The β-Globin LCR is Not The β-Globin LCR is Not Necessary for an Open Chromatin Necessary for an Open Chromatin Structure or Developmentally Structure or Developmentally Regulated Transcription of the Regulated Transcription of the Native Mouse β-Globin LocusNative Mouse β-Globin LocusElliot Epner, Andreas Reik, Daniel Cimbora, Agnes Telling, M. A. Elliot Epner, Andreas Reik, Daniel Cimbora, Agnes Telling, M. A. Bender, Steve Fiering, Tariq Enver, David I. K. Martin, Marion Bender, Steve Fiering, Tariq Enver, David I. K. Martin, Marion Kennedy, Gordon Keller, and Mark GroudineKennedy, Gordon Keller, and Mark Groudine

BIO 4751: Advanced Molecular BIO 4751: Advanced Molecular and Cellular Biologyand Cellular Biology

Locus Control Regions (LCRs)Locus Control Regions (LCRs) DNA regulatory sequences DNA regulatory sequences

that regulate the accessibility that regulate the accessibility and expression of distant and expression of distant genes or gene clusters (ex. genes or gene clusters (ex. globin genes)globin genes)

β-gβ-globin one of best-lobin one of best-understood genes under LCR understood genes under LCR regulationregulation

Β-globin expressed only in red Β-globin expressed only in red blood cells (erythrocytes) and blood cells (erythrocytes) and at specific time in developmentat specific time in development

Part of a cluster of globin genesPart of a cluster of globin genes εε, , γγGG, , γγAA, , δδ, , ββ

Models of LCR actionModels of LCR action LoopingLooping

- Based on LCR as an integral unit to stimulate Based on LCR as an integral unit to stimulate transcription of individual globin genes by “looping” transcription of individual globin genes by “looping” through the nucleoplasm to recruit transcriptional through the nucleoplasm to recruit transcriptional apparatus assembled at globin gene promotersapparatus assembled at globin gene promoters

- However, a holocomplex would be very limiting with However, a holocomplex would be very limiting with only one activation center, thus competition for the only one activation center, thus competition for the activity of the LCRactivity of the LCR

TrackingTracking TopologicTopologic AlterationsAlterations Chromatin associated protein modificationsChromatin associated protein modifications

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

locationlocation

Natural ocurring deletions in human Natural ocurring deletions in human ββ--globin seen in thalassemia patientsglobin seen in thalassemia patients- Intact regulatory regions, but transcriptionally Intact regulatory regions, but transcriptionally

silent in erythroid cellssilent in erythroid cells- Chromatin fails to undergo decondensation Chromatin fails to undergo decondensation

during development, and is thus DNaseI during development, and is thus DNaseI resistantresistant

Experiments arguing LCR’s Experiments arguing LCR’s dominant role in locus activation dominant role in locus activation

Five DNaseI hypersensitive sites (HSs) found 5’ of the Five DNaseI hypersensitive sites (HSs) found 5’ of the embryonic embryonic ββ-like globin gene-like globin gene

Hispanic thalassemia removes ~35 kb of DNA upstream Hispanic thalassemia removes ~35 kb of DNA upstream resulting in failure to activate the resulting in failure to activate the ββ-globin locus at the -globin locus at the level of transcriptionlevel of transcription

All five HSs form when chromosome 11 is transferred to All five HSs form when chromosome 11 is transferred to an erythroid environmentan erythroid environment

5’HSs increase expression of 5’HSs increase expression of ββ-globin genes in -globin genes in transfection and transgenic analysestransfection and transgenic analyses

All these observations suggest that these HSs play All these observations suggest that these HSs play dominant role in activation of the dominant role in activation of the ββ-globin locus-globin locus

LCRs as Multi-taskersLCRs as Multi-taskers Transfection assays reveal that isolated Transfection assays reveal that isolated ββ-globin -globin

components can have multiple properties:components can have multiple properties: Transcriptional enhancersTranscriptional enhancers InsulatorsInsulators Mediators of chromatin “opening”Mediators of chromatin “opening” Suppressors of position effect variegationSuppressors of position effect variegation

These properties support notion of LCR as an These properties support notion of LCR as an element capable of creating domain independent element capable of creating domain independent of flanking chromatin influenceof flanking chromatin influence

However, no discrete element conferring such properties has However, no discrete element conferring such properties has been isolated from complete LCR regionbeen isolated from complete LCR region

Full LCR is unable to counteract repressive influences of Full LCR is unable to counteract repressive influences of flanking sequencesflanking sequences

Can expression occur without the Can expression occur without the LRC?LRC?

Transgene and multigenic experiments Transgene and multigenic experiments have revealed that low-level globin gene have revealed that low-level globin gene expression occurs even in the absence of expression occurs even in the absence of the the ββ-g-globin LCRlobin LCR

Developmental regulations of globins can Developmental regulations of globins can occur in transgene lacking an LCRoccur in transgene lacking an LCR

Suggests that some inherent properties of Suggests that some inherent properties of the globin locus are independent of the the globin locus are independent of the LCRLCR

Alternate views…Alternate views…

Current views of LCR functionCurrent views of LCR function- Establish and maintain transcriptional activity Establish and maintain transcriptional activity

in a locusin a locus- Shield locus from repressive effect of flanking Shield locus from repressive effect of flanking

chromatinchromatin- Determine which genes in a locus will be Determine which genes in a locus will be

transcribedtranscribedThese views predict that complete LCR These views predict that complete LCR

deletion will render locus inactivedeletion will render locus inactive

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

Embryonic stem (ES) Embryonic stem (ES) cells homozygous for cells homozygous for deletion of murine deletion of murine ββ--globin LCR does not globin LCR does not inactivate inactivate ββ-globin locus -globin locus on level of chromatin on level of chromatin structure or transcriptionstructure or transcription

Locus is “open” chromatin Locus is “open” chromatin conformation (DNaseI conformation (DNaseI sensitivity)sensitivity)

Reduced levels of Reduced levels of expression by 5%-25%, expression by 5%-25%, but developmentally but developmentally regulated regulated

Homologus Recombination

Southern Analysis

What do the results say?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?What do the results say?

DNaseI-resistant conformation

ControlControl 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?What do the results say? Removal of LCR Removal of LCR

results in general results in general decrease in levels of decrease in levels of expression, but expression, but frequency of frequency of nonexpressing cells nonexpressing cells remains the sameremains the same

LCR determines level LCR determines level of expression per cell of expression per cell and does not affect and does not affect probability of probability of expression of globin expression of globin gene in given cellgene in given cell

(WT)

∆LCR: prior to transfer

What does all this mean?What does all this mean? Findings provide evidence against studies indicating Findings provide evidence against studies indicating

LCR vital to transcription and regulation of LCR vital to transcription and regulation of ββ-globin locus-globin locus LCR is necessary for normal levels of LCR is necessary for normal levels of ββ-globin -globin

transcriptiontranscription LCR properties resemble those of enhancersLCR properties resemble those of enhancers Determines that LCR provides contributory rather then Determines that LCR provides contributory rather then

dominant functions for its native locationdominant functions for its native location Regulatory sequences in addition to the LCR may Regulatory sequences in addition to the LCR may

function to establish chromatin structure and function to establish chromatin structure and transcriptiontranscription

LCR in its native location seems only contributory rather LCR in its native location seems only contributory rather then dominant in determining chromatin structurethen dominant in determining chromatin structure