The

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The ß-Globin LCR is Not Necessary for an Open
Chromatin Structure or Developmentally Regulated
Transcription of the Native Mouse ß-Globin
LocusElliot 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

• BIO 4751 Advanced Molecular and Cellular Biology

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Locus Control Regions (LCRs)

• DNA regulatory sequences that regulate the
  accessibility and expression of distant genes or
  gene clusters (ex. globin genes)
• ß-globin one of best-understood genes under LCR
  regulation
• ?-globin expressed only in red blood cells
  (erythrocytes) and at specific time in
  development
• Part of a cluster of globin genes
• e, ?G, ?A, d, ß

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Models of LCR action

• Looping
• 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

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Clues to the existence and functions of ß-globin
LCR in native location

• Natural ocurring deletions in human ß-globin seen
  in thalassemia patients
• Intact regulatory regions, but transcriptionally
  silent in erythroid cells
• Chromatin fails to undergo decondensation during
  development, and is thus DNaseI resistant

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Experiments arguing LCRs dominant role in locus
activation

• Five DNaseI hypersensitive sites (HSs) found 5
  of the embryonic ß-like globin gene
• Hispanic thalassemia removes 35 kb of DNA
  upstream resulting in failure to activate the
  ß-globin locus at the level of transcription
• All five HSs form when chromosome 11 is
  transferred to an erythroid environment
• 5HSs increase expression of ß-globin genes in
  transfection and transgenic analyses
• All these observations suggest that these HSs
  play dominant role in activation of the ß-globin
  locus

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LCRs as Multi-taskers

• Transfection assays reveal that isolated ß-globin
  components can have multiple properties
• 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
• 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

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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 ß-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

• Current views of LCR function
• 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

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What if we should delete the LCR controlling a
multigene locus?

• Embryonic stem (ES) cells homozygous for deletion
  of murine ß-globin LCR does not inactivate
  ß-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
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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

Non-erythroid cells
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What do the results say?
DNaseI-resistant conformation
Control
Control comparison for assay
Evidence that LCR does not dictate initiation of
open conformation
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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
ß-globin transcription in its native position
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What do the results say?

• 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
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What does all this mean?

• Findings provide evidence against studies
  indicating LCR vital to transcription and
  regulation of ß-globin locus
• LCR is necessary for normal levels of ß-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