream (Ac?) to the promoter. This explanation is akin to the one described by Ptashne and collaborators, which showed that a transcription activator (Gal4p) works from downstream on telomere-linked (not internal) genes (de Bruin et al., 2001).
Molecular Cell  , DOI: ( /S (03) )