Reactivation of Developmentally Silenced Globin Genes by Forced Chromatin Looping

Abstract

Summary Distal enhancers commonly contact target promoters via chromatin looping. In erythroid cells, the locus control region (LCR) contacts β-type globin genes in a developmental stage-specific manner to stimulate transcription. Previously, we induced LCR-promoter looping by tethering the self-association domain (SA) of Ldb1 to the β-globin promoter via artificial zinc fingers. Here, we show that targeting the SA to a developmentally silenced embryonic globin gene in adult murine erythroblasts triggered its transcriptional reactivation. This activity depended on the LCR, consistent with an LCR-promoter looping mechanism. Strikingly, targeting SA to the fetal γ-globin promoter in primary adult human erythroblasts increased γ-globin promoter-LCR contacts, stimulating transcription to approximately 85% of total β-globin synthesis with a reciprocal reduction in adult β-globin expression. Our findings demonstrate that forced chromatin looping can override a stringent developmental gene expression program and suggest a novel approach to control the balance of globin gene transcription for therapeutic applications.

Enhancer-bound LDB1 regulates a corticotrope promoter-pausing repression program

“…Because LDB1 also has been reported to interact with other transcription factors, it is tempting to suggest a potentially general role for LDB1 in regulating cell typespecific, enhancer-dependent gene-expression programs. LDB1-mediated promoter:enhancer looping has been reported in the β-globin locus based on the homodimerization of LDB1 occupying both enhancer and promoter sites (22)(23)(24). Our data show that, in corticotropes, LDB1 can mediate looping to promoters without detectable promoter LDB1 binding.…”

“…LDB1 was postulated to play an important role in promoter:enhancer looping events and as a coactivator in several systems (21)(22)(23)(24)(33)(34)(35). To investigate its roles in gene transcription and in promoter:enhancer looping events in the POMC lineage, we performed GRO-seq after Ldb1 knockdown and observed that, of 3,378 genes down-regulated by siLdb1, 927 transcription units are located in direct proximity to the LDB1-occupied enhancers.…”

“…Lim domain-binding (LDB)/nuclear LIM interactor (NLI)/ cofactor of LIM homeodomain protein (CLIM), the mammalian homolog of CHIP, has been demonstrated to be a critical factor for multiple developmental systems and to be a component of LIM homeodomain codes that determine neuronal subtype specification in the spinal cord (3,20,21). Recently, using an engineered transcription unit as a model, LIM domain-binding protein 1 (LDB1) has been reported to be capable of mediating promoter:enhancer looping through LDB1 homodimerization (22)(23)(24).…”

-thalassemias: paradigmatic diseases for scientific discoveries and development of innovative therapies

“…27 In addition, the sequential looping of the LCR is also responsible for the switch between embryonic, fetal (HbF) and HbA ( Figure 2A). [31][32][33][34] The pattern of expression of the b-globin gene has also been a subject of intense investigations since the switching between HbF and HbA represents an important biological phenomenon and an exemplary model to understand how gene expression is regulated during development. In humans, the switching between the expression of g-globin and b-globin gene occurs in the first three months after birth ( Figure 1B).…”

“…[61][62][63][64][65] In particular, the important role of Ldb1 in globin gene regulation has been emphasized by the observation that this protein is able to reactivate the silenced mouse embryonic globin and the human g-globin genes when fused to an artificial zinc finger tethering Ldb1 onto their promoters ( Figure 2B). 32,34 It has been shown that this artificial zinc finger-Ldb1 fusion protein is able to force the LCR-holocomplex to loop onto the promoter recognized by the zinc finger moiety. 32,34 This repositioning of the LCR is sufficient to re-activate the expression of otherwise silenced globin genes.…”