The β-globin Locus Dr. Ann Dean

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1 , PhD Chief, Section on Gene Regulation and Development Laboratory of Cellular and Developmental Biology, NIDDK, NIH Bethesda, MD, USA 1 α and β-globin loci encode hemoglobin subunits 64 kda tetrameric protein 2 β polypeptide chains and 2 α polypeptide chains HbA α 2 β 2 Each chain contains a heme group with a central iron atom 2 The iron atom reversibly binds oxygen Erythrocytes carry hemoglobin in blood 3 Disc shaped erythrocytes visualized by scanning electron microscopy Oxygen is bound to hemoglobin as red cells pass through the alveoli of the lung Oxygen is released from hemoglobin when red cells circulate to peripheral tissues 1

2 Human globin genes and hemoglobins Two different gene loci encode the α and β globin families α-globin locus ζ α2 α1 5 3 Chr16 β-globin locus 5 3 Chr11 Embryonic Fetal Adult Hb Gower ζ 2 ε 2 Hb F α 2 γ 2 HbA α 2 β 2 HbA2 α 2 δ 2 4 The globin genes are expressed sequentially in the order of their 5 to 3 arrangement in the genome Different α and β chains combine to form different hemoglobin species during development Globin gene expression during development 5 The changes in gene expression coincide with changes in the site of erythropoiesis Globin gene structure 5 UTR 3 UTR Promoter GATA1 EKLF TATA 0 6 The globin genes encode a 16 kda protein within three exons separated by a small first intron and a large second intron The genes have short 5 and 3 untranslated regions A TATA box appears about 30 nt upstream of the transcription start site Motifs for EKLF and GATA1 are shared by all the promoters 2

3 Sickle cell disease s β-globin locus X Embryonic Fetal Adult First genetic disease for which the molecular basis was understood A single base pair mutation that results in a glutamic acid to valine amino acid change at position 6 in the β chain of hemoglobin HbS tetramers are less soluble than HbA tetramers and polymerize within red cells distorting their shape (sickle cells) 7 Reactivation of γ-globin production in adults is therapeutically beneficial because HbF is a potent inhibitor of polymerization of HbS and elevated HbF results in relatively mild clinical manifestations β-thalassemia β-globin locus X Embryonic Fetal Adult Rather than a structural mutation, this disease is due to deletion or mutation of regulatory sequences, often in the promoter, of the β-globin gene which result in decreased synthesis of β globin The resulting chain imbalance leads to formation of α-globin tetramers which precipitate and damage red cells leading to anemia Increased expression of γ-globin production in adults could diminish the chain imbalance Gene therapy by transfer of a properly regulated normal β-globin gene could result in adequate β-globin production 8 The mammalian β-globin loci Human 10 kb Odorant receptor genes Embryo Fetus Adult Odorant receptor genes Mouse εy βh1 Embryo βmaj βmin Adult 9 Erythroid specific DNase I hypersensitive sites (HS1-4) are 6-60 kb distant and act collectively as an enhancer for all the genes - known as the locus control region or Widely detected DHS separate the locus from odorant receptor genes, bind and are potential insulators and chromatin organizers 3

4 The is necessary and sufficient for β-globin expression Human Transgene Mouse εy βh1 βmaj βmin Mouse Deletion in the endogenous locus 10 Grosveld et al., Cell (1987) Bender et al., Mol. Cell (2000) Transcriptional regulation by recruitment Enhancers Silencers Silent H1 H1 H1 Yeast gene promoter Factors Insulators H1 loss H1 ADP ATP SWI/SNF BRG1 Factors UAS HAT/ HMT RNA pol II complex TATA Yeast gene promoter Active 11 Nucleosome eviction, sliding? Enhancers are regulatory DNA sequence elements that increase the transcriptional output of target genes Models for how enhancers exert their effect at a distance E P Enhancer Promoter E Recruitment P Activators Pol II HATS Remodelers Looping Tracking 12 P E An enhancer could contact a promoter directly, looping out intervening chromatin E P Factors recruited to an enhancer could track along intervening DNA to a promoter 4

5 Chromatin conformation capture (3C) assay RE cutting site Digestion Cross-linked by formaldehyde Ligation Reverse crosslink 13 Detect ligated products by PCR Dekker et al., Science (2002) Chromatin looping in the β-globin locus interaction not cell specific Human Embryo Fetus Adult Erythroid cell specific 14 Tolhuis et al., Genes and Dev. (2002) Palstra et al., Nat. Gen. (2003) Chromatin looping in the β-globin locus is developmentally regulated interaction not cell specific Human Embryo Fetus Adult Fetal stage Adult stage Erythroid cell and developmental stage specific 15 Tolhuis et al., Genes and Dev. (2002) Palstra et al., Nat. Gen. (2003) 5

6 Important questions raised by 3C looping studies How are chromatin loops established and maintained? How are loops modulated during development to change transcription? What transcription factors are involved in mediating proximity? 16 Loops are mediated by factors binding to the sites involved Mouse HS5 10 kb εy βh1 βmaj 3 HS1 βmin Factors that are required for β-globin looping according to RNAi and 3C studies TAL1 GATA1 FOG1 Adult globins expressed 17 Drissen et al., Genes and Dev. (2004) Vakoc et al., Mol. Cell (2005) Song et al., Mol. Cell (2007) Yun et al., Nucl. Acids Res. (2013) GATA1 FOG1 Essential erythroid ZN finger transcription factor that binds to virtually all erythroid enhancers and promoters. Is assisted in binding chromatin at some sites by Friend of GATA (FOG1) TAL1 Essential basic-helix-loop-helix transcription factor that heterodimerizes with E box proteins. Found at erythroid regulatory elements in conjunction with GATA1 EKLF ZN finger transcription essential for adult β-globin transcription. Binding sites are found in close proximity to GATA1 sites 18 LDB1 Highly conserved, widely expressed protein with no DNA-binding or enzymatic activity. Mammalian orthologue of Drosophila Chip that was isolated in a genetic screen for enhancer facilitators 6

7 LDB1 is central to the development of erythroid cells N C Dimerization domain NLS LIM interaction domain LDB1 null embryos have severe patterning defects by gastrulation and absence of primitive erythropoiesis and conditional knock out studies show it is required for definitive erythropoiesis WT Ldb1 null E Mukhopadhyay et al., Development (2003) Li et al., JEM (2011) The multimeric LDB1 complex in erythroid cells LIM interaction domain N C Dimerization domain E47 LID LMO2 GATA-1 LDB1 complex E box (N) GATA N 9 LDB1 interacts with the with erythroid LIM-only protein LMO2 that bridges DNA-binding GATA1 and the TAL1/E47 heterodimer The LDB1 complex binds to a composite E box-gata motif with a 9 nt separation 20 Wadman et al., PNAS (1997) Meier, et al, Genes. Dev. (2005) LDB1 complex ldb1 alone can tether an /β globin chromatin loop Normal erythroid cells LDB1 complex LDB1 dimerization Promoter GATA-1 null erythroid cells LDB1 complex - GATA1 ZF Promoter 21 GATA-1 ZF β-globin-binding Zn finger peptide Krivega at al., Genes Dev Deng et al., Cell,

8 3D FISH β-globin loci Nuclear re-location of the β-globin locus to transcription factories for activation Globin transcription 3D immunofish RNA pol II transcription factory β-globin loci Stages of differentiation of fetal liver erythroid cells Nuclear migration of the β-globin locus requires the and also requires LDB1 This raises the possibility that enhancer looping is required for the relocation to transcription factories 22 Osborne et al., Nat. Genet. 2004; Ragoczy et al., Genes Dev. 2006; Song et al., Blood, 2010 Co-association of active erythroid genes in shared transcription factories Erythroid genes EKLF dependent Not EKLF dependent Transcription factory Specialized transcription factory 23 Osborne et al., Nat. Genet Schoenfelder et al., Nat. Gen LDB1 complexes have a global role in erythroid gene expression LDB1/TAL1/GATA1 co-incident sites 24% 29% 69% 24 Li et al., Blood,

9 LDB1 complexes bind known and putative erythroid enhancers Runx1 Zdhhc19 25 Li et al., Blood, 2013 The LDB1 may function in concert with EKLF/Klf1 26 Li et al., Blood (2013) Transcription and chromatin looping in the β-globin locus are developmentally regulated HS5 3 HS1 Human Embryo Fetus Adult Fetal stage γ-globin transcribed Adult stage β-globin transcribed 27 Re-activation of γ-globin transcription in adults would be clinically valuable to ameliorate severe symptoms of SCD and β-thalassemia 9

10 Natural human mutations are important to understand hemoglobin switching No factors specifically activating or repressing γ-globin transcription had been identified over many years of study An important clue finally emerged from genome-wide association studies (GWAS) of natural human genetic variation Two loci harboring common variants outside the β-globin locus that influenced HbF levels were identified: - Chr6 between the genes HBSIL and MYB - Chr2 within the BCL11A gene This suggested that BCL11A might be a regulator of γ-globin expression 28 BCL11A binds to sites in the β-globin locus in adult erythroid cells Human HS5 3 HS1 29 Chromatin immunoprecipitation Sankaran et al., Science (2008) BCL11A represses γ-globin expression Reduction of BCL11A in erythroid cells results in elevation of γ-globin as a percentage of total β-like globin gene expression Developmental silencing of γ-globin is impaired in BCL11A null mice carrying a complete human β-locus transgene Loss of BCL11A reconfigures loops in the β-locus in erythroid cells of human β-locus transgenic mice BCL11A -/- HS5 3 HS1 Embryo Fetus Adult 30 BCL11A +/+ Sankaran et al., Science (2008) Sankaran et al., Nature (2009) Xu et al., Genes and Development (2010) 10

11 Interfering with BCL11A silencing of γ-globin corrects sickle cell disease in a mouse model Peripheral blood cells of mice that are a model for sickle cell disease show the abnormal sickle shaped cells compared to cells in control mice SCD mice that are null for BCL11A show restoration of normal red cells Discovery of an erythroid enhancer of BCL11A raises hopes of manipulating its expression in an erythroid-specific fashion 31 Xu et al., Science (2011) Bauer et al., Science (2013) 32 The β-globin locus: many firsts Sickle cell disease was the first genetic disease to be molecularly explained (Linus Pauling, 1949; Vernon Ingram, 1957) The x-ray crystallographic structure of the globin gene protein product hemoglobin was the first to be solved for a large protein (Max Perutz, 1959) Globin genes were among the first examples showing the existence of introns within mammalian genes (Philip Leder, 1978) The globin genes were among the first genes to be molecularly cloned and sequenced long in advance of the genomic age (early 1980s) The first was described in the globin locus (Frank Grosveld, 1987) Intra-locus looping in chromosomes between an enhancer and gene was first demonstrated in the globin locus by 3C and RNA-TRAP (Peter Fraser, 2002 and Frank Grosveld, 2002) Proof of principle for targeted reactivation of γ-globin as a cure for sickle cell disease by reduction of a γ-globin inhibitor (Stuart Orkin, 2011) Suggested reviews Bulger, M. and Groudine, M. (2011). Functional and mechanistic diversity of distal transcription enhancers. Cell 144, Edelman, L.B. and Fraser, P. (2012). Transcription factories: genetic programming in three dimensions. Curr. Opin. Genet. Dev. 22, Krivega, I. and Dean, A. (2012). Enhancer and promoter interactions-long distance calls. Curr. Opin. Genet. Dev. 22, 1-7 Sankaran, V.G. and Orkin, S.H. (2013). The switch from fetal to adult hemoglobin. Cold Spring. Harb. Perspect. Med. 2013;doi /cshperspect. a Plank, J.L. and Dean, A. (2014). Enhancer Function: Mechanistic and Genome-wide Insights Come Together. Mol. Cell 55, 5-14 Reference list Bender, M.A., Bulger, M., Close, J., and Groudine, M. (2000). β-globin gene switching and DNase I sensitivity of the endogenous b-globin locus in mice do not require the locus control region. Mol. Cell 5, Bauer, D.E., Kamran, S.C., Lessard, S., Xu, J., Fujiwara, Y., Lin, C., Shao, Z., Canver, M. C., Smith, E. C., Pinello, L., Sabo, P. J., Vierstra, J., Voit, R. A., Yuan, G.-C., Porteus, M. H., Stamatoyannopoulos, J., Lettre, G. and Orkin, S. H. (2013). An erythroid enhancer of BCL11A subject to genetic variation determines fetal hemoglobin level. Science 342, Dekker, J., Rippe, K., Dekker, M., and Kleckner, N. (2002). Capturing chromosome conformation. Science 295, Deng, W., Lee, J., Wang, H., Miller, J., Reik, A., Gregory, P.D., Dean, A., and Blobel, G.A. (2012). Controlling long-range genomic interactions at a native locus by targeted tethering of a looping factor. Cell 149,

12 Reference list (2) 34 Grosveld, F., van Assendelft, G.B., Greaves, D.R., and Kollias, G. (1987). Position-independent, high-level expression of the human β-globin gene in transgenic mice. Cell 51, Krivega, I., Dale, R.K. and Dean, A. Role of LDB1 in the transition from chromatin looping to transcription activation. Genes Dev. 28, Li, L., Freudenberg, J., Cui, K., Dale, R., Song, S.H., Dean, A., Zhao, K., Jothi, R., and Love, P.E. (2013). Ldb1-nucleated transcription complexes function as primary mediators of global erythroid gene activation. Blood. 121, Li, L., Jothi, R., Cui, K., Lee, J.Y., Cohen, T., Gorivodsky, M., Tzchori, I., Zhao, Y., Hayes, S.M., Bresnick, E.H., Zhao, K., Westphal, H., and Love, P.E. (2011). Nuclear adaptor Ldb1 regulates a transcriptional program essential for the maintenance of hematopoietic stem cells. Nat Immunol 12, Meier, N., Krpic, S., Rodriguez, P., Strouboulis, J., Monti, M., Krijgsveld, J., Gering, M., Patient, R., Hostert, A., and Grosveld, F. (2006). Novel binding partners of Ldb1 are required for haematopoietic development. Development 133, Mukhopadhyay, M., Teufel, A., Yamashita, T., Agulnick, A.D., Chen, L., Downs, K.M., Schindler, A., Grinberg, A., Huang, S.P., Dorward, D., and Westphal, H. (2003). Functional ablation of the mouse Ldb1 gene results in severe patterning defects during gastrulation. Development 130, Osborne, C.S., Chakalova, L., Brown, K.E., Carter, D., Horton, A., Debrand, E., Goyenechea, B., Mitchell, J.A., Lopes, S., Reik, W., and Fraser, P. (2004). Active genes dynamically colocalize to shared sites of ongoing transcription. Nat. Genet. 36, Palstra, R.J., Tolhuis, B., Splinter, E., Nijmeijer, R., Grosveld, F., and de Laat, W. (2003). The β-globin nuclear compartment in development and erythroid differentiation. Nat. Genet. 35, Reference list (3) Ragoczy, T., Bender, M.A., Telling, A., Byron, R., and Groudine, M. (2006). The locus control region is required for association of the murine beta-globin locus with engaged transcription factories during erythroid maturation. Genes Dev. 20, Sankaran, V.G., Menne, T.F., Xu, J., Akie, T.E., Lettre, G., Van, H.B., Mikkola, H.K., Hirschhorn, J.N., Cantor, A.B., and Orkin, S.H. (2008). Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A. Science 322, Sankaran, V.G., Xu, J., Ragoczy, T., Ippolito, G.C., Walkley, C.R., Maika, S.D., Fujiwara, Y., Ito, M., Groudine, M., Bender, M.A., Tucker, P.W., and Orkin, S.H. (2009). Developmental and species-divergent globin switching are driven by BCL11A. Nature 460, Schoenfelder, S., Sexton, T., Chakalova, L., Cope, N.F., Horton, A., Andrews, S., Kurukuti, S., Mitchell, J.A., Umlauf, D., Dimitrova, D.S., Eskiw, C.H., Luo, Y., Wei, C.L., Ruan, Y., Bieker, J.J., and Fraser, P. (2010). Preferential associations between co-regulated genes reveal a transcriptional interactome in erythroid cells. Nat Genet 42, Song, S.-H., Hou, C., and Dean, A. (2007). A positive role for NLI/Ldb1 in long-range β-globin locus control region function. Mol. Cell 28, Song, S.-H., Kim, A., Ragoczy, T., Bender, M.A., Groudine, M., and Dean, A. (2010). Multiple functions of Ldb1 required for β-globin activation during erythroid differentiation. Blood 116, Tolhuis, B., Palstra, R.J., Splinter, E., Grosveld, F., and de Laat, W. (2002). Looping and interaction between hypersensitive sites in the active β-globin locus. Mol. Cell 10, Vakoc, C.R., Letting, D.L., Gheldof, N., Sawado, T., Bender, M.A., Groudine, M., Weiss, M.J., Dekker, J., and Blobel, G.A. (2005). Proximity among distant regulatory elements at the β-globin locus requires GATA-1 and FOG-1. Mol. Cell 17, Reference list (4) Wadman, I.A., Osada, H., Grutz, G.G., Agulnick, A.D., Westphal, H., Forster, A., and Rabbitts, T.H. (1997). The LIM-only protein Lmo2 is a bridging molecule assembling an erythroid, DNA-binding complex which includes the TAL1, E47, GATA-1 and Ldb1/NLI proteins. EMBO J. 16, Xu, J., Peng, C., Sankaran, V.G., Shao, Z., Esrick, E.B., Chong, B.G., Ippolito, G.C., Fujiwara, Y., Ebert, B.L., Tucker, P.W., and Orkin, S.H. (2011). Correction of Sickle Cell Disease in Adult Mice by Interference with Fetal Hemoglobin Silencing. Science Xu, J., Sankaran, V.G., Ni, M., Menne, T.F., Puram, R.V., Kim, W., and Orkin, S.H. (2010). Transcriptional silencing of {gamma}-globin by BCL11A involves long-range interactions and cooperation with SOX6. Genes Dev 24, Yun, W. J., Kim, Y. W., Kang, Y., Lee, J. Dean, A. and Kim, A. The hematopoietic regulator TAL1 is required for chromatin looping between the beta-globin and human gamma-globin genes to activate transcription (2014). Nucl. Acids Res. 42,

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