Lewin Benjamin (ed.) Genes IX
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an anri-BEAF-32
antibody
srains
-50%
of rhe
interbands
of the
polytene
chromosomes.
This
suggests
that there are
many insulators
in the
genome,
and that BEAF-32
is
a common
part
of the insulating
apparatus. It
would imply
that
the band is
a functional
unit, and
that inter-
bands
often have insulators
that
block the
prop-
agation
of activating
or inactivating
effects.
Another
example of
an insulator
that
defines
a domain
is found in
the chick
p-globin
LCR
(the
group
of hypersensitive
sites that con-
trols
expression
of all
B-globin
genes;
see Sec-
lion 29.20,
An LCR
May
Control a Domain).
The leftmost
hypersensitive
site
of the chick
p-globin
LCR
(HS4)
is
an
insulator
rhar marks
the 5'end
of the functional
domain.
This restricts
the LCR
to acting
only on
the
globin
genes
in
the
domain.
A
gene
that is
surrounded
by insulators
is
usually
protected
against
the
propagation
of
inactivating
effects from
the surrounding
regions.
The
test is
to insert DNA
into
a
genome
at ran-
dom locations
by transfection.
The expression
of
a
gene
in
the inserted
sequence is
often erratic;
in
some instances
it
is
properly
expressed,
but
in others
it is
extinguished.
When insulators
that
have
a barrier function
are
placed
on either
side
of the
gene
in the inserted
DNA,
however,
its
expression
typically
is uniform
in
every case.
@
Insulators
May
Act
in
0ne
Direction
.
Some insutators
have
directionatity,
and may
stop
passage
of effects
in one direction
but
not
the
other.
Insulators
may have
directional properties.
Insertions
of
the transposon gypsy
into the
yel-
low
(y)
locus
of D. melanogaster
cause loss
of
gene
function
in
some
tissues,
but not in
others. The
reason
is
that the
y
locus is
regulated
by four
enhancers,
as shown
in tISl,JRf
PS.4S.
Wherever
gypsy
is
inserted,
it blocks
expression
of all
enhancers
that it
separates
from
the
promoter,
but not
those
that lie
on
the other
side. The
sequence
responsible
for this
effect
is an insu-
lator
that lies
at one
end of
the transposon.
The
insulator
works
irrespective
of its
orientation
of insertion.
Some
of the
enhancers
are
upstream
of the
promoter
and others
are
downstream,
so
the
effect
cannot
depend
on
position
with regard
to
the
promoter,
nor
can
it require
transcrip-
CHAPTER
29 Nucteosomes
Positions
of enhancers for soecific
tissues
wing
body
bristles
tarsal
blade cuticle
claws
Exon
1
Exon 2
A
Insertion
of
insulator
and
expression
pattern
FIGURil
t$.46 The insutator
ofthe
gypsy
transposon
blocks
the action of an
enhancer when it is
placed
between
tne
enhancer and
the
promoter.
tion
to occur through
the insulator.
This
is dif-
ficult
to explain
in terms
of
looping
models for
enhancer-promoter
interaction,
which
essen-
tially
predict
the irrelevance
of the
intervening
DNA. The
obvious model
to invoke
is
a track-
ing mechanism,
in
which some
component
must
move
unidirectionally
from
the enhancer
to
the
promoter,
but this is
difficult to
reconcile
with
previous
characterizations
of the independence
of enhancers
from such
effects.
Proteins
that act
upon the insulator
have
been identified
through
the
existence
of two
other loci
that
affect insulator
function
in
a
trans-acring
manner.
Mutations
in
su(Hw)
abol-
ish insulation: y
is
expressed
in
all tissues
in
spite
of the
presence
of the insulator.
This
sug-
gests
that
su(Hw)
codes for a
protein
that rec-
ognizes
the insulator
and is necessary
for its
action.
Su(Hw) has
a zinc finger
DNA-motif;
mapping
to
polytene
chromosomes
shows
that
it is
bound at a large
number
of
sites. The insu-
lator
contains
twelve copies
of a26
bp
sequence
that is
bound
by Su(Hw).
Manipularions
show
that
the strength
of the
insulator
is determined
by the number
of copies
of
the binding
sequence.
The
second
locus is
mod(mdg4),
in
which
mutations
have the
opposite
effect.
This
is
observed
by the loss
of directionality.
These
mutations
increase
the
effectiveness
of the insu-
lator
by
extending its
effects
so
that it
blocks
utilization
of
enhancers
on both
sides.
su(Hw)
is
epistatic
to mod(mdg4);
this means
rhat
in a
double
mutant
we
see only
the effectof
su(Hw).
This
implies
that
mod(mdg4)
acts
through
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snorrerr
Digest
chromatin
with
DNAase I
Itt
Electrophorese
fragments
and denature
DNA;
probe
lor expressed
and nonexpressed
genes
Probe.l
*
Compare intensities
of bands in
preparations
in which
chromatin was
digested with
increasing
concentrations of DNAase
Extract
DNA and
cleave with restriction
enzvme
U'.i4;11.:i.
iF.:-!;
Sensitivity
to
DNAase
I can
be measured by
determining the rate
of disappearance
of the materiaI
hybridizing
with a
particutar
probe.
actually engaged
in transcription
at any
moment;
this
implies
that
the sensitivity
to
DNAase I does not result
from
the act of tran-
scription,
but instead is
a feature
of
genes
that
are able to be transcribed.
What is
the extent
of the
preferentially
sen-
sitive region? This
can be determined
by using
a series
of
probes
representing
the flanking
regions
as well as the
transcription
unit itself.
The sensitive region
always
extends over
the
entire transcribed region;
an additional region
of several kb on either
side may show
an
inter-
mediate level
of sensitivity
(probably
as the
result of spreading
effects).
The critical concept implicit
in the descrip-
tion of the domain is
that a region
of high sen-
lli.,;.j$if.
':l:i,i::
In
adutt
erythroid cetts. the adutt
B-gtobin
gene
is highLy sensitjve to DNAase
I
digestion;
the embry-
onic
B-gtobin
gene
is
partiatly
sensitive
(probabty
due to
spreading effects), but ovalbumin
js
not sensitjve.
Photos
courtesy
of
Harotd
Weintraub,
Fred Hutchinson Cancer
Research
Center. Used with
permission
of
Mark T.
Groudine.
sitivity to DNAase
I
extends
over a considerable
distance. Often we think of
regulation as resid-
ing in
events that
occur at a discrete
site in
DNA-for
example,
in
the
ability to
initiate tran-
scription at
the
promoter. Even if this is true,
such
regulation must determine, or
must be
accompanied by, a
more wide-ranging change
in
structure. This
is
a difference
between
eukary-
otes and
prokaryotes.
An LCR May Control
a Domain
r
An LCR
is located at the
5' end of the
domain and
consists of severaI
hypersensitive sites.
Every
gene
is
controlled
by
its
promoter,
and
some
genes
also respond
to enhancers
(con-
taining similar control
elements
but located
far-
ther away), as discussed
in
Chapter
24,
Promoters
and
Enhancers.
These local controls
are not sufficient
for all
genes,
though.
In some
cases, a
gene
lies within
a domain of
several
genes,
all of
which
are
influenced by
regulatory
elements that act
on the whole
domain.
The
existence
of
these elements
was identified
by
the inability of a
region of DNA
including a
gene
and all its known regulatory
elements
to be
29.20 An LCR Mav Control a
Domain
5' hypersensitive sites 3' hypersensitive site
HS4 HS1 Globin
genes
60 80
FI*IJRE i9.54
A
gtobin
domain is marked by
hypersen-
sitive sites at either end. The
group
of sites at the 5' side
constitutes the LCR and is essentiaI for the function of atl
oenes in the cluster.
properly
expressed
when
introduced into an
animal
as a transgene.
The best characterized example of a regu-
Iated
gene
cluster is
provided
by the mammalian
B-globin
genes.
Recall from Figure 6.1 that the
o- and
B-globin
genes
in mammals
each exist
as clusters of related
genes
that are expressed at
different times during embryonic and adult
development. These
genes
are
provided
with a
large number
of
regulatory
elements, which
have
been analyzed
in
detail.
In
the case of
the
adult human
B-globin
gene,
regulatory
sequences are located both 5'and 3'to the
gene.The
regulatory
sequences include both
positive
and negative elements in the
promoter
region,
as well as additional
positive
elements
within
and downstream of the
gene.
A human
B-globin
gene
containing all of
these
control
regions,
however, is never
expressed in a transgenic mouse
within an order
of magnitude
of
wild-type levels.
Some further
regulatory
sequence
is required.
Regions that
provide
the additional regulatory function are
identified
by DNAase I hypersensitive sites that
are
found
at the ends
of
the
cluster.
The map
of
FI**qr
f *.54
shows that the
20
kb upstream of
the
e
gene
contains a
group
of five
sites,
and
that there is
a single site 30 kb downstream of
the
p
gene.
Ttansfecting
various constructs into
mouse
erythroleukemia
cells shows that
sequences
between the individual hypersensi-
tive
sites in the 5'region
can be removed with-
out much effect, but
that
removal
of any of the
sites
reduces
the overall level
of expression.
The
5'regulatory
sites are the
primary
reg-
ulators, and the
cluster of hypersensitive sites
is called
the locus control region
(LCR).
We
do not know if
the 3'site has any function. The
LCR is
absolutely required
for expression of
each of the
globin genes
in the cluster. Each
gene
is
then further regulated
by its own spe-
cific controls. Some of these controls
are
autonomous:
Expression oI the e and
y
genes
appears
intrinsic to those loci in conjunction
with
the LCR. Other controls appear to rely
upon
position
in the cluster, which
provides
a
suggestion IhaI
gene
order in a cluster is impor-
tant for regulation.
The entire region containing the
globin
genes,
and extending
well beyond them, con-
stitutes a chromosomal
domain. It shows
increased sensitivity to digestion by
DNAase I
(see
Figure 29.52\. Deletion of the 5'LCR
restores
normal resistance to DNAase over the
whole
region. TWo
models for how
an LCR
works
propose
that its action is required in
order to activate the
promoter,
or alternatively,
is required to increase the rate of transcrip-
tion from the
promoter.
The exact nature of
the
interactions between the LCR and the indi-
vidual
promoters
has not
yet
been fully
defined.
Does this model apply to other
gene
clus-
ters? The cr-globin
locus has
a similar organi-
zation of
genes
that are expressed at different
times,
with
a
group
of hypersensitive sites at
one end of
the cluster, and increased
sensitiv-
ity to DNAase I throughout the region. Only a
small
number
of
other cases are known in
which
an LCR controls a
group
of
genes.
One of
these cases involves
an
LCR
that
controls
genes
on
more
than one chromosome.
The Ts2 LCR coordinately regulates a
group
of
genes
that are spread out over
I20
kb on chro-
mosome I I by interacting with their
promot-
ers.
It
also
interacts
with
the
promoter
of the
IFNygene on chromosome
10.
The two types of
interaction are alternatives that comprise
two
different cell fates, that is, in one
group
of cells
the LCR causes expression
of the
genes
on chro-
mosome
I
l,
whereas
in
the other
group
it
causes
the
gene
on chromosome l0 to
be expressed.
What Constitutes
a Regulatory Domain?
r
A domain may have an
insutator,
an LCR,
a
matrix
attachment
site, and transcription unit(s).
If we
put
together the
various types of
struc-
tures that have been found in
different sys-
tems,
we can think about the
possible
nature
of a chromosomal domain. The
basic feature
of a regulatory
domain is that regulatory
ele-
CHAPTER 29 Nucleosomes