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Luis M. Hernández - One of the best experts on this subject based on the ideXlab platform.
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The LDB1 mutant of Saccharomyces cerevisiae is defective in Pmr1p, the yeast secretory pathway/Golgi Ca2+/Mn2+-ATPase
FEMS microbiology letters, 2003Co-Authors: Isabel Olivero, Isaac Corbacho, Luis M. HernándezAbstract:The LDB1 gene of Saccharomyces cerevisiae was identified by complementation of the LDB1 mutant phenotype with a genomic library. We found that the LDB1 defect is complemented by PMR1 which codes for the yeast secretory pathway/Golgi Ca2+/Mn2+-ATPase. Besides that, the analysis of a null mutation of the PMR1 gene revealed a phenotype identical to that of LDB1 mutant. Thus, LDB1 must be considered a synonym of PMR1.
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the LDB1 mutant of saccharomyces cerevisiae is defective in pmr1p the yeast secretory pathway golgi ca2 mn2 atpase
Fems Microbiology Letters, 2003Co-Authors: Isabel Olivero, Isaac Corbacho, Luis M. HernándezAbstract:The LDB1 gene of Saccharomyces cerevisiae was identified by complementation of the LDB1 mutant phenotype with a genomic library. We found that the LDB1 defect is complemented by PMR1 which codes for the yeast secretory pathway/Golgi Ca2+/Mn2+-ATPase. Besides that, the analysis of a null mutation of the PMR1 gene revealed a phenotype identical to that of LDB1 mutant. Thus, LDB1 must be considered a synonym of PMR1.
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Proteolytic processing of a secreted glycoprotein and O-glycosylation of mannoproteins are affected in the N-glycosylation mutant Saccharomyces cerevisiae LDB1
Biochimica et biophysica acta, 1998Co-Authors: Paula Mañas, Isabel Olivero, Luis M. HernándezAbstract:In a previous work [P.I. Manas, I. Olivero, M. Avalos, L.M. Hernandez, Glycobiology, 7 (1997) 487–497], we described the isolation and characterization of the Saccharomyces cerevisiae LDB1 mutant which is affected in several steps of the N-glycosylation of mannoproteins probably due to a malfunction of the Golgi apparatus. Here, we found that two further functions assigned to the Golgi cisternae are also affected in the mutant: proteolytic processing of a secreted protein and O-glycosylation. We found that around 70% of the exoglucanase activity that is secreted into the culture medium by LDB1 bears an extra tetrapeptide in its NH2-terminus due to incomplete proteolytic processing. The O-linked oligosaccharides from LDB1 mnn1 were indistinguishable from those synthesized by the parental strain mnn1. However, when the O-oligosaccharides from the wild type and LDB1 were compared, we found a significant decrease in the tetrasaccharide in the latter, as well as a concomitant increase in the disaccharide, suggesting a defect in the Kre2p/Mnt1p involved in the transfer of the third mannose of these residues.
Isabel Olivero - One of the best experts on this subject based on the ideXlab platform.
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The LDB1 mutant of Saccharomyces cerevisiae is defective in Pmr1p, the yeast secretory pathway/Golgi Ca2+/Mn2+-ATPase
FEMS microbiology letters, 2003Co-Authors: Isabel Olivero, Isaac Corbacho, Luis M. HernándezAbstract:The LDB1 gene of Saccharomyces cerevisiae was identified by complementation of the LDB1 mutant phenotype with a genomic library. We found that the LDB1 defect is complemented by PMR1 which codes for the yeast secretory pathway/Golgi Ca2+/Mn2+-ATPase. Besides that, the analysis of a null mutation of the PMR1 gene revealed a phenotype identical to that of LDB1 mutant. Thus, LDB1 must be considered a synonym of PMR1.
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the LDB1 mutant of saccharomyces cerevisiae is defective in pmr1p the yeast secretory pathway golgi ca2 mn2 atpase
Fems Microbiology Letters, 2003Co-Authors: Isabel Olivero, Isaac Corbacho, Luis M. HernándezAbstract:The LDB1 gene of Saccharomyces cerevisiae was identified by complementation of the LDB1 mutant phenotype with a genomic library. We found that the LDB1 defect is complemented by PMR1 which codes for the yeast secretory pathway/Golgi Ca2+/Mn2+-ATPase. Besides that, the analysis of a null mutation of the PMR1 gene revealed a phenotype identical to that of LDB1 mutant. Thus, LDB1 must be considered a synonym of PMR1.
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Proteolytic processing of a secreted glycoprotein and O-glycosylation of mannoproteins are affected in the N-glycosylation mutant Saccharomyces cerevisiae LDB1
Biochimica et biophysica acta, 1998Co-Authors: Paula Mañas, Isabel Olivero, Luis M. HernándezAbstract:In a previous work [P.I. Manas, I. Olivero, M. Avalos, L.M. Hernandez, Glycobiology, 7 (1997) 487–497], we described the isolation and characterization of the Saccharomyces cerevisiae LDB1 mutant which is affected in several steps of the N-glycosylation of mannoproteins probably due to a malfunction of the Golgi apparatus. Here, we found that two further functions assigned to the Golgi cisternae are also affected in the mutant: proteolytic processing of a secreted protein and O-glycosylation. We found that around 70% of the exoglucanase activity that is secreted into the culture medium by LDB1 bears an extra tetrapeptide in its NH2-terminus due to incomplete proteolytic processing. The O-linked oligosaccharides from LDB1 mnn1 were indistinguishable from those synthesized by the parental strain mnn1. However, when the O-oligosaccharides from the wild type and LDB1 were compared, we found a significant decrease in the tetrasaccharide in the latter, as well as a concomitant increase in the disaccharide, suggesting a defect in the Kre2p/Mnt1p involved in the transfer of the third mannose of these residues.
Masanori Taira - One of the best experts on this subject based on the ideXlab platform.
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Selective degradation of excess LDB1 by Rnf12/RLIM confers proper LDB1 expression levels and Xlim-1/LDB1 stoichiometry in Xenopus organizer functions.
Development (Cambridge England), 2003Co-Authors: Ichiro Hiratani, Naoko Yamamoto, Toshiaki Mochizuki, Shin-ya Ohmori, Masanori TairaAbstract:The Xenopus LIM homeodomain (LIM-HD) protein, Xlim-1, is expressed in the Spemann organizer and cooperates with its positive regulator, LDB1, to activate organizer gene expression. While this activation is presumably mediated through Xlim-1/LDB1 tetramer formation, the mechanisms regulating proper Xlim-1/LDB1 stoichiometry remains largely unknown. We isolated the Xenopus ortholog (XRnf12) of the RING finger protein Rnf12/RLIM and explored its functional interactions with Xlim-1 and LDB1. Although XRnf12 functions as a E3 ubiquitin ligase for LDB1 and causes proteasome-dependent degradation of LDB1, we found that co-expression of a high level of Xlim-1 suppresses LDB1 degradation by XRnf12. This suppression requires both the LIM domains of Xlim-1 and the LIM interaction domain of LDB1, suggesting that LDB1, when bound to Xlim-1, escapes degradation by XRnf12. We further show that a high level of LDB1 suppresses the organizer activity of Xlim-1/LDB1, suggesting that excess LDB1 molecules disturb Xlim-1/LDB1 stoichiometry. Consistent with this, LDB1 overexpression in the dorsal marginal zone suppresses expression of several organizer genes including postulated Xlim-1 targets, and importantly, this suppression is rescued by co-expression of XRnf12. These data suggest that XRnf12 confers proper LDB1 protein levels and Xlim-1/LDB1 stoichiometry for their functions in the organizer. Together with the similarity in the expression pattern of LDB1 and XRnf12 throughout early embryogenesis, we propose Rnf12/RLIM as a specific regulator of LDB1 to ensure its proper interactions with LIM-HD proteins and possibly other LDB1-interacting proteins in the organizer as well as in other tissues.
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selective degradation of excess LDB1 by rnf12 rlim confers proper LDB1 expression levels and xlim 1 LDB1 stoichiometry in xenopus organizer functions
Development, 2003Co-Authors: Ichiro Hiratani, Naoko Yamamoto, Toshiaki Mochizuki, Shin-ya Ohmori, Masanori TairaAbstract:The Xenopus LIM homeodomain (LIM-HD) protein, Xlim-1, is expressed in the Spemann organizer and cooperates with its positive regulator, LDB1, to activate organizer gene expression. While this activation is presumably mediated through Xlim-1/LDB1 tetramer formation, the mechanisms regulating proper Xlim-1/LDB1 stoichiometry remains largely unknown. We isolated the Xenopus ortholog (XRnf12) of the RING finger protein Rnf12/RLIM and explored its functional interactions with Xlim-1 and LDB1. Although XRnf12 functions as a E3 ubiquitin ligase for LDB1 and causes proteasome-dependent degradation of LDB1, we found that co-expression of a high level of Xlim-1 suppresses LDB1 degradation by XRnf12. This suppression requires both the LIM domains of Xlim-1 and the LIM interaction domain of LDB1, suggesting that LDB1, when bound to Xlim-1, escapes degradation by XRnf12. We further show that a high level of LDB1 suppresses the organizer activity of Xlim-1/LDB1, suggesting that excess LDB1 molecules disturb Xlim-1/LDB1 stoichiometry. Consistent with this, LDB1 overexpression in the dorsal marginal zone suppresses expression of several organizer genes including postulated Xlim-1 targets, and importantly, this suppression is rescued by co-expression of XRnf12. These data suggest that XRnf12 confers proper LDB1 protein levels and Xlim-1/LDB1 stoichiometry for their functions in the organizer. Together with the similarity in the expression pattern of LDB1 and XRnf12 throughout early embryogenesis, we propose Rnf12/RLIM as a specific regulator of LDB1 to ensure its proper interactions with LIM-HD proteins and possibly other LDB1-interacting proteins in the organizer as well as in other tissues.
Jacqueline M Matthews - One of the best experts on this subject based on the ideXlab platform.
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Solution structure of a tethered Lmo2LIM2/LDB1LID complex
Protein science : a publication of the Protein Society, 2012Co-Authors: Siavoush Dastmalchi, Roland Gamsjaeger, Joel P. Mackay, Ann H. Kwan, Lorna Wilkinson-white, Jacqueline M MatthewsAbstract:LIM-only protein 2, Lmo2, is a regulatory protein that is essential for hematopoietic development and inappropriate overexpression of Lmo2 in T-cells contributes to T-cell leukemia. It exerts its functions by mediating protein–protein interactions and nucleating multicomponent transcriptional complexes. Lmo2 interacts with LIM domain binding protein 1 (LDB1) through the tandem LIM domains of Lmo2 and the LIM interaction domain (LID) of LDB1. Here, we present the solution structure of the LIM2 domain of Lmo2 bound to LDB1LID. The ordered regions of LDB1 in this complex correspond well with binding hotspots previously defined by mutagenic studies. Comparisons of this Lmo2LIM2–LDB1LID structure with previously determined structures of the Lmo2/LDB1LID complexes lead to the conclusion that modular binding of tandem LIM domains in Lmo2 to tandem linear motifs in LDB1 is accompanied by several disorder-to-order transitions and/or conformational changes in both proteins.
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Solution structure of the LIM-homeodomain transcription factor complex Lhx3/LDB1 and the effects of a pituitary mutation on key Lhx3 interactions.
PloS one, 2012Co-Authors: Mugdha Bhati, Joel P. Mackay, Ann H. Kwan, Christopher Lee, Cy M. Jeffries, M.s. Gadd, Andrew E. Whitten, Jill Trewhella, Jacqueline M MatthewsAbstract:Lhx3 is a LIM-homeodomain (LIM-HD) transcription factor that regulates neural cell subtype specification and pituitary development in vertebrates, and mutations in this protein cause combined pituitary hormone deficiency syndrome (CPHDS). The recently published structures of Lhx3 in complex with each of two key protein partners, Isl1 and LDB1, provide an opportunity to understand the effect of mutations and posttranslational modifications on key protein-protein interactions. Here, we use small-angle X-ray scattering of an LDB1-Lhx3 complex to confirm that in solution the protein is well represented by our previously determined NMR structure as an ensemble of conformers each comprising two well-defined halves (each made up of LIM domain from Lhx3 and the corresponding binding motif in LDB1) with some flexibility between the two halves. NMR analysis of an Lhx3 mutant that causes CPHDS, Lhx3(Y114C), shows that the mutation does not alter the zinc-ligation properties of Lhx3, but appears to cause a structural rearrangement of the hydrophobic core of the LIM2 domain of Lhx3 that destabilises the domain and/or reduces the affinity of Lhx3 for both LDB1 and Isl1. Thus the mutation would affect the formation of Lhx3-containing transcription factor complexes, particularly in the pituitary gland where these complexes are required for the production of multiple pituitary cell types and hormones.
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solution structure of the lim homeodomain transcription factor complex lhx3 LDB1 and the effects of a pituitary mutation on key lhx3 interactions
PLOS ONE, 2012Co-Authors: Mugdha Bhati, Joel P. Mackay, Ann H. Kwan, Christopher Lee, Cy M. Jeffries, M.s. Gadd, Andrew E. Whitten, Jill Trewhella, Jacqueline M MatthewsAbstract:Lhx3 is a LIM-homeodomain (LIM-HD) transcription factor that regulates neural cell subtype specification and pituitary development in vertebrates, and mutations in this protein cause combined pituitary hormone deficiency syndrome (CPHDS). The recently published structures of Lhx3 in complex with each of two key protein partners, Isl1 and LDB1, provide an opportunity to understand the effect of mutations and posttranslational modifications on key protein-protein interactions. Here, we use small-angle X-ray scattering of an LDB1-Lhx3 complex to confirm that in solution the protein is well represented by our previously determined NMR structure as an ensemble of conformers each comprising two well-defined halves (each made up of LIM domain from Lhx3 and the corresponding binding motif in LDB1) with some flexibility between the two halves. NMR analysis of an Lhx3 mutant that causes CPHDS, Lhx3(Y114C), shows that the mutation does not alter the zinc-ligation properties of Lhx3, but appears to cause a structural rearrangement of the hydrophobic core of the LIM2 domain of Lhx3 that destabilises the domain and/or reduces the affinity of Lhx3 for both LDB1 and Isl1. Thus the mutation would affect the formation of Lhx3-containing transcription factor complexes, particularly in the pituitary gland where these complexes are required for the production of multiple pituitary cell types and hormones.
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structural basis of simultaneous recruitment of the transcriptional regulators lmo2 and fog1 zfpm1 by the transcription factor gata1
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Lorna Wilkinsonwhite, Roland Gamsjaeger, Beeke Wienert, Philippa H Stokes, Merlin Crossley, Joel P. Mackay, Siavoush Dastmalchi, Jacqueline M MatthewsAbstract:The control of red blood cell and megakaryocyte development by the regulatory protein GATA1 is a paradigm for transcriptional regulation of gene expression in cell lineage differentiation and maturation. Most GATA1-regulated events require GATA1 to bind FOG1, and essentially all GATA1-activated genes are cooccupied by a TAL1/E2A/LMO2/LDB1 complex; however, it is not known whether FOG1 and TAL1/E2A/LMO2/LDB1 are simultaneously recruited by GATA1. Our structural data reveal that the FOG1-binding domain of GATA1, the N finger, can also directly contact LMO2 and show that, despite the small size (< 50 residues) of the GATA1 N finger, both FOG1 and LMO2 can simultaneously bind this domain. LMO2 in turn can simultaneously contact both GATA1 and the DNA-binding protein TAL1/E2A at bipartite E-box/WGATAR sites. Taken together, our data provide the first structural snapshot of multiprotein complex formation at GATA1-dependent genes and support a model in which FOG1 and TAL1/E2A/LMO2/LDB1 can cooccupy E-box/WGATAR sites to facilitate GATA1-mediated activation of gene activation.
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Tandem LIM domains provide synergistic binding in the LMO4:LDB1 complex.
The EMBO journal, 2004Co-Authors: Janet E Deane, Jane E Visvader, Daniel P Ryan, Margaret Sunde, J. Mitchell Guss, Megan J. Maher, Jacqueline M MatthewsAbstract:Nuclear LIM-only (LMO) and LIM-homeodomain (LIM-HD) proteins have important roles in cell fate determination, organ development and oncogenesis. These proteins contain tandemly arrayed LIM domains that bind the LIM interaction domain (LID) of the nuclear adaptor protein LIM domain-binding protein-1 (LDB1). We have determined a high-resolution X-ray crystal structure of LMO4, a putative breast oncoprotein, in complex with LDB1-LID, providing the first example of a tandem LIM:LDB1-LID complex and the first structure of a type-B LIM domain. The complex possesses a highly modular structure with LDB1-LID binding in an extended manner across both LIM domains of LMO4. The interface contains extensive hydrophobic and electrostatic interactions and multiple backbone–backbone hydrogen bonds. A mutagenic screen of LDB1-LID, assessed by yeast two-hybrid and competition ELISA analysis, identified key features at the interface and revealed that the interaction is tolerant to mutation. These combined properties provide a mechanism for the binding of LDB1 to numerous LMO and LIM-HD proteins. Furthermore, the modular extended interface may form a general mode of binding to tandem LIM domains.
Ann Dean - One of the best experts on this subject based on the ideXlab platform.
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Crystal structure of human LDB1 in complex with SSBP2.
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Hongyang Wang, Juhyun Kim, Zhizhi Wang, Xiao-xue Yan, Ann DeanAbstract:The Lim domain binding proteins (LDB1 and LDB2 in human and Chip in Drosophila) play critical roles in cell fate decisions through partnership with multiple Lim-homeobox and Lim-only proteins in diverse developmental systems including cardiogenesis, neurogenesis, and hematopoiesis. In mammalian erythroid cells, LDB1 dimerization supports long-range connections between enhancers and genes involved in erythropoiesis, including the β-globin genes. Single-stranded DNA binding proteins (SSBPs) interact specifically with the LDB/Chip conserved domain (LCCD) of LDB proteins and stabilize LDBs by preventing their proteasomal degradation, thus promoting their functions in gene regulation. The structural basis for LDB1 self-interaction and interface with SSBPs is unclear. Here we report a crystal structure of the human LDB1/SSBP2 complex at 2.8-A resolution. The LDB1 dimerization domain (DD) contains an N-terminal nuclear transport factor 2 (NTF2)-like subdomain and a small helix 4–helix 5 subdomain, which together form the LDB1 dimerization interface. The 2 LCCDs in the symmetric LDB1 dimer flank the core DDs, with each LCCD forming extensive interactions with an SSBP2 dimer. The conserved linker between LDB1 DD and LCCD covers a potential ligand-binding pocket of the LDB1 NTF2-like subdomain and may serve as a regulatory site for LDB1 structure and function. Our structural and biochemical data provide a much-anticipated structural basis for understanding how LDB1 and the LDB1/SSBP interactions form the structural core of diverse complexes mediating cell choice decisions and long-range enhancer–promoter interactions.
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Enhancer long-range contacts: The multi-adaptor protein LDB1 is the tie that binds.
Biochimica et biophysica acta. Gene regulatory mechanisms, 2019Co-Authors: Guoyou Liu, Ann DeanAbstract:Abstract The eukaryotic genome is organized at varying levels into chromosome territories, transcriptional compartments and topologically associating domains (TADs), which are architectural features largely shared between different cell types and across species. In contrast, within TADs, chromatin loops connect enhancers and their target genes to establish unique transcriptomes that distinguish cells and tissues from each other and underlie development and differentiation. How these tissue-specific and temporal stage-specific long-range contacts are formed and maintained is a fundamental question in biology. The widely expressed L im d omain b inding 1 protein, LDB1, plays a critical role in connecting enhancers and genes by forming complexes with cell-type specificity across diverse developmental pathways including neurogenesis, cardiogenesis, retinogenesis and hematopoiesis. Here we review the multiple roles of LDB1 in cell fate determination and in chromatin loop formation, with an emphasis on mammalian systems, to illuminate how LDB1 functions in normal cells and in diseases such as cancer.
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the LDB1 complex co opts ctcf for erythroid lineage specific long range enhancer interactions
Cell Reports, 2017Co-Authors: Jongjoo Lee, Ryan K. Dale, Ivan Krivega, Ann DeanAbstract:Lineage-specific transcription factors are critical for long-range enhancer interactions, but direct or indirect contributions of architectural proteins such as CCCTC-binding factor (CTCF) to enhancer function remain less clear. The LDB1 complex mediates enhancer-gene interactions at the β-globin locus through LDB1 self-interaction. We find that an LDB1-bound enhancer upstream of carbonic anhydrase 2 (Car2) activates its expression by interacting directly with CTCF at the gene promoter. Both LDB1 and CTCF are required for enhancer-Car2 looping, and the domain of LDB1 contacted by CTCF is necessary to rescue Car2 transcription in LDB1-deficient cells. Genome-wide studies and CRISPR/Cas9 genome editing indicate that LDB1-CTCF enhancer looping underlies activation of a substantial fraction of erythroid genes. Our results provide a mechanism by which long-range interactions of architectural protein CTCF can be tailored to achieve a tissue-restricted pattern of chromatin loops and gene expression.
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LDB1-mediated enhancer looping can be established independent of mediator and cohesin
Nucleic acids research, 2017Co-Authors: Ivan Krivega, Ann DeanAbstract:Mechanistic studies in erythroid cells indicate that LDB1, as part of a GATA1/TAL1/LMO2 complex, brings erythroid-expressed genes into proximity with enhancers for transcription activation. The role of co-activators in establishing this long-range interaction is poorly understood. Here we tested the contributions of the RNA Pol II pre-initiation complex (PIC), mediator and cohesin to establishment of locus control region (LCR)/β-globin proximity. CRISPR/Cas9 editing of the β-globin promoter to eliminate the RNA Pol II PIC by deleting the TATA-box resulted in loss of transcription, but enhancer-promoter interaction was unaffected. Additional deletion of the promoter GATA1 site eliminated LDB1 complex and mediator occupancy and resulted in loss of LCR/β-globin proximity. To separate the roles of LDB1 and mediator in LCR looping, we expressed a looping-competent but transcription-activation deficient form of LDB1 in LDB1 knock down cells: LCR/β-globin proximity was restored without mediator core occupancy. Further, Cas9-directed tethering of mutant LDB1 to the β-globin promoter forced LCR loop formation in the absence of mediator or cohesin occupancy. Moreover, ENCODE data and our chromatin immunoprecipitation results indicate that cohesin is almost completely absent from validated and predicted LDB1-regulated erythroid enhancer-gene pairs. Thus, lineage specific factors largely mediate enhancer-promoter looping in erythroid cells independent of mediator and cohesin.
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the LDB1 complex co opts ctcf for erythroid lineage specific long range enhancer interactions
bioRxiv, 2017Co-Authors: Jongjoo Lee, Ryan K. Dale, Ivan Krivega, Ann DeanAbstract:Lineage-specific transcription factors are critical for long-range enhancer interactions but direct or indirect contributions of architectural proteins such as CTCF to enhancer function remain less clear. The LDB1 complex mediates enhancer-gene interactions at the β-globin locus through LDB1 self-interaction. We find that a novel LDB1-bound enhancer upstream of carbonic anhydrase 2 (Car2) activates its expression by interacting directly with CTCF at the gene promoter. Both LDB1 and CTCF are required for enhancer-Car2 looping and the domain of LDB1 contacted by CTCF is necessary to rescue Car2 transcription in LDB1 deficient cells. Genome wide studies and CRISPR/Cas9 genome editing indicate that LDB1-CTCF enhancer looping underlies activation of a substantial fraction of erythroid genes. Our results provide a mechanism by which long-range interactions of architectural protein CTCF can be tailored to achieve a tissue-restricted pattern of chromatin loops and gene expression.