The beta -globin locus control region (LCR) functions primarily by enhancing the transition from transcription initiation to elongation.

To investigate the molecular basis of beta-globin gene activation, we analyzed factor recruitment and histone modification at the adult beta-globin gene in wild-type (WT)/locus control region knockout (DeltaLCR) heterozygous mice and in murine erythroleukemia (MEL) cells. Although histone acetylation and methylation (Lys 4) are high before and after MEL differentiation, recruitment of the erythroid-specific activator NF-E2 to the promoter and preinitiation complex (PIC) assembly occur only after differentiation. We reported previously that targeted deletion of the LCR reduces beta-globin gene expression to 1%-4% of WT without affecting promoter histone acetylation. Here, we report that NF-E2 is recruited equally efficiently to the adult beta-globin promoters of the DeltaLCR and WT alleles. Moreover, the LCR deletion reduces PIC assembly only twofold, but has a dramatic effect on Ser 5 phosphorylation of RNA polymerase II and transcriptional elongation. Our results suggest at least three distinct stages in beta-globin gene activation: (1) an LCR-independent chromatin opening stage prior to NF-E2 recruitment to the promoter and PIC assembly; (2) an intermediate stage in which NF-E2 binding (LCR-independent) and PIC assembly (partially LCR-dependent) occur; and (3) an LCR-dependent fully active stage characterized by efficient pol II elongation. Thus, in its native location the LCR functions primarily downstream of activator recruitment and PIC assembly.

[1]  L. Madisen,et al.  The Immunoglobulin Heavy Chain Locus Control Region Increases Histone Acetylation along Linked c-myc Genes , 1998, Molecular and Cellular Biology.

[2]  N. Andrews,et al.  The transcriptional integrator CREB-binding protein mediates positive cross talk between nuclear hormone receptors and the hematopoietic bZip protein p45/NF-E2 , 1997, Molecular and cellular biology.

[3]  Dimitris Thanos,et al.  Ordered Recruitment of Chromatin Modifying and General Transcription Factors to the IFN-β Promoter , 2000, Cell.

[4]  J. Greenblatt,et al.  Opposing effects of Ctk1 kinase and Fcp1 phosphatase at Ser 2 of the RNA polymerase II C-terminal domain. , 2001, Genes & development.

[5]  G. Prelich.  RNA Polymerase II Carboxy-Terminal Domain Kinases: Emerging Clues to Their Function , 2002, Eukaryotic Cell.

[6]  M. Groudine,et al.  β-globin Gene Switching and DNase I Sensitivity of the Endogenous β-globin Locus in Mice Do Not Require the Locus Control Region , 2000 .

[7]  Michael R. Green,et al.  Enhancement of TBP binding by activators and general transcription factors , 1999, Nature.

[8]  J. T. Kadonaga,et al.  Going the distance: a current view of enhancer action. , 1998, Science.

[9]  Mark Groudine,et al.  A Functional Enhancer Suppresses Silencing of a Transgene and Prevents Its Localization Close to Centromeric Heterochromatin , 1999, Cell.

[10]  Paul Tempst,et al.  Erythroid transcription factor NF-E2 is a haematopoietic-specific basic–leucine zipper protein , 1993, Nature.

[11]  I. Talianidis,et al.  Coordination of PIC Assembly and Chromatin Remodeling During Differentiation-Induced Gene Activation , 2002, Science.

[12]  J. Lis,et al.  Promoter-associated pausing in promoter architecture and postinitiation transcriptional regulation. , 1998, Cold Spring Harbor symposia on quantitative biology.

[13]  M. Groudine,et al.  ChIPs of the β-globin locus: unraveling gene regulation within an active domain , 2002 .

[14]  E. Bresnick,et al.  Direct interaction of NF-E2 with hypersensitive site 2 of the beta-globin locus control region in living cells. , 2000, Blood.

[15]  M. Reitman,et al.  Developmental regulation of topoisomerase II sites and DNase I-hypersensitive sites in the chicken beta-globin locus. , 1990, Molecular and cellular biology.

[16]  M. Groudine,et al.  Looping versus linking: toward a model for long-distance gene activation. , 1999, Genes & development.

[17]  Kirby D. Johnson,et al.  Distinct mechanisms control RNA polymerase II recruitment to a tissue-specific locus control region and a downstream promoter. , 2001, Molecular cell.

[18]  B. Peterlin,et al.  Control of RNA initiation and elongation at the HIV-1 promoter. , 1994, Annual review of biochemistry.

[19]  T. Ley,et al.  Targeted deletion of 5'HS2 of the murine beta-globin LCR reveals that it is not essential for proper regulation of the beta-globin locus. , 1995, Genes & development.

[20]  Tae Hoon Kim,et al.  Efficient recruitment of TFIIB and CBP-RNA polymerase II holoenzyme by an interferon-beta enhanceosome in vitro. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Robert K. Davis,et al.  The myoD gene family: nodal point during specification of the muscle cell lineage. , 1991, Science.

[22]  K. Nasmyth,et al.  Ordered Recruitment of Transcription and Chromatin Remodeling Factors to a Cell Cycle– and Developmentally Regulated Promoter , 2016, Cell.

[23]  D. Martin,et al.  Transcriptional enhancers--on/off gene regulation as an adaptation to silencing in higher eukaryotic nuclei. , 2001, Trends in genetics : TIG.

[24]  M. Groudine,et al.  The block to transcriptional elongation within the human c-myc gene is determined in the promoter-proximal region. , 1992, Genes & development.

[25]  Ken Itoh,et al.  Regulation of transcription by dimerization of erythroid factor NF-E2 p45 with small Maf proteins , 1994, Nature.

[26]  E. Sekinger,et al.  Silenced Chromatin Is Permissive to Activator Binding and PIC Recruitment , 2001, Cell.

[27]  G. Blobel,et al.  Stimulation of NF-E2 DNA Binding by CREB-binding Protein (CBP)-mediated Acetylation* , 2001, The Journal of Biological Chemistry.

[28]  M. Dahmus.  Reversible Phosphorylation of the C-terminal Domain of RNA Polymerase II* , 1996, The Journal of Biological Chemistry.

[29]  B. Peterlin,et al.  NF-κB Binds P-TEFb to Stimulate Transcriptional Elongation by RNA Polymerase II , 2001 .

[30]  J. D. Engel,et al.  The world according to Maf. , 1997, Nucleic acids research.

[31]  Jung-Hua Yeh,et al.  Promoter activation by enhancer-dependent and -independent loading of activator and coactivator complexes. , 2002, Molecular cell.

[32]  Michael Hampsey,et al.  Molecular Genetics of the RNA Polymerase II General Transcriptional Machinery , 1998, Microbiology and Molecular Biology Reviews.

[33]  N. Gavva,et al.  Distinction between AP1 and NF-E2 factor-binding at specific chromatin regions in mammalian cells , 1999, Oncogene.

[34]  D. Eick,et al.  Elongation and premature termination of transcripts initiated from c-fos and c-myc promoters show dissimilar patterns. , 1995, Oncogene.

[35]  M. Groudine,et al.  The murine β-globin locus control region regulates the rate of transcription but not the hyperacetylation of histones at the active genes , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[36]  S. Jane,et al.  The activation domain of the enhancer binding protein p45NF-E2 interacts with TAFII130 and mediates long-range activation of the alpha- and beta-globin gene loci in an erythroid cell line. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[37]  V. Orlando,et al.  General transcription factors bind promoters repressed by Polycomb group proteins , 2001, Nature.

[38]  K. Senger,et al.  Mechanism by which the IFN-beta enhanceosome activates transcription. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[39]  E. Cho,et al.  Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription. , 2000, Genes & development.

[40]  Stuart L. Schreiber,et al.  Active genes are tri-methylated at K4 of histone H3 , 2002, Nature.

[41]  M. Groudine,et al.  Activation of β-major globin gene transcription is associated with recruitment of NF-E2 to the β-globin LCR and gene promoter , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[42]  S. Orkin,et al.  Purification of the human NF-E2 complex: cDNA cloning of the hematopoietic cell-specific subunit and evidence for an associated partner , 1993, Molecular and cellular biology.

[43]  M. Groudine,et al.  The β-Globin LCR Is Not Necessary for an Open Chromatin Structure or Developmentally Regulated Transcription of the Native Mouse β-Globin Locus , 1998 .

[44]  K. Yamamoto,et al.  The glucocorticoid receptor inhibits NFkappaB by interfering with serine-2 phosphorylation of the RNA polymerase II carboxy-terminal domain. , 2000, Genes & development.

[45]  S. Orkin,et al.  The ubiquitous subunit of erythroid transcription factor NF-E2 is a small basic-leucine zipper protein related to the v-maf oncogene. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[46]  J. R. Morris,et al.  Spt5 and spt6 are associated with active transcription and have characteristics of general elongation factors in D. melanogaster. , 2000, Genes & development.

[47]  K. Struhl,et al.  Binding of TBP to promoters in vivo is stimulated by activators and requires Pol II holoenzyme , 1999, Nature.

[48]  R. Tjian,et al.  Orchestrated response: a symphony of transcription factors for gene control. , 2000, Genes & development.

[49]  P. Sharp,et al.  HIV‐1 Tat protein promotes formation of more‐processive elongation complexes. , 1991, The EMBO journal.

[50]  Steven A. Brown,et al.  Activator-dependent regulation of transcriptional pausing on nucleosomal templates. , 1996, Genes & development.

[51]  Myles Brown,et al.  Cofactor Dynamics and Sufficiency in Estrogen Receptor–Regulated Transcription , 2000, Cell.

[52]  Mark Groudine,et al.  A block to elongation is largely responsible for decreased transcription of c-myc in differentiated HL60 cells , 1986, Nature.

[53]  Y. Kan,et al.  Isolation of cDNA encoding the human NF-E2 protein. , 1993, Proceedings of the National Academy of Sciences of the United States of America.