The Ldb1/NLI complex, including GATA-1 and TAL1, mediates long range interaction between the beta-globin locus control region (LCR) and gene in adult mouse erythroid cells, but whether this complex mediates chromatin interactions at other developmental stages or in human cells is unknown. Fetal gamma-globin genes can be robustly re-activated in adult human erythroid cells by cytokines. We investigated activator, repressor and co-repressor occupancy and chromatin conformation of the beta-globin locus in these cells when gamma-globin is either repressed or re-activated. In cells transcribing primarily beta-globin, the gamma-globin repressor BCL11A occupies a site downstream of the A-gamma globin gene, which is within sequences of BGL3, an intergenic RNA transcript. The Ldb1/NLI complex also occupies BGL3 sequences as well as the LCR together with the co-repressor ETO2. In these conditions, long range interactions between the LCR and the beta-globin gene are observed. In contrast, when gamma-globin is re-activated, BCL11A occupancy at BGL3 is lost and both BGL3 and gamma-globin are transcribed. ETO2 no longer participates in the Ldb1/NLI complex and proximity between the gamma-globin/BGL3 region and LCR is established. These results implicate diverse Ldb1/NLI complexes in mediating gamma-globin transcription or silencing through long range LCR interactions involving an intergenic site of non-coding RNA transcription. Furthermore, ETO2 joins gamma-globin repressor BCL11A as a therapeutic target to ameliorate Sickle Cell Disease and beta-thalassemia. How individual globin genes establish stage specific enhancer communication is unknown and most studies have been performed in a non-chromosomal environment. We are using homologous recombination in mouse ES cells to address this issue. We targeted a region upstream of the mouse embryonic epsilon y gene and a second region upstream of beta-major in ES cells and then used recombinase mediated cassette exchange to insert the chicken HS4 insulator in these two positions which are (1) between the LCR and the globin genes and (2) between the embryonic and adult genes. We differentiated ES cells with erythropoietin along erythroid lines and then monitored globin gene expression. The cHS4 insertion between the LCR and downstream genes reduced transcription of the embryonic ey, beta-h1 and beta major genes and resulted in non-genic transcript accumulation in the insulator. Currently, we are investigating localization of RNA polymerase II and other factors such as GATA1 and Ldb1 from the LCR to 3HS1 using chromatin immunoprecipitation to understand the molecular mechanism underlying insulation. In addition, we plan to monitor globin gene transcription of ES cells that have cHS4 between the embryonic globin genes and the beta major gene. Furthermore, we are using blastocyst injection of our ES cell clones to mouse lines with insertions of cHS4 at these two positions in the beta-globin locus. These experiments are novel since they alter transcription factor recruitment and chromatin organization in a normal chromosomal setting and will provide information on how the LCR and globin genes communicate in vivo during development and during globin gene switches from embryonic to adult types.