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BIO 4751: Advanced Molecular and Cellular Biology

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BIO 4751: Advanced Molecular and Cellular Biology

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

  2. Locus Control Regions (LCRs) • DNA regulatory sequences that regulate the accessibility and expression of distant genes or gene clusters (ex. globin genes) • β-globin one of best-understood 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 • ε, γG, γA, δ, β

  3. 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

  4. Clues to the existence and functions of β-globin LCR in native location • Natural ocurring deletions in human β-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

  5. Experiments arguing LCR’s dominant role in locus activation • 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

  6. LCRs as Multi-taskers • Transfection assays reveal that isolated β-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

  7. 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

  8. 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

  9. What if we should delete the LCR controlling a multigene locus? • Embryonic stem (ES) cells homozygous for deletion of murine β-globin 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

  10. 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

  11. What do the results say? DNaseI-resistant conformation Control Control comparison for assay Evidence that LCR does not dictate initiation of open conformation

  12. 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

  13. 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

  14. What does all this mean? • 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

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