Transcript The β-Globin LCR is Not Necessary for an Open Chromatin

BIO 4751: Advanced Molecular
and Cellular Biology
The β-Globin LCR is Not
Necessary for an Open Chromatin
Structure or Developmentally
Regulated Transcription of the
Native Mouse β-Globin Locus
Elliot Epner, Andreas Reik, Daniel Cimbora, Agnes Telling, M. A.
Bender, Steve Fiering, Tariq Enver, David I. K. Martin, Marion
Kennedy, Gordon Keller, and Mark Groudine
Locus Control Regions (LCRs)
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DNA regulatory sequences
that regulate the accessibility
and expression of distant
genes or gene clusters (ex.
globin genes)
β-globin one of bestunderstood genes under LCR
regulation
Β-globin expressed only in red
blood cells (erythrocytes) and
at specific time in development
Part of a cluster of globin genes
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ε, γG, γA, δ, β
Models of LCR action
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Looping
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Based on LCR as an integral unit to stimulate
transcription of individual globin genes by “looping”
through the nucleoplasm to recruit transcriptional
apparatus assembled at globin gene promoters
However, a holocomplex would be very limiting with
only one activation center, thus competition for the
activity of the LCR
Tracking
Topologic
Alterations
Chromatin associated protein modifications
Clues to the existence and
functions of β-globin LCR in native
location
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Natural ocurring deletions in human βglobin seen in thalassemia patients
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Intact regulatory regions, but transcriptionally
silent in erythroid cells
Chromatin fails to undergo decondensation
during development, and is thus DNaseI
resistant
Experiments arguing LCR’s
dominant role in locus activation
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Five DNaseI hypersensitive sites (HSs) found 5’ of the
embryonic β-like globin gene
Hispanic thalassemia removes ~35 kb of DNA upstream
resulting in failure to activate the β-globin locus at the
level of transcription
All five HSs form when chromosome 11 is transferred to
an erythroid environment
5’HSs increase expression of β-globin genes in
transfection and transgenic analyses
All these observations suggest that these HSs play
dominant role in activation of the β-globin locus
LCRs as Multi-taskers
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Transfection assays reveal that isolated β-globin
components can have multiple properties:
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Transcriptional enhancers
Insulators
Mediators of chromatin “opening”
Suppressors of position effect variegation
These properties support notion of LCR as an
element capable of creating domain
independent of flanking chromatin influence
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However, no discrete element conferring such properties has
been isolated from complete LCR region
Full LCR is unable to counteract repressive influences of
flanking sequences
Can expression occur without the
LRC?
Transgene and multigenic experiments
have revealed that low-level globin gene
expression occurs even in the absence of
the β-globin LCR
 Developmental regulations of globins can
occur in transgene lacking an LCR
 Suggests that some inherent properties of
the globin locus are independent of the
LCR
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Alternate views…
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Current views of LCR function
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Establish and maintain transcriptional activity
in a locus
Shield locus from repressive effect of flanking
chromatin
Determine which genes in a locus will be
transcribed
These views predict that complete LCR
deletion will render locus inactive
What if we should delete the LCR
controlling a multigene locus?
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Embryonic stem (ES)
cells homozygous for
deletion of murine βglobin LCR does not
inactivate β-globin locus
on level of chromatin
structure or transcription
Locus is “open” chromatin
conformation (DNaseI
sensitivity)
Reduced levels of
expression by 5%-25%,
but developmentally
regulated
Homologus Recombination
Southern
Analysis
What do the results say?
Human locus is open
Mouse locus is also
open with DLCR
Results suggest
that LCR is not
vital to chromatin
structure and
expression
Nonerythroid
cells
What do the results say?
DNaseI-resistant
conformation
Control comparison
for assay
Evidence that
LCR does not
dictate initiation
of open
conformation
Control
What do the results say?
Northern Assay
Controls
LCR in ES cells deleted prior to and after
chromosome transfer to K562 cells
RT-PCR analysis shows that LCR not required for
either initiation or maintenance of mouse β-globin
transcription in its native position
What do the results say?
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Removal of LCR
results in general
decrease in levels of
expression, but
frequency of
nonexpressing cells
remains the same
LCR determines level
of expression per cell
and does not affect
probability of
expression of globin
gene in given cell
(WT)
∆LCR:
prior to
transfer
What does all this mean?
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Findings provide evidence against studies indicating
LCR vital to transcription and regulation of β-globin locus
LCR is necessary for normal levels of β-globin
transcription
LCR properties resemble those of enhancers
Determines that LCR provides contributory rather then
dominant functions for its native location
Regulatory sequences in addition to the LCR may
function to establish chromatin structure and
transcription
LCR in its native location seems only contributory rather
then dominant in determining chromatin structure