Controlling Long-Range Genomic Interactions at a Native Locus by Targeted Tethering of a Looping Factor

“The use of chromosome conformation capture (3C) and its derivatives has revealed that distant chromosomal elements can be juxtaposed to form chromatin loops, thus providing one mechanism of long-range enhancer function (Cullen et al., 1993; Dekker et al., 2002).”

“…arose from studies of the mammalian b-globin locus, which is among the first gene clusters at which long-range chromosomal interactions between a powerful distal enhancer, the locus control region (LCR), and the target b-globin promoters were described (Carter et al., 2002; Tolhuis et al., 2002).”

“GATA1 is a DNA-binding protein essential for normal erythroid differentiation and b-globin gene expression (Evans and Felsenfeld, 1989; Pevny et al., 1991; Tsai et al., 1989).”

“GATA1 is a DNA-binding protein essential for normal erythroid differentiation and b-globin gene expression (Evans and Felsenfeld, 1989; Pevny et al., 1991; Tsai et al., 1989).”

“ZFs have previously been linked to activation domains to successfully activate gene expression (Klug, 2010).”

“Fundamental insights into these issues arose from studies of the mammalian b-globin locus, which is among the first gene clusters at which long-range chromosomal interactions between a powerful distal enhancer, the locus control region (LCR), and the target b-globin promoters were described (Carter et al., 2002; Tolhu…”

“However, physical interactions among all of these proteins have been reported (Cantor and Orkin, 2002), making it difficult to distinguish whether they function in linear or parallel pathways.”

“GATA1 is a DNA-binding protein essential for normal erythroid differentiation and b-globin gene expression (Evans and Felsenfeld, 1989; Pevny et al., 1991; Tsai et al., 1989).”

“Using HS2 as the anchor region, we found that, in parental G1E cells, the 3C signals generally declined with increasing distance (Figure 3A), which is consistent with our previous observations (Vakoc et al., 2005).”

“Although looping can occur at genes prior to their full activation, the onset of transcription is tightly associated with additional looped interactions (Palstra et al., 2003; Spilianakis and Flavell, 2004; Vernimmen et al., 2007).”

“The R1 (Ter119- and CD71-/low) populations were isolated, infected with desired retrovirus, and cultured for 24 hr in proliferation medium containing Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 15% fetal bovine serum, 1% penicillin-streptomycin, 1% glutamine, 10 ng/ml mIL3, 20 ng/ml m/h IL6, 50 ng/ml…”

“Indeed, a widely used and evolutionarily conserved looping function for Ldb1 is suggested by studies in diverse organisms and cell lineages (Matthews and Visvader, 2003; Morcillo et al., 1997; Thaler et al., 2002).”

“, 2005), KLF1 (also known as EKLF) (Drissen et al., 2004), and the more broadly expressed protein Ldb1 (Song et al.”

“Cell Culture G1E andG1E-ER4 cells were cultured as described (Weiss et al., 1997).”

“…the gene were impaired in the absence of GATA1, suggesting GATA1 impacts on P-TEFb regulation at multiple levels, perhaps via direct interaction (Bottardi et al., 2011; Elagib et al., 2008) or indirectly via proteins of the bromodomain and extra terminal domain (BET) family (Lamonica et al., 2011).”

“Chromatin looping has been discovered at numerous gene loci and reflects a widespread organizing principle of the chromatin fiber (for review, see Dean, 2011; Kadauke and Blobel, 2009; Miele and Dekker, 2008; Schoenfelder et al., 2010; Sexton et al., 2009).”

“GATA1 and Ldb1 display a highly overlapping genomic occupancy pattern, but notably, Ldb1 association strongly favors sites at which GATA1 functions as a transcriptional activator, such as the b-globin locus (Cheng et al., 2009; Kassouf et al., 2010; Soler et al., 2010; Tripic et al., 2009; Wu et al., 2011).”

“Third, like GATA1, Ldb1 cooccupies the b-globin promoter and LCR and might therefore function by physically linking the two (Song et al., 2007; Tripic et al., 2009).”

“However, based on studies using pharmacological inhibitors of transcription elongation, it has become clear that ongoing transcription is dispensable for sustaining preformed chromatin loops (Mitchell and Fraser, 2008; Palstra et al., 2008).”

“The strong induction of b-globin transcription by ZF-Ldb1 or ZFSA implicates an involvement of the LCR and, hence, chromatin looping, because in the absence of the LCR, b-globin transcription is very low (Bender et al., 2000).”

“GATA1 and Ldb1 display a highly overlapping genomic occupancy pattern, but notably, Ldb1 association strongly favors sites at which GATA1 functions as a transcriptional activator, such as the b-globin locus (Cheng et al., 2009; Kassouf et al., 2010; Soler et al., 2010; Tripic et al., 2009; Wu et al., 2011).”

“On the other hand, our observation that ZF-Ldb1 proteins completely rescued chromatin looping but only partially restored transcription elongation agrees with the notion that full transcription is not required for loop formation (Jing et al., 2008; Mitchell and Fraser, 2008; Palstra et al., 2008).”

“Indeed, a widely used and evolutionarily conserved looping function for Ldb1 is suggested by studies in diverse organisms and cell lineages (Matthews and Visvader, 2003; Morcillo et al., 1997; Thaler et al., 2002).”

“, 2008), and placing an enhancer and promoter on separate loops can isolate the enhancer to render it inactive (Ameres et al., 2005; Hou et al., 2008).”

“The use of chromosome conformation capture (3C) and its derivatives has revealed that distant chromosomal elements can be juxtaposed to form chromatin loops, thus providing one mechanism of long-range enhancer function (Cullen et al., 1993; Dekker et al., 2002).”

“Chromatin looping has been discovered at numerous gene loci and reflects a widespread organizing principle of the chromatin fiber (for review, see Dean, 2011; Kadauke and Blobel, 2009; Miele and Dekker, 2008; Schoenfelder et al., 2010; Sexton et al., 2009).”

“, 2007), as well as studies in eukaryotic cells using plasmid constructs, have succeeded in influencing gene expression through forced looping among regulatory elements (Ameres et al., 2005; Mahmoudi et al., 2002; Nolis et al., 2009; Petrascheck et al., 2005).”

“…Ldb1 display a highly overlapping genomic occupancy pattern, but notably, Ldb1 association strongly favors sites at which GATA1 functions as a transcriptional activator, such as the b-globin locus (Cheng et al., 2009; Kassouf et al., 2010; Soler et al., 2010; Tripic et al., 2009; Wu et al., 2011).”

“Third, like GATA1, Ldb1 cooccupies the b-globin promoter and LCR and might therefore function by physically linking the two (Song et al., 2007; Tripic et al., 2009).”

“Fourth, Ldb1 can form homodimers and even higher-order oligomers (Cross et al., 2010; Jurata and Gill, 1997), which might underlie its role in loop formation.”

“Indeed, a widely used and evolutionarily conserved looping function for Ldb1 is suggested by studies in diverse organisms and cell lineages (Matthews and Visvader, 2003; Morcillo et al., 1997; Thaler et al., 2002).”

“phosphorylation at serine 5 of its C-terminal domain, a modification associated with early transcription elongation (Sawado et al., 2003).”

“Chromatin looping has been discovered at numerous gene loci and reflects a widespread organizing principle of the chromatin fiber (for review, see Dean, 2011; Kadauke and Blobel, 2009; Miele and Dekker, 2008; Schoenfelder et al., 2010; Sexton et al., 2009).”

“, 2007), as well as studies in eukaryotic cells using plasmid constructs, have succeeded in influencing gene expression through forced looping among regulatory elements (Ameres et al., 2005; Mahmoudi et al., 2002; Nolis et al., 2009; Petrascheck et al., 2005).”

“However, based on studies using pharmacological inhibitors of transcription elongation, it has become clear that ongoing transcription is dispensable for sustaining preformed chromatin loops (Mitchell and Fraser, 2008; Palstra et al., 2008).”

“Is Sufficient for b-Globin Activation Ldb1 contains an N-terminal self-association (SA) domain that mediates the assembly of higher-order molecular complexes and might account for its looping function (Cross et al., 2010; Xu et al., 2003).”

“Chromatin looping has been discovered at numerous gene loci and reflects a widespread organizing principle of the chromatin fiber (for review, see Dean, 2011; Kadauke and Blobel, 2009; Miele and Dekker, 2008; Schoenfelder et al., 2010; Sexton et al., 2009).”

“GATA1 and Ldb1 display a highly overlapping genomic occupancy pattern, but notably, Ldb1 association strongly favors sites at which GATA1 functions as a transcriptional activator, such as the b-globin locus (Cheng et al., 2009; Kassouf et al., 2010; Soler et al., 2010; Tripic et al., 2009; Wu et al., 2011).”

“Prior studies in prokaryotes (for review, see Marenduzzo et al., 2007), as well as studies in eukaryotic cells using plasmid constructs, have succeeded in influencing gene expression through forced looping among regulatory elements (Ameres et al., 2005; Mahmoudi et al., 2002; Nolis et al., 2009;…”

“, 2007), as well as studies in eukaryotic cells using plasmid constructs, have succeeded in influencing gene expression through forced looping among regulatory elements (Ameres et al., 2005; Mahmoudi et al., 2002; Nolis et al., 2009; Petrascheck et al., 2005).”

“Chromatin looping has been discovered at numerous gene loci and reflects a widespread organizing principle of the chromatin fiber (for review, see Dean, 2011; Kadauke and Blobel, 2009; Miele and Dekker, 2008; Schoenfelder et al., 2010; Sexton et al., 2009).”

“In particular, forced association between the LCR and the b-globin gene sufficed to exert two functions ascribed to the LCR, the formation of a preinitiation complex at the promoter and the generation of early elongating Pol II, as reflected in Ser5ph (Sawado et al., 2003; Song et al., 2010).”

“Binding of the ZFs to their designated DNA sequences was characterized by using a previously described ELISA-based assay (data not shown) (Bartsevich et al., 2003).”

“The failure to fully restore transcription elongation can be explained by the lack of GATA1 and its cofactors that exert additional looping-independent functions, possibly including the recruitment and activation of P-TEFb complex (Bottardi et al., 2011; Elagib et al., 2008; see Discussion).”

“, 2008), and placing an enhancer and promoter on separate loops can isolate the enhancer to render it inactive (Ameres et al., 2005; Hou et al., 2008).”

“Fourth, Ldb1 can form homodimers and even higher-order oligomers (Cross et al., 2010; Jurata and Gill, 1997), which might underlie its role in loop formation.”

“The failure to fully restore transcription elongation can be explained by the lack of GATA1 and its cofactors that exert additional looping-independent functions, possibly including the recruitment and activation of P-TEFb complex (Bottardi et al., 2011; Elagib et al., 2008; see Discussion).”

“Further, the looping interaction directly stimulates transcription by facilitating recruitment and phosphorylation of RNA polymerase II 39 .”

“Nonetheless, most recent study of eukaryotic enhancer-promoter interactions has focused on cofactors that lack DNA binding capabilities and bridge enhancer-bound transcription factors and promoter-bound transcription apparatus (Allen and Taatjes, 2015; Deng et al., 2012; Jeronimo et al., 2016; Kagey et al., 2010; Mali…”