One of the models of long range gene activation hypothesizes that facilitator proteins might function to link enhancers to distant genes. We found that Ldb1/NLI, a ubiquitously expressed nuclear factor meets the criteria for an enhancer facilitator and is required for long range gene activation and chromatin looping in the beta-globin locus. A complex of proteins including Ldb1, NLI, GATA-1, SCL and Lmo2 binds in vivo to the beta-globin LCR in erythroid cells and after induction of transcription the NLI complex is detected at the gene promoter. Using mice with an endogenous deletion of the LCR we have recently determined that the Ldb1 conplex binds independently to the LCR and beta-promoter. We are currently testing the hypothesis that the dimerization domain of Ldb1 is required for the long range interactions. ? ? In erythroid cells Ldb1/NLI binds directly to and stabilizes Lmo2 an erythroid specific member of the LIM only protein family: in different cells or tissues Ldb1/NLI might interact with a different LIM only or LIM-HD proteins to acquire regulatory functions through different DNA binding partners. To investigate the repertoire of Ldb1/NLI functions in long range gene activation, we are taking a two pronged approach. First, we are using chromatography, co-IP and mass spectrometry to identify new partners of Ldb1/NLI. We have established stable cell lines expressing HA-tagged NLI. Second, we have performed chromatin immunoprecipitation-on-chip analysis using NimbleGen tiled microarrays representing sequences of about 2% of the human genome. We found that about 50% of the NLI sites were co-occupied by GATA-1. These data provide both GATA-1 mediated and non-GATA-1 mediated sites of NLI interaction with chromatin that we will validate by ChIP and analyze with respect to novel NLI partners at those sites. A third approach has employed expression profiling on microarrays in cells in normal and Ldb1/NLI knock down cells. Taken together we anticipate that these investigations will expand understanding of the general role of Ldb1/NLI in long range gene activation.? ? Chromatin insulators are thought to interrupt long range enhancer-gene interactions but the mechanism underlying enhancer blocking is unclear. We explored enhancer blocking by human beta-globin HS5, the orthologue of the CTCF dependent chicken HS4 insulator. An extra copy of HS5 placed between the beta-globin LCR and downstream genes on a transgene fulfilled the classic predictions for an enhancer blocker. Ectopic HS5 did not perturb the LCR but blocked gene activation by interfering with RNA pol II, activator and co-activator recruitment and epigenetic modification at the downstream beta-globin gene. Underlying these effects, ectopic HS5 disrupted chromatin loop formation between beta-globin and the LCR and instead formed a new loop with endogenous HS5 that topologically isolated the LCR. Both enhancer blocking and insulator loop formation depend on an intact CTCF site in ectopic HS5 and were sensitive to knock down of CTCF protein by siRNA. Thus, intrinsic looping activity of CTCF sites can nullify LCR function.
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