Chromatin Insulators and Boundary Elements

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Chromatin Insulators and Boundary Elements
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Confining Gene Expression
Some of the organisational properties of the
eukaryotic genome reside in the ability of
chromatin to establish autonomous units that
specify levels and patterns of gene
expression. i.e. enhancers act on a promoter in a
specific domain, but are unable to act on a
promoter in a separate domain. The candidates
charged with the function of establishing and
delimiting domains of expression are boundary or
insulator elements. These set up independent
territories of gene activity. A few of these
sequences have been characterised in Drosophila
and vertebrates and the gypsy retrotransposon is
one that has been well characterised.
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The gypsy Retrotransposon of Drosophila

• Boundary or insulator elements have two
  characteristic effects on gene expression
• They confer position independent transcription to
  transgenes stably integrated in a chromosome.
• They buffer a promoter from activation by
  enhancers when located between the two.
• The effects of gypsy on the expression of the
  yellow gene has been studied in detail.
• yellow is a non-essential gene - it pigments
  embryonic, larval and adult cuticular structures.
  yellow is regulated by a series of
  tissue-specific transcriptional enhancers.
• gypsy is an infectious retrovirus. It has two
  long terminal repeat regions for viral
  replication and integration and three retroviral
  genes gag, pol and env encoding nucleocapsid,
  reverse transcriptase and envelope proteins.

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Influence of gypsy in the yellow Locus

• The yellow gene is regulated by 5 enhancer
  elements wing blade, body cuticle, larval
  tissues, bristles and tarsal claws.
• Two of the enhancer elements (bristles and tarsal
  claws) are present in the intron of the yellow
  gene.
• Insertion of the gypsy element at different
  positions of the yellow gene results in a variety
  of different phenotypes affecting only certain
  tissues at specific times during development.
• Insertion of gypsy in the 5' region of yellow
  inhibits the interaction between the upstream
  wing and body cuticle enhancers but not the
  larval enhancer located proximal to the promoter.
• Insertion of gypsy in the first intron upstream
  of the bristles and tarsal claws only affects
  these two enhancers.
• Suppression by gypsy is because it has 12
  sequence elements at that bind an insulator
  protein called suppressor of hairy wing su(Hw).

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The gypsy Retrotransposon of Drosophila

• The gypsy insulator is therefore polar only
  enhancers distal from the promoter relative to
  gypsy are affected. Thus it is a unidirectional
  insulator element.
• Mutation of the gene su(Hw) results in a
  non-functional insulator allowing upstream
  enhancers to function in the presence of
  insulator sequences. Therefore su(Hw) is required
  for the insulating properties of gypsy.
• modifier of mdg4 mod(mdg4) was discovered
  through mutations that alter the ability of
  su(Hw) to interfere with enhancer-promoter
  interactions. Mutations in mod(mdg4) cause an
  enhancement of the yellow phenotype, (i.e. flies
  in which every cuticular structure is
  unpigmented). In this case gypsy acts as a
  silencer rather than insulator.
• The mod(mdg4) gene encodes at least 3 different
  proteins arising from alternatively spliced RNAs
  that contain a BTB domain (a motif found in a
  unique class of transcription factors).

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The gypsy Retrotransposon of Drosophila

• Mod(mdg4) and Su(Hw) interact. Mod(mdg4) protein
  lacks a recognizable DNA binding domain and is
  unable to bind to insulator sequences of gypsy.
  Nevertheless Mod(mdg4) is present on polytene
  chromosomes, in 2-3 times the number of bands
  containing Su(Hw), suggesting that Mod(mdg4)
  might also interact with other proteins.
• In the absence of Mod(mdg4), gypsy loses its
  uni-directional effects on distal enhancers and
  instead silences both upstream and downstream
  enhancers. Thus the function of Mod(mdg4) appears
  to be establishment of uni-directionality on the
  gypsy insulator.
• This is thought to be due to heterochromatisation
  of sequences surrounding the insulator and may be
  due to recruitment of heterochromatin proteins by
  Su(Hw).

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Figure 1 Structure and function of the gypsy
insulator. (a) Insulator sequences (ins) are
composed of 12 copies of the binding site for the
su(Hw) protein (su), which interacts in turn with
the mod(mdg4) protein (mo). The complex of both
proteins binds to insulator sequences and
interferes with the function of enhancers present
distally from the promoter with respect to the
location of the insulator. Enhancers are
diagrammed as ellipsoid bodies on the DNA. In the
case shown here, the enhancers that control
expression of yellowin the wings (wng) and body
cuticle (bc) of the fly are affected (represented
by an X over the enhancer), whereas those
responsible for expression in the larval tissues
(lv), bristles (br) or tarsal claws (tc) can
function normally. Exons of the yellowgene are
indicated by open bars, and the intron by a thin
line. The direction of transcription is also
indicated. (b) In a mod(mdg4) mutant, the protein
product of this gene is missing, and only the
su(Hw) protein is bound to insulator sequences.
In this case, repression of transcription is
bi-directional, the insulator behaves as a
silencer, and none of the enhancers can act on
the promoter.
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Figure 2 Long-range and trans-effects of the
gypsy insulator (symbols and organization of the
diagram are as in Fig. 1). (a) Representation of
the yellow locus (left) and the adjacent (1520
kb) achaete (ac) gene (right) of the AS-C of
Drosophila. The gypsy element is inserted in the
complex causing a scute (sc) phenotype, whereas
expression of the yellow gene is unaffected. In
the absence of mod(mdg4) protein, the effect of
the gypsy insulator becomes bi-directional,
silencing the bristle (br) enhancer located in
the intron of the yellow gene. (b) A female
heterozygous for the gypsy-induced y2 mutation
shows a wild-type cuticle coloration the gypsy
insulator represses the wing (wng) and body
cuticle (bc) enhancers in the y2 chromosome
whereas expression of the gene in the other
homolog is normal. (c) In a mod(mdg4) mutant, the
su(Hw) protein causes a bi-directional silencing
effect in the y2 chromosome. In addition, the
flies show a bristle mutant phenotype, suggesting
that the silencing effect can be transmitted in
trans to repress the bristle enhancer in the
other homolog.
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Figure 3 Mechanism of insulator effect on
enhancer function. (a) Diagram of two genes, X
and Y, located within a chromosomal domain
defined by two insulator sequences (ins) and
their associated proteins (ibp). Enhancers
located between the two genes (en1and en2) can
activate transcription from the promoter of
either gene. (b) If a boundary element such as
the gypsy insulator (gyp) is inserted between the
two enhancers, a new chromosomal domain forms,
leaving gene X in one domain and gene Y outside.
One of the insulators forming the original domain
is now free to form other domains with
alternative boundary elements (in this case
containing genes Z1and Z2). Enhancer 1(en1) is
now unable to act on the promoter of gene Y
because of the new location of the gypsy
insulator. Nevertheless, this enhancer is still
functional and competent to activate
transcription from the promoter of gene X,
located within the same chromosomal domain.
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Fig. 2. Transvection at yellow. In the y2
mutation, the insertion of the gypsy element
between the upstream enhancers and the promoter
acts as an insulator to block the action of the
enhancers on the promoter. A homologously paired
yellow gene that lacks its own enhancers,
promoter, and part of the coding region can still
provide enhancer action by inducing the y2 gene
to fold, looping out the gypsy enhancer and
bringing the y2 enhancers close to the promoter.
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Locations of SU(HW) and MOD(MDG4)
Distribution of Su(Hw) is shown in yellow on the
left in a gypsy negative strain. DNA is stained
blue. Distribution of Mod(mdg4) on the right
shows it is present in many more sites than
Su(Hw). Note Su(Hw) is found at all the
Mod(mdg4) sites
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Other Information

• mod(mdg4) mutations only affect the expression of
  yellow if it contains a gypsy element.
• If a gypsy element lacks insulator sequence
  elements, then neither su(hw) nor mod(mdg4) have
  any effect on yellow.
• Insulator sequence elements alone are sufficient
  for the effects of both proteins.
• The mod(mdg4) gene encodes a protein that had
  already been identified as an enhancer of
  variegation E(var)3-93D.
• mod(mdg4) null mutants are lethal whereas su(hw)
  null mutants are female sterile. This suggests
  that Mod(mdg4) has a broader function than
  Su(Hw), probably through binding to other
  proteins involved in recognising boundary
  elements.

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Further Reading
Gdula et al., (1996) PNAS 93, 9378-9383. Tatiana
et al., (1996) Curr. Opin. Gen. Dev. 6,
185-192. Cai and Levine (1997) EMBO J. 16,
1732-1741. Tatiana et al., (1998) Cell 92,
511-521.