The β-globin nuclear compartment in development and erythroid differentiation

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1 The β-gloin nucler comprtment in development nd erythroid differentition Roert-Jn Plstr 1,2, Bs Tolhuis 1,2, Erik Splinter 1, Rin Nijmeijer 1, Frnk Grosveld 1 & Wouter de Lt 1 Efficient trnscription of genes requires high locl concentrtion of the relevnt trns-cting fctors. Nucler comprtmentliztion cn provide n effective mens to loclly increse the concentrtion of rpidly moving trns-cting fctors; this my e chieved y sptil clustering of chromtinssocited inding sites for such fctors 1 5. Here we nlyze the structure of n erythroid-specific sptil cluster of cisregultory elements nd ctive β-gloin genes, the ctive chromtin hu (ACH; ref. 6), t different stges of development nd in erythroid progenitors. We show, in mice nd humns, tht core ACH is developmentlly conserved nd consists of the hypersensitive sites (HS1 HS6) of the locus control region (LCR), the upstrem 5 HS 60/ 62 nd downstrem 3 HS1. Gloin genes switch their interction with this cluster during development, correlting with the switch in their trnscriptionl ctivity 7. In mouse erythroid progenitors tht re committed to ut do not yet express β-gloin, only the interctions etween 5 HS 60/ 62, 3 HS1 nd hypersensitive sites t the 5 side of the LCR re stly present. After induction of differentition, these sites cluster with the rest of the LCR nd the gene tht is ctivted. We conclude tht during erythroid differentition, cis-regultory DNA elements crete developmentlly conserved nucler comprtment dedicted to RNA polymerse II trnscription of β-gloin genes. f c d e Figure 1 Sptil orgniztion of the mouse β-gloin locus. () Schemtic presenttion of the mouse locus. Arrows indicte the individul hypersensitive sites, tringles indicte gloin genes nd oxes indicte the olfctory receptor (OR) genes. ( e) Southern lots show tht in definitive erythrocytes, digestion efficiency of crosslinked chromtin depends on formldehyde concentrtion nd is comprle etween hypersensitive site in the LCR (), trnscried gene in the locus (c), nonexpressed gene in the locus (d) nd nonexpressed gene on different chromosome (Chr. 4; e). Percentge formldehyde crosslinking (FA %) is shown t the top of ech lot ( indictes genomic DNA not treted with formldehyde), nd the yield of specificlly cut frgments is shown t the ottom (in percentges). Arrowheds indicte prtil digests, nd sterisks indicte crosshyridiztion signls with other genes. (f,g) Erythroidspecific nd developmentlly stle clustering of cis-regultory elements. Reltive crosslinking frequencies oserved in primitive erythrocytes re shown in red, definitive erythrocytes in green nd nonexpressing rin in lck. Gry shding indictes position nd size of the nlyzed frgments, nd lck shding represents the fixed frgments HS4 HS5 (f) nd 5 HS 60/ 62 (g). In ech grph, the highest crosslinking frequency vlue ws set to 1. The x xis shows position in the locus. Errors rs represent s.e.m. g 1 Deprtment of Cell Biology nd Genetics, ErsmusMC, PO Box 1738, 3000 DR Rotterdm, The Netherlnds. 2 These uthors contriuted eqully to this work. Correspondence should e ddressed to W.d.L. (w.delt@ersmusmc.nl). Pulished online 21 Septemer 2003; doi: /ng VOLUME 35 NUMBER 2 OCTOBER 2003 NATURE GENETICS

2 The mouse nd humn β-gloin loci contin n upstrem LCR nd multiple β-like genes rrnged from 5 to 3 in order of their developmentl expression (Fig. 1). In ddition there re severl distl hypersensitive sites, including downstrem 3 HS1 (pproximtely 20-k 3 of the β-gloin genes) nd two upstrem hypersensitive sites, 60 k (mouse) nd 110 k (humn) wy from the genes 8. The loci re emedded in n olfctory receptor gene cluster tht is inctive in erythroid cells 9. To investigte the sptil orgniztion of β-gloin loci in mice nd humns during development nd erythroid differentition, we pplied chromosome conformtion cpture (3C) technology 6,10. 3C technology involves quntittive PCR nlysis of crosslinking frequencies etween two given DNA restriction frgments, which gives mesure of their proximity in the nucler spce. Locl chromtin configurtion hs no effect on digestion efficiency, implying tht the ssy is not ised owing to preferentil restriction enzyme digestion of one site over the other (Fig. 1 e; for other controls, see Methods nd ref. 6). First, we nlyzed the sptil orgniztion of the mouse β-gloin locus in primitive erythroid cells present in lood from emryos t 10.5 d post-coitum (d.p.c.), which predominntly express the emryonic gloin genes H-y nd H-h1 (ref. 11). We determined crosslinking frequencies for 66 pirs of HindIII restriction frgments, spred over 170 k of DNA encompssing the mouse β-gloin gene cluster. The 3C mesurements indicte sic structurl orgniztion in primitive cells very similr to tht oserved previously in definitive lood cells isolted from 14.5-d.p.c. fetl liver 6. This is est illustrted y compring the locus-wide crosslinking frequencies of restriction frgment tht contins HS4 HS5 of the LCR. Two peks of high crosslinking frequency with this genomic site stnd out in primitive lood cells: one with the upstrem HS 60/ 62 nd nother with 3 HS1 downstrem of the genes (Fig. 1f). We found significntly lower crosslinking frequencies with frgments in etween, suggesting tht the LCR intercts with these distl hypersensitive sites through looping. We oserved the sme interctions in definitive lood cells tht exclusively express the dult gloin genes H-1 nd H-2 (ref. 11), where H-1 is lso found in close proximity (Fig. 1f). In contrst, in nonexpressing rin cells, HS4 HS5 hs no peks of interction with distl DNA frgments, suggesting liner conformtion of the trnscriptionlly inctive locus 6. We otined similr results when nlyzing the locus-wide crosslinking frequencies of frgments crrying 5 HS 60/ 62 (Fig. 1g) nd other hypersensitive sites (dt not shown): interctions mong the cis-regultory elements of the β-gloin locus were conserved etween primitive nd definitive erythroid cells. We conclude tht the cis-regultory elements of the mouse β-gloin locus sptilly cluster to form trnscription regultory comprtment tht is conserved etween primitive nd definitive erythroid cells, two developmentlly different types of cells tht express different suset of β-like gloin genes. This core ACH includes the two hypersensitive sites t 60 k, ll hypersensitive sites of the LCR nd 3 HS1. The min differences in conformtion etween the two expressing cell types seem to e confined to interctions etween the gloin genes nd the regultory DNA elements. We confirmed this y mesuring crosslinking frequencies with HS2 nd HS3 of the LCR, the two most prominent trnscriptionl ctivting elements The emryonic gloin genes H-y nd H-h1 intercted frequently with these elements in primitive erythroid cells, wheres in definitive red lood cells interction frequencies etween these sites dropped to levels similr to those oserved in the inctive rin (Fig. 2). We found the opposite pttern for the dult genes H-1 nd H-2, which intercted most Figure 2 A developmentl switch occurs in contcts etween individul β-gloin genes nd the core ACH of the mouse β-gloin locus. (,) Crosslinking frequencies of HS2 nd the β-gloin genes were mesured. An exmple of PCR-mplified ligtion products on 2% grose gel () nd the quntified dt of ll experiments (t lest five in duplicte per primer set; ) re shown. (c,d) Similr to nd ut for HS3 nd the β- gloin genes. Error rs in nd d represent s.e.m., nonexpressing rin; 10.5, primitive erythrocytes; 14.5, definitive erythrocytes. Control is PCR-mplified ligtion product of two restriction frgments in the Ercc3 locus. Crosslinking frequencies shown in nd d re not corrected for PCR mplifiction efficiency; therefore, only signls otined with the sme primer set cn e compred. frequently with HS2 nd HS3 in definitive erythroid cells. Crosslinking frequencies etween these sites in 10.5-d.p.c. emryonic lood were not s low s in rin, proly owing to the fct tht H1 nd H-2 re lredy trnscriptionlly ctive t this stge, leit t less thn 10% of the levels oserved in definitive cells 11. Alterntively, it my merely e the result of 3 HS1 intercting with the LCR nd the dult genes eing drgged long, s we previously found tht the region etween H-2 nd 3 HS1, which is full of repetitive sequences, cts s rigid region 6. These dt show tht there is developmentl switch in contcts etween the different gloin genes nd core ACH creted y regultory elements tht surround the genes in cis. This structurl chnge correltes with the developmentl switch in expression of the genes. We next nlyzed the conformtion of the humn β-gloin locus t different stges of development. The mouse nd humn β-gloin gene loci hve high degree of nucleotide sequence conservtion, prticulrly t regions implicted in gene regultion 9,18. We used trnsgenic mice crrying single copy of 185-k PAC (Fig. 3) c d NATURE GENETICS VOLUME 35 NUMBER 2 OCTOBER

3 spnning the humn β-gloin locus tht hd norml expression pttern (refs. 19,20 nd G. Ptrinos nd F.G., unpulished dt). Though lrge, this PAC does not include the humn equivlent of the mouse 5 HS 60/ 62, which is locted 110 k upstrem of the humn gloin genes 8,9. We nlyzed the conformtion of the trnsgenic humn gloin locus in 10.5-d.p.c. emryonic lood, d.p.c. fetl liver nd 14.5-d.p.c. fetl rin, mesuring lmost ll of the 120 site pirs tht cn e formed etween the 16 EcoRI frgments tht we nlyzed. The locus-wide crosslinking frequencies of frgment corresponding to 3 HS1 illustrte tht the trnsgenic humn locus lso forms core ACH, consisting of the 3 HS1 nd the hypersensitive site of the LCR, tht is conserved in primitive nd definitive erythroid cells (Fig. 3). The structurl chnges we oserved primrily concerned the position of the genes reltive to this core ACH, correlting with trnscriptionl ctivity. Thus, the emryonic gene HBE1 nd the two HBG genes most frequently interct with HS2 HS4 (Fig. 3c e) nd 3 HS1 (Fig. 3) in primitive erythroid c Figure 3 Sptil orgniztion of the humn β-gloin locus. () Schemtic presenttion of the humn locus. () Locus-wide crosslinking frequencies of 3 HS1 frgment show erythroid cell specific clustering with the LCR throughout development. (c) Developmentl switching in contcts of the LCR etween the different β-gloin genes s shown y locus-wide crosslinking frequencies of HS2 HS4. (d,e) The contcts etween HS2 HS4 of the LCR nd individul β-gloin genes lter during development in erythroid cells, s shown y n exmple on grose gel (d) nd quntified dt (e; t lest five experiments in duplicte per primer set). Controls, symols, color ptterns nd numering re s in Figures 1 nd 2. d e cells, nd the dult gene HBB primrily contcts the ACH in definitive cells (Fig. 3c e). We found identicl results for HindIII digest nd for different trnsgenic PAC line (dt not shown). Results otined with definitive erythroid cells isolted from dult one mrrow (Ter119 + ) were identicl to those found for 14.5-d.p.c. fetl liver cells (dt not shown). The lower crosslinking frequency of HS5 in the definitive cells is notle, s we hve recently shown tht this element hs LCR-locking ctivity in primitive ut not definitive erythroid cells 21. We conclude tht the overll sptil orgniztion of the β-gloin gene cluster is conserved from mice to humns. Next we determined β-gloin genomic site interctions in I/11 erythroid progenitor cells tht re committed to ut do not yet express the β-gloin genes. When exposed to physiologiclly relevnt stimuli, I/11 cells synchronously undergo the norml in vivo differentition progrm to mture terminlly into enucleted erythrocytes 22,23. As expected, in differentiting I/11 cells tht ctively trnscrie the dult β-like gloin genes, the locus dopted sptil orgniztion very similr to tht oserved previously in definitive erythroid cells isolted from fetl livers 6 (Fig. 4). In uninduced proliferting I/11 cells tht do not yet express the β-gloin genes, however, we oserved different structure. Locus-wide crosslinking frequencies of frgment corresponding to the gene H-1 were lower thn those oserved in erythroid cells expressing the gene (Fig. 4). We oserved similr reduction in locus-wide crosslinking frequencies for frgment contining HS2 nd for most other frgments (dt not shown). The structure of the locus poised for trnscription ws clerly different from tht of the inctive locus in rin cells. This structure is etter resolved y looking t the locus-wide crosslinking frequencies of the restriction frgment tht contins HS4 HS5 of the LCR. Two peks of high crosslinking frequency with this frgment stnd out in erythroid progenitor cells: one with 5 HS 60/ 62 nd nother with 3 HS1 (Fig. 4). Interctions mong these three sites occur lmost s frequently in proliferting progenitors s in differentiting erythroid cells, wheres ll other interctions exmined etween gloin site pirs re much less frequent in progenitor cells (Fig. 4 nd dt not shown). We conclude tht the β-gloin locus tht is poised for trnscription in progenitor cells dopts looped conformtion through frequent interctions etween the two distl regultory elements t either end of the locus (5 HS 60/ 62 nd 3 HS1) nd hypersensitive sites t the 5 side of the LCR (HS4, HS5 or HS6; it is not cler which of these hypersensitive sites is responsile for direct interction). After induction of differentition, clustering with the ctive genes nd the complete LCR is estlished nd the β-gloin genes re expressed (Fig. 5). 192 VOLUME 35 NUMBER 2 OCTOBER 2003 NATURE GENETICS

4 Figure 4 Sptil orgniztion of the mouse β-gloin locus in erythroid progenitors. Controls, symols nd numering re s in Figure 1, lck lines represent rin, red lines represent proliferting I/11 erythroid progenitor cells nd green lines represent differentited I/11 cells. () Locus-wide crosslinking frequencies of H-1. () Locus-wide crosslinking frequencies of HS4 HS5. Only the interctions mong HS4 HS5, 5 HS 60/ 62 nd 3 HS1 re lredy fully estlished in nonexpressing progenitor cells. In summry, our dt strongly suggest tht regultory elements surrounding the β-gloin genes in cis crete n erythroid-specific developmentlly stle nucler comprtment dedicted to RNA polymerse II trnscription (Fig. 5). A sustructure is lredy present in erythroid progenitors tht do not express gloin, nd notly, the three sites involved in this structure ll ind CTCF 24. The 3 HS1 nd 5 HS 60/ 62 re dispensle for norml gloin gene expression in trnsgenic mice 19,21, suggesting tht these sites hve more generl structurl role not relted to trnscription per se. Sptil clustering of trnscription regultory elements results in high locl concentrtion of DNA inding sites for cognte trnscription fctors, which consequently ccumulte t the site. Efficiency of trnscription is proportionl to the concentrtion of trnscription fctors involved, nd in greement, we found tht proximity of β-gloin genes to the ACH correlted with trnscriptionl ctivity. The prdigm of chromtin-ssocited nucler comprtment is the nucleolus, dedicted to RNA polymerse I trnscription of riosoml RNA genes 3. No polymerse II dependent gene-specific comprtments hve een descried efore, ut precedent for this ws provided y electron microscopy studies showing tht RNA polymerse II clusters in discrete trnscription fctories in the nucleus 25,26. The fct tht the density of RNA polymerses on ctive β-gloin nd riosoml RNA genes is much higher thn on most other ctive genes 27,28 suggests tht such nucler comprtments formed y numerous chromtin-ssocited regultory elements primrily function to increse the efficiency of trnscription. METHODS Chromosome conformtion cpture (3C). We crried out isoltion nd formldehyde fixtion of primry cells, restriction enzyme digestion of crosslinked DNA in the nucleus, intrmoleculr ligtion, reversl of crosslinks, PCR nlysis of ligtion products nd clcultion of reltive crosslinking frequencies s descried efore 6,10, with some modifictions. Before fixtion, we forced cells otined from emryonic lood (from d.p.c. emryos) nd from fetl liver nd fetl rin (oth from 14.5-d.p.c. emryos; cells per tissue) through cell-striner cp (Flcon #352235) to otin homogeneous single-cell suspension. To correct for differences in qulity nd quntity of templte, we normlized ligtion frequencies etween gloin site pirs to those detected etween two restriction frgments (with the sites nlyzed 8.3 k prt) in the Ercc3 locus (insted of the previously used Clr locus 6 ). Ercc3 encodes suunit of the sl trn- Figure 5 Cis-regultory elements of the β-gloin locus crete nucler comprtment dedicted to RNA polymerse II trnscription: the ctive chromtin hu. Two-dimensionl presenttion of three-dimensionl interctions tht occur etween regultory DNA elements 130 k prt (red ovls) nd β-gloin genes (ctive, red nd green rectngles; inctive, lck rectngles) in erythroid progenitors (left) nd in differentited primitive nd definitive erythroid cells (right). In erythroid progenitors not expressing gloin, sustructure (gry sphere) is present tht is formed through interctions etween the upstrem 5 HS 60/ 62, the downstrem 3 HS1 nd hypersensitive sites t the 5 side of the LCR (HS4 HS6; it is not yet cler which of these hypersensitive sites is directly responsile for this interction). During erythroid differentition, the β-gloin gene tht gets ctivted nd the rest of the LCR stly interct with this sustructure to form functionl ACH (yellow sphere); β-gloin gene expression is ctivted. Clustering of inding sites for trnscription fctors in the ACH cuses locl ccumultion of cognte proteins nd ssocited positive chromtin modifiers, required to drive efficient trnscription of the gloin genes. The core of the ACH is erythroid-specific nd developmentlly stle; developmentl switch occurs in gloin genes entering this nucler comprtment, s indicted y the rrows. Inctive gloin nd olfctory receptor genes (gry squres) loop out. NATURE GENETICS VOLUME 35 NUMBER 2 OCTOBER

5 scription fctor TFIIH, nd we ssumed tht expression levels nd sptil conformtion of this gene were similr in ll nlyzed tissues. To compre signl intensities otined with different primer sets in quntittive mnner, we included control templte contining ll possile ligtion products in equimolr mounts to correct for the PCR mplifiction efficiency of ech set. For this purpose we used BAC nd PAC clones spnning the complete loci (insted of the previously used PCR frgments tht spn the restriction sites of interest 6 ). For the mouse β-gloin locus we used 214-k BAC (#RP23-370E12, Enseml Genome Browser), nd for the humn β-gloin locus we used 185-k PAC 20. In ddition, we used 60- to 70-k PAC contining the mouse Ercc3 locus (PAC Clone #443-C18, MRC geneservice). We mixed either the mouse gloin BAC or the humn gloin PAC with the Ercc3 PAC t equimolr mounts. We then digested nd ligted the mixes s descried 6. We could not otin control PCR products with primers designed to nlyze frgments contining H-y nd H-2 owing to polymorphisms in the BAC clone #RP23-370E12. As consequence, these frgments were not included in the locus-wide crosslinking frequency nlysis (Fig. 1). We crried out ll niml experiments ccording to institutionl nd ntionl guidelines (Committee on Experiments with Lortory Animls (DEC-Consult); Ministry of Agriculture, Nture nd Food Qulity, The Hgue, The Netherlnds). Southern lotting. We treted fetl liver cells (from 14.5-d.p.c. emryos) s descried ove (with indicted formldehyde concentrtions), ut we omitted ligtion nd nlyzed 10 µg of purified DNA y Southern lotting. We used the following proes: H-h1, 255-p HinfI frgment, which hyridizes to 2.7- k HindIII H-h1 frgment nd H-h0 (5.5 k) nd H-h2 (6.4 k) pseudogene frgments; H-1, 700-p HindIII/NcoI frgment, which hyridizes to 1.0-k HindIII frgment nd H-2 (8.6 k) frgment; HS3, 300-p PCR frgment, which hyridizes to 2.0-k HindIII frgment; Pou3f1, 100-p PCR frgment, which hyridizes to 4.0-k HindIII frgment. Cell culture. We cultured I/11 cells s descried previously 22,23. Briefly, we mintined proliferting I/11 cells in StemPro-34 contining 2 units ml 1 humn recominnt erythropoietin, 100 ng ml 1 murine recominnt SCF, 10 6 M dexmethsone nd 40 ng ml 1 insulin-like growth fctor. We expnded cells y dily prtil medium chnges nd ddition of fresh fctors, keeping cell density etween nd cells per ml. To induce differentition, we removed continuously proliferting I/11 cells from the culture, wshed them twice in phosphte-uffered sline nd seeded them t cells per ml in differentition medium contining 10 units ml 1 erythropoietin, IE insulin per ml, the dexmethsone ntgonist ZK ( M) nd 1 mg ml 1 iron-sturted humn trnsferrin. We mintined differentiting erythrolsts t densities of cells per ml. For 3C nlysis of differentiting I/11 cells, we fixed cells with formldehyde 40 h fter induction nd processed them s descried ove. URLs. Enseml Genome Browser is ville t MRC gene service is ville t ACKNOWLEDGMENTS We thnk K. Hussin nd R. Hendriks for technicl ssistnce nd M. von Lindern for I/11 cells. This work is supported y The Netherlnds Orgnistion for Scientific Reserch to W.d.L. s prt of the Innovtionl Reserch Incentives Scheme nd y grnts from The Netherlnds Orgnistion for Scientific Reserch nd Europen Community to F.G. 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