The eukaryotic genome is non-randomly organized in the interphase nucleus. Critical control elements can physically contact each other to form chromatin loops. Looped configurations of the chromatin fiber have been described at numerous gene loci, however, the mechanism by which these structures are established and their functional relationship with gene expression have remained unclear. The transcription co-factor Ldb1 is critical for establishing looped chromatin interactions at the murine ?-globin locus. Specifically, we showed that tethering Ldb1 to the locus via artificial zinc finger proteins in immature erythroid cells is sufficient to promote a looped interaction between the ?-globin enhancer and promoter and potently activate transcription. This suggested for the first time that chromatin looping causally underlies gene expression and raised the possibility that forced chromatin looping might be employed to effectively manipulate gene expression. This proposal builds on these findings by investigating in Specific Aim 1 the mechanisms of Ldb1 function and its broad role in genome organization.
In Specific Aim 2 we will further develop the approach of forced chromatin looping to enhance and broaden its usefulness.
In Specific Aim 3 we will examine forced chromatin looping as an approach to developmentally reprogram the murine and human ?-globin locus.
In Specific Aim 4 proof-of-concept studies will examine whether reactivation of fetal hemoglobin via chromatin looping can ameliorate sickle cell anemia in a humanized mouse model. To our knowledge, the manipulation of higher order chromatin structure for the purpose of regulating gene expression is unique and novel in its design. The juxtaposition of complex regulatory elements promises more dramatic changes in gene activation when compared to conventional approaches, and might also be exploited to repress gene transcription for exploratory or therapeutic purposes.
This application seeks to explore the function of higher order chromatin folding in gene regulation. We combine basic research into the mechanisms of higher scale chromatin organization with approaches to efficiently manipulate chromatin loops. We will carry out proof- of-principle experiments that explore whether altering the folding of the chromatin fiber can be exploited for therapeutic purposes using sickle cell anemia as a model disease.
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|Grevet, Jeremy D; Lan, Xianjiang; Hamagami, Nicole et al. (2018) Domain-focused CRISPR screen identifies HRI as a fetal hemoglobin regulator in human erythroid cells. Science 361:285-290|
|Breda, Laura; Motta, Irene; Lourenco, Silvia et al. (2016) Forced chromatin looping raises fetal hemoglobin in adult sickle cells to higher levels than pharmacologic inducers. Blood 128:1139-43|
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|Traxler, Elizabeth A; Yao, Yu; Wang, Yong-Dong et al. (2016) A genome-editing strategy to treat ?-hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition. Nat Med 22:987-90|
|Hsiung, Chris C-S; Bartman, Caroline R; Huang, Peng et al. (2016) A hyperactive transcriptional state marks genome reactivation at the mitosis-G1 transition. Genes Dev 30:1423-39|
|Paralkar, Vikram R; Taborda, Cristian C; Huang, Peng et al. (2016) Unlinking an lncRNA from Its Associated cis Element. Mol Cell 62:104-10|
|Deng, Wulan; Blobel, Gerd A (2014) Manipulating nuclear architecture. Curr Opin Genet Dev 25:1-7|
|Deng, Wulan; Rupon, Jeremy W; Krivega, Ivan et al. (2014) Reactivation of developmentally silenced globin genes by forced chromatin looping. Cell 158:849-860|
|Deng, Wulan; Lee, Jongjoo; Wang, Hongxin et al. (2012) Controlling long-range genomic interactions at a native locus by targeted tethering of a looping factor. Cell 149:1233-44|
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