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 BIO 4751: Advanced Molecular and Cellular Biology

Locus Control Regions (LCRs) DNA regulatory sequences that regulate the accessibility and expression of distant genes or gene clusters (ex. globin genes) DNA regulatory sequences that regulate the accessibility and expression of distant genes or gene clusters (ex. globin genes) β-g lobin one of best- understood genes under LCR regulation β-g lobin one of best- understood genes under LCR regulation Β-globin expressed only in red blood cells (erythrocytes) and at specific time in development Β-globin expressed only in red blood cells (erythrocytes) and at specific time in development Part of a cluster of globin genes Part of a cluster of globin genes ε, γ G, γ A, δ, β ε, γ G, γ A, δ, β

Models of LCR action Looping 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 Tracking Topologic Topologic Alterations Alterations Chromatin associated protein modifications Chromatin associated protein modifications

Clues to the existence and functions of β-globin LCR in native location Natural ocurring deletions in human β- globin seen in thalassemia patients 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

Experiments arguing LCR’s dominant role in locus activation Five DNaseI hypersensitive sites (HSs) found 5’ of the embryonic β-like globin gene 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 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 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 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 All these observations suggest that these HSs play dominant role in activation of the β-globin locus

LCRs as Multi-taskers Transfection assays reveal that isolated β-globin components can have multiple properties: Transfection assays reveal that isolated β-globin components can have multiple properties: Transcriptional enhancers Transcriptional enhancers Insulators Insulators Mediators of chromatin “opening” Mediators of chromatin “opening” Suppressors of position effect variegation Suppressors of position effect variegation These properties support notion of LCR as an element capable of creating domain independent of flanking chromatin influence 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 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 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-g lobin LCR Transgene and multigenic experiments have revealed that low-level globin gene expression occurs even in the absence of the β-g lobin LCR Developmental regulations of globins can occur in transgene lacking an 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 Suggests that some inherent properties of the globin locus are independent of the LCR

Alternate views… Current views of LCR function 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 These views predict that complete LCR deletion will render locus inactive

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 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) Locus is “open” chromatin conformation (DNaseI sensitivity) Reduced levels of expression by 5%-25%, but developmentally regulated 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  LCR Results suggest that LCR is not vital to chromatin structure and expression Non- erythroid cells

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

What do the results say? RT-PCR analysis shows that LCR not required for either initiation or maintenance of mouse β-globin transcription in its native position Controls LCR in ES cells deleted prior to and after chromosome transfer to K562 cells Northern Assay

What do the results say? Removal of LCR results in general decrease in levels of expression, but frequency of nonexpressing cells remains the same 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 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? Findings provide evidence against studies indicating LCR vital to transcription and regulation of β-globin locus 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 is necessary for normal levels of β-globin transcription LCR properties resemble those of enhancers LCR properties resemble those of enhancers Determines that LCR provides contributory rather then dominant functions for its native location 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 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 LCR in its native location seems only contributory rather then dominant in determining chromatin structure