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OCR for page 57
Colloquium
The insulation of genes from external enhancers and
silencing chromatin
Bonnie Burgess-Beusse, Catherine Farrell, Miklos Gaszner, Michael Litt, Vesco Mutskov, Felix Recillas-Targa,
Melanie Simpson, Adam West, and Gary Felsenfeld*
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0540
Insulators are DNA sequence elements that can serve in some cases
as barriers to protect a gene against the encroachment of adjacent
inactive condensed chromatin. Some insulators also can act as
blocking elements to protect against the activating influence of
distal enhancers associated with other genes. Although most of
the insulators identified so far derive from Drosophila, they also
are found in vertebrates. An insulator at the 5' end of the chicken
,l]-globin locus marks a boundary between an open chromatin
domain and a region of constitutively condensed chromatin. De-
tailed analysis of this element shows that it possesses both en-
hancer blocking activity and the ability to screen reporter genes
against position effects. Enhancer blocking is associated with
binding of the protein CTCF; sites that bind CTCF are found at other
critical points in the genome. Protection against position effects
involves other properties that appear to be associated with control
of histone acetylation and methylation. Insulators thus are com-
plex elements that can help to preserve the independent function
of genes embedded in a genome in which they are surrounded by
regulatory signals they must ignore.
A [though DNA methylation has been considered the primary
chromosomal modification involved in transmission of epi-
genetic information, it is becoming clear that the chromatin
proteins, especially the histones, are also involved in this process.
It is understood that the histones bound to transcriptionally
active and inactive regions of the genome are chemically mod-
ified in different ways, and mechanisms have been proposed by
which these modified states can be propagated along the chro-
mosome and perhaps transmitted during replication.
We have for some time been interested in the boundary
elements called insulators. Detailed study of these elements has
led us to mechanisms that are coupled to allele-specific expres-
sion at an imprinted locus, as well as to mechanisms by which
propagation of inactive chromatin states may be modulated.
An Insulator as an Enhancer-Blocking Element
Within the vertebrate genome, transcriptionally active genes are
embedded in an environment containing in some cases extensive
regions of condensed chromatin. In other cases active genes may
be located near other, silent, genes that have a different program
of expression. The possibilities thus arise that the active gene will
be inappropriately silenced by the condensed chromatin or will
inappropriately activate the adjacent silent gene. It is equally
possible that in other tissues or at other developmental stages,
where this gene is inactive, signals from adjacent extraneous
enhancers could cause incorrect patterns of expression.
During the past several years studies begun initially in Dro-
sophila and now extended to vertebrates have identified DNA
sequence elements called insulators that appear to function as
blocks against both kinds of signals from the outside. Two kinds
of assay have been developed to measure these properties (Fig.
1~. The first assay measures "enhancer blocking," the ability to
shield a promoter from the action of a distal enhancer without
www.pnas.org/cgi/doi/10.1073/pnas.162342499
preventing the enhancer from working on a proximal promoter.
The second assay measures the "barrier" activity that prevents
the advance of adjacent condensed chromatin. We have applied
both of these assays in the study of vertebrate insulators.
Our attention was first drawn to this problem through our
interest in the role of chromatin structure in the regulation of
gene expression in the chicken I3-globin locus (Fig. 2~. The gene
cluster contains four members of the ,B-globin family, expressed
at different developmental stages. The regulatory elements of
these genes are marked by a series of erythroid-specific DNase
hypersensitive sites (HSs), but at the 5' end of the locus there is
a "constitutive" HS (5'HS4) present in all tissues that have been
examined (1-3~. We speculated that this HS might mark the 5'
boundary of the "open" globin chromatin domain, and indeed
subsequent work (4) has shown (Fig. 2) that there is an abrupt
change from a chromatin structure characterized by general
heightened nuclease sensitivity and a high level of histone
acetylation (signs of an active globin chromatin locus), to a
region of condensed chromatin further upstream, that is nucle-
ase resistant and underacetylated.
To test whether the DNA at 5'HS4 had properties of an
insulator we devised a method to assay enhancer blocking
activity (Fig. 3A), based on the ability of a 1.2-kb element that
includes the HS to shield a reporter expressing a neomycin
resistance gene from the action of a strong enhancer. As shown
in Fig. 3B, the 1.2-kb element is quite effective in reducing the
number of G418-resistant colonies, a measure of the strength of
the blockade. Placing the 1.2-kb element outside the region
between enhancer and promoter resulted in little blocking (2, 3),
confirming that 5'HS4 has insulating properties. We dissected
this region further and found that a 250-bp "core" sequence
containing the HS was equally effective in enhancer blocking.
Further subdivision, making use of the DNase I footprint
patterns generated on the core by nuclear extracts, revealed that
a single binding site (footprint II) was also active. This finding
led to the identification of a known regulatory protein, CTCF,
as the DNA binding factor responsible for enhancer blocking
activity (5~.
We asked where else CTCF sites with enhancer blocking
activity could be found. It seemed plausible that if these were
truly associated with boundaries there might also be one at the
3' end of the ,8-globin locus. Indeed a constitutive HS with this
activity, and binding CTCF, is found just upstream of the 3'
condensed chromatin region (ref. 6, Fig. 4~. This region also
harbors a gene for an odorant receptor (7~. It seems possible that
This paper results from the Arthur M. Sackler Colloquium of the National Academy of
Sciences, "Self-Perpetuating Structural States in Biology, Disease, and Genetics," held
March 22-24, 2002, at the National Academy of Sciences in Washington, DC.
Abbreviations: HS, hypersensitive site; FR, folate receptor; ICR, imprinted control region;
IL-2R, IL-2 receptor.
*To whom reprint requests should be addressed. E-mail: gary.felsenfeld~nih.gov.
PNAS 1 December 10, 2002 1 vol. 99 1 suppl. 4 1 16433-16437
OCR for page 58
A
Fig. 1. Two kinds of insulator functions. (A) Some insulators may function as
barriers against the encroachment of adjacent genomic condensed chroma-
tin. (B) Some insulators may serve as positional enhancer-blocking elements
that prevent enhancer action when placed between enhancer and promoter,
but not otherwise.
the 3' CTCF site could block cross-interaction between the
regulatory elements of this receptor and those of the ,B-globin
locus. CTCF sites are also present at conserved locations in the
mouse and human loci that are embedded within clusters of
odorant receptor genes (8~. Further examination of the DNA
upstream of the chicken 5'HS4 insulator reveals a somewhat
different arrangement: Here, the upstream sequence is packaged
as condensed chromatin containing CR1 repeat sequences. This
sequence extends for about 16 kb and is followed by a gene (ref.
9, Fig. 4) for an erythroid-specific folate receptor (FR) that is
expressed at a developmental stage preceding the point at which
the globin genes are switched on. The FR gene and the globin
genes are not expressed at the same stages, so that the presence
of an enhancer blocking activity at 5'HS4 might once again be
useful in avoiding cross-talk between the two gene systems.
A role for CTCF-mediated enhancer blocking activity has
been demonstrated most clearly at the I~f2/H19 locus in mouse
LCR elements p
and human (10-12~. In this imprinted locus the maternally
transmitted allele expresses H19 but not Igf2, whereas the
BaFl~iet paternally transmitted allele expresses Igf2 but not H19. Fur-
thermore the paternal allele is methylated at a site (the ICR or
imprinted control region) located between the two genes (Fig.
54. Earlier work had suggested that the ICR might contain an
enhancer blocking activity that would prevent downstream
endodermal enhancers from activating If. Direct examination
reveals that the ICR contains four CTCF binding sites in the
mouse ICR and seven in human. Methylation of these sites
abolishes CTCF binding. These results indicate that CTCF plays
Enhancer an important role as an insulator protein in allele-specific
B' k regulation at this imprinted locus, and that the insulator function
OC Ing can be modulated by DNA methylation, thus making the CTCF
sites susceptible to epigenetic regulation. Quite recently a cluster
of differentially methylated CTCF sites has been identified at the
Xist gene promoter, and it has been suggested that these are
enhancer-blocking elements important for X chromosome in-
activation (13~.
Insulators as Barriers
A second property that some insulators possess is the ability to
protect against silencing caused by formation of condensed
chromatin. This is the barrier function, which can be detected by
assays designed to measure protection of stably integrated
transgenes against position effects (Fig. 1~. We established an
assay to test barrier function by constructing a reporter express-
ing a fragment of the IL-2 receptor (IL-2R) (Fig. 6) driven by an
erythroid-specific promoter and enhancer, and integrating it into
a chicken erythroid cell line, 6C2 (14~. Typically after 80-100
days in culture expression was extinguished in most lines. This
extinction is a manifestation of position effects, i.e., the depen-
dence of expression on the site of integration. We repeated the
experiment with the same reporter, but flanked on each side by
two copies of the 1.2-kb 5'HS4 element; now expression was
maintained in nearly all lines even after 80-100 days of incuba-
tion. The 5'HS4 element thus protects against position effects,
a second property possessed by some insulators.
Silencing of gene expression can involve a number of chro-
matin modifications, and insulators might interfere with some of
these. We turned again to the chicken ,B-globin locus and
examined the state of modification of the histones over the entire
BH 13 A Enh
e ~
.. 1.2 kB
chicken ,l3~1obin domain ~
~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ 1
~ ....
Dnase insensitive DNase sensitive
tow histone acety~afion
High histone acety/ation
Fig. 2. The chicken ,B-giobin iocus (Upper) showing the four genes, the strong enhancer between the adult ,B gene (pA) and the E gene, and the constitutive HS, 5'HS4.
The bounclary between the open chromatin clomain anc] the conclensec] chromatin clomain further 5', as determined by Hebbes et a/. (4), is shown (Lower).
16434 1 www.pnas.org/cgi/doi/10.1073/pnas.162342499
Burgess-Beusse eta/.
OCR for page 59
A
B
a.
Control
Test for enhancer blocking activity ,>11
I
Relative colony number
0;5 1;0
,>
Fig. 3. (A) Construction used in an assay for enhancer (ENH) blocking activity (2, 3). Expression of a gene conferring G418 resistance (neo) is driven by an
erythroid-specific enhancer and promoter. This plasmid is stably transfected into K562 human erythroleukemia cells, and G41 8-resistant colonies are counted.
Typically transfection produces tandem integrants (Control). The test for the putative insulator (I) is to insert it so that it can block enhancer action, and again
to count colonies. (B) Results of inserting a 1.2-kb fragment (see Fig. 2) containing 5'HS4 on colony number in the above assay. The control has an approximately
equal length of A phage DNA on either side of the reporter to keep distances constant. The 5'HS4 element strongly reduces enhancer (ENH) activity. Other
experiments show that it has a much smaller effect when placed on the other side of the enhancer, confirming the positional enhancer blocking activity (2, 3).
54-kb region containing the globin genes and the upstream FR
(15, 16~. Chromatin immunoprecipitation (CHIP) experiments
with antibodies to acetylated histones H3 and H4 revealed
elevated levels of acetylation associated with activation of the
individual genes. Thus there was strong acetylation over the FR
gene in 6C2 cells (Fig. 7), which express this gene, but in 10-day
embryonic erythrocytes, corresponding to a later developmental
stage, high levels of acetylation are shifted to the alobin gene
cluster (15~. At all stages, the approximately 16-kb condensed
chromatin region upstream of the globin genes remains unac-
etylated. Furthermore, there is a peak of acetylation over the
5'HS4 insulator element (as well as over the HSA enhancer of
the FR gene) in all cells we examined, including a DT40
lymphocyte line in which neither globin nor FR genes are active
Burgess-Beusse et a/.
(15~. Recent evidence (16-20) implicates histone methylation as
well in the regulation of expression: Methyl group modifications
at histone H3 lysines 4 or 9 are associated with active or inactive
chromatin, respectively. We again used ChIP methods to mea-
sure patterns of histone methylation over the same region (Fig.
7~. We found a striking correlation between previously observed
patterns of histone acetylation and lysine 4 methylation and
anticorrelation between acetylation and methylation of lysine 9.
The clearest example of such distinct regions of modification is
found in the mating type locus of Schizosaccharomyces pombe,
where the heterochromatic and euchromatic domains of the
mating type locus are distinguished by similar patterns of meth-
ylation and acetylation (18~.
Work in a number of laboratories has led to models for
propagation of the condensed chromatin state (15, 17, 18~. These
PNAS | December so, 2002 | vow. 99 | suppl. 4 | ~6435
OCR for page 60
HS4 3'HS
A
3' enc/
of domain
Fo/ate Rec-~p,for
B _~L~_
5' enc/
of domain
~ L T~
-10 0 10,..' 20 30"~ Amp::
,
,-' BOUNDARY ELEMENT ,`
,~' 39HS `~`
in'
,.'
- Sensitive Resistant
20 25 30
5' Insulator
Condensed Chromatin J~ [CR elements Globin locus
HSA'
~16kb ~ ~~`
'I' ~I'T'I'
_ 1 -
5' HS
Fig. 4. The ends of the open ,B-globin domain. (A) mend. A second constitutive HS that binds CTCF and has enhancer blocking activity is found upstream of
the beginning of a condensed chromatin region containing a gene for an odorant receptor (8). (B) 5' end. A condensed chromatin region extends for about 16
kb upstream of 5'HS4, and beyond that is an erythroid-specific FR gene (9).
are based on the following observations: (i) the heterochromatin
protein HP1 for in S. pombe its homolog Swi6 (18~] binds
selectively to histone H3 that is methylated at lysine 9 in the
amino terminal tail, and (ii) the enzyme responsible for meth-
ylating lysine 9, Suv39H1, interacts with HP1. This finding leads
to the proposal (15, 17, 18) that a nucleosome methylated at that
site will indirectly recruit Suv39H1 and promote methylation of
sites on the adjacent nucleosome. Our observation that the
5'HS4 insulator is a major center of histone acetylation suggests
that one of its roles at the 5' end of the ,B-globin locus is to
continually acetylate the adjacent upstream nucleosome, in
particular lysine 9 of H3. By doing so, it prevents methylation of
that residue and thus terminates the propagation of the con-
densation signal. A related mechanism has been proposed for
barrier activity in Saccharomyces cerevisiae, where a site that
binds histone acetylases is sufficient to prevent extension of
silencing from HMR-E (21~.
These results suggest that in vivo the 5'HS4 insulator has two
functions: it serves as an enhancer blocking element to screen
out upstream signals, and it also acts as a barrier against the
advance of the condensed chromatin structure immediately
upstream. In fact, recent results indicate that these two functions
are separable (22~. The enhancer blocking function, as discussed
16436 1 www.pnas.org/cgi/doi/10.1073/pnas.162342499
~ *
CTCF
~ ^~_l -LW^Y-I.~.I-~Y. ~
+++
Fig. 5. The mouse Igf2/H19 imprinted locus. In the maternally transmitted
allele, Igf2 is silent, but in the paternal allele it is expressed, and the ICR is
methylated. The ICR has been shown to contain four CTCF binding sites, which
have strong enhancer blocking properties; enhancer blocking is abolished by
methylation of cytosines at CpG sites within the ICR. These and other results
lead to a model in which the maternal ICR blocks the action of downstream
endodermal enhancers (E) on the Igf2 promoter. Methylation of the paternal
ICR abolishes enhancer blocking and permits Igf2 activation (10-12).
Burgess-Beusse et a/.
OCR for page 61
13A '6/6 Globin
Promoter Enhancer
IL2R _
.
2 x 1~2 kb insulator
Fig. 6. Constructions to measure protection against position effects (barrier
function). A reporter containing a fragment of the IL-2R driven by the chicken
adult,B-globin promoter and the downstream p/ enhancer (see Fig. 2) is stably
transformed into the avian erythroid line 6C2. Expression of IL-2R on the cell
surface is monitored by FACS analysis. (Upper) The control construction. (Lower)
The control reporter is surrounded by two copies of the 1.2-kb chicken 5'HS4
insulator on each side. In most lines transformed with the control construction,
IL-2R expression was extinguished after 80-100 days in culture. Almost all lines
carrying the insulated construction still expressed IL-2R after 80-100 days (14).
above, depends on the CTCF site, whereas the barrier function
depends on the other four subregions of the 250-bp insulator
core and does not require CTCF. We note that the CTCF site at
the 3' end of the globin locus is not accompanied by the other
subregions present in 5'HS4 and is also not a site of strong
acetylation. The condensed chromatin at the 3' end of the locus
is facilitative, i.e., it must open for expression of the odorant
receptor, and it may therefore be distinct in properties from the
constitutive condensed chromatin at the 5' end and may not
require an acetylated barrier.
How widely are insulators distributed in the genome? A
considerable number of elements have been identified in Dro-
sophila, each with its own characteristic site and associated
proteins (for a recent general review of insulators see ref. 234.
Barrier functions have been identified in both S. pombe and S.
cerevisiae (20, 21~. In vertebrates, the CTCF site appears to be
widely distributed and to function in many cases as an enhancer
blocking agent. However, no other barrier (position effect)
elements have yet been identified similar to that found at the
f-globin locus. Evidently many genes will not require insulators
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6C2 Cells
cllc~ty' H3 80un~
~ ~ tatroacty~ H~ Boun~
H^S3 HS2 HS1
FR :~e ~ ~ ~ ~ ~ ~ ~ ~ ~1 ~ ~ ~ 1 ~D
1 ! ,~ 11 ~ ~ 1 i 1 1 1 11 I ~ 1 ~ 1 ! 1 I 1 1 1
TagrllProb: Z ~ ~ ~ ~ ~ ~ ~ ~ ~4 ~ 3 ~ ~ ~ ~ ~
dl-c~ty' H3 Bound 10 Day Embyronic Red Blood Cells
^_ Mthyl Ly4 H3 Bound4~
~ .oo -
~ u M.~ ~ ~y. 9 H3 Bound; / ~ ~
I!o,~e, ~ Y ~ 1~) ,,,,,,,\
o.oo - ~ ~ ~ ~ ~ j ~
000 _ _ ~
O ~ 1 0 1 6 20 Kb s. ~O ^6 oo ms
Fig. 7. Chromatin immunoprecipitation of modified histones across the
,8-globin locus. (Top) Diacetylated histone H3 and tetraacetylated histone H4 in
6C2 cells, arrested atthe CFU-E stage of chicken erythroid development. (Middle)
Map of the locus showing positions of HSs above the line and of probes used in
PCR detection below the line. (Bottom) Patterns of histone H3 methylation at
Iysines 4 and 9 and diacetylated (Iysines 9 and 14) histone H3 across the locus in
1 O-day embryonic chicken erythrocytes. (Adapted from refs. 1 5 and 16.)
as a protection against inappropriate interaction of neighboring
signals, but it seems reasonable to look for them in cases (such
as the Ig and T cell receptor gene loci) where multiple regulatory
elements are clustered. As the results in Drosophila and yeast
have shown, diverse proteins and sites may be involved. There is
no reason to think that vertebrates will be less complex, so it is
likely that many novel insulators remain to be identified.
Conclusion
Insulator elements are clearly an important family of regulatory
sequences, likely to be distributed widely in the genome. The
connection of enhancer blocking activity with imprinted loci, and
tentatively with the X-inactivation locus (13), suggests a role in
the establishment of epigenetic imprinting marks. The barrier
function is connected with the maintenance of boundaries that
may also be established through epigenetic signals such as
histone methylation. We are still not completely certain of
detailed mechanisms of insulator action, but as we learn more,
we will also understand more about how the cell controls and
exploits epigenetic signals.
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PNAS 1 December 10, 2002 1 vol. 99 1 suppl. 4 1 16437
Representative terms from entire chapter:
condensed chromatin
