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Required enhancer-matrin-3 network interactions for a homeodomain transcription program.

Skowronska-Krawczyk D, Ma Q, Schwartz M, Scully K, Li W, Liu Z, Taylor H, Tollkuhn J, Ohgi KA, Notani D, Kohwi Y, Kohwi-Shigematsu T, Rosenfeld MG - Nature (2014)

Bottom Line: Here investigation of a developmentally required POU-homeodomain transcription factor, Pit1 (also known as Pou1f1), has revealed that, unexpectedly, binding of Pit1-occupied enhancers to a nuclear matrin-3-rich network/architecture is a key event in effective activation of the Pit1-regulated enhancer/coding gene transcriptional program.The matrin-3 network-tethered R271W Pit1 mutant, but not the untethered protein, restores Pit1-dependent activation of the enhancers and recruitment of co-activators, exemplified by p300, causing both enhancer RNA transcription and target gene activation.These studies have thus revealed an unanticipated homeodomain factor/β-catenin/Satb1-dependent localization of target gene regulatory enhancer regions to a subnuclear architectural structure that serves as an underlying mechanism by which an enhancer-bound homeodomain factor effectively activates developmental gene transcriptional programs.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
Homeodomain proteins, described 30 years ago, exert essential roles in development as regulators of target gene expression; however, the molecular mechanisms underlying transcriptional activity of homeodomain factors remain poorly understood. Here investigation of a developmentally required POU-homeodomain transcription factor, Pit1 (also known as Pou1f1), has revealed that, unexpectedly, binding of Pit1-occupied enhancers to a nuclear matrin-3-rich network/architecture is a key event in effective activation of the Pit1-regulated enhancer/coding gene transcriptional program. Pit1 association with Satb1 (ref. 8) and β-catenin is required for this tethering event. A naturally occurring, dominant negative, point mutation in human PIT1(R271W), causing combined pituitary hormone deficiency, results in loss of Pit1 association with β-catenin and Satb1 and therefore the matrin-3-rich network, blocking Pit1-dependent enhancer/coding target gene activation. This defective activation can be rescued by artificial tethering of the mutant R271W Pit1 protein to the matrin-3 network, bypassing the pre-requisite association with β-catenin and Satb1 otherwise required. The matrin-3 network-tethered R271W Pit1 mutant, but not the untethered protein, restores Pit1-dependent activation of the enhancers and recruitment of co-activators, exemplified by p300, causing both enhancer RNA transcription and target gene activation. These studies have thus revealed an unanticipated homeodomain factor/β-catenin/Satb1-dependent localization of target gene regulatory enhancer regions to a subnuclear architectural structure that serves as an underlying mechanism by which an enhancer-bound homeodomain factor effectively activates developmental gene transcriptional programs.

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β-catenin and SATB1 influence transcription of genes regulated by Pit1 enhancersa, Genes containing Pit1 enhancers (991) are significantly downregulated upon knockdown of β-catenin and Satb1. RPKM – reads per kilobase per million b, GRO-seq tag distribution on both strands of Pit1 bound enhancers before and after treatment of GC cells with β-catenin and Satb1 siRNAs. c, ChIP-qPCR analysis of Satb1 association with Pit1 enhancers upon either Pit1 or β-catenin knockdown. Experiments were repeated 3 times, and p-values calculated using student’s two tailed t-test. (+/− SD; *p<0.05, **p<0.01, ***p<0.001) d, ChIP-qPCR analysis of matrin-3 association with Pit1 enhancers upon Pit1 knockdown. Experiments repeated 2–4 times, p-values calculated using student’s two tailed t-test. (+/− SD; *p<0.05, **p<0.01, ***p<0.001)
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Figure 3: β-catenin and SATB1 influence transcription of genes regulated by Pit1 enhancersa, Genes containing Pit1 enhancers (991) are significantly downregulated upon knockdown of β-catenin and Satb1. RPKM – reads per kilobase per million b, GRO-seq tag distribution on both strands of Pit1 bound enhancers before and after treatment of GC cells with β-catenin and Satb1 siRNAs. c, ChIP-qPCR analysis of Satb1 association with Pit1 enhancers upon either Pit1 or β-catenin knockdown. Experiments were repeated 3 times, and p-values calculated using student’s two tailed t-test. (+/− SD; *p<0.05, **p<0.01, ***p<0.001) d, ChIP-qPCR analysis of matrin-3 association with Pit1 enhancers upon Pit1 knockdown. Experiments repeated 2–4 times, p-values calculated using student’s two tailed t-test. (+/− SD; *p<0.05, **p<0.01, ***p<0.001)

Mentions: Global run-on (GRO-seq) analysis using GC cells before and after simultaneous knockdown of both β-catenin and Satb1 revealed that in total, 1350 coding gene nascent transcripts were downregulated and 916 genes were upregulated under these double-knockdown conditions (Fig. S3a). Out of the 1350 genes positively regulated by both β-catenin and SATB1 (Supplementary Table 8), 991 had Pit1 bound enhancer elements within 200kbp of their start sites (Fig. S3a), and all 991 were significantly downregulated (Fig. 3a). Consistent with their functional importance22 the expression of the eRNAs associated with these 991 Pit1-bound enhancers was significantly affected in the simultaneous knockdown of β-catenin and Satb1 (Fig. 3b). ChIP-seq analysis revealed that Satb1 and β-catenin proteins were located at the center of Pit1-positive enhancers (Fig. S3b–d), strongly supporting the co-binding model of these three proteins. Based on this analysis, we selected several highly down-regulated genes that contain Pit1 enhancers (Fig. S3c) and observed that knockdown of either of the two proteins significantly diminished target gene expression (Fig. S3g). Additionally, knockdown of either Pit1 or β-catenin affected Satb1 association with enhancer elements, as assessed by ChIP-qPCR (Fig. 3c, Fig. S3e). Association of matrin-3 with Pit-1 dependent enhancers was significantly reduced when either β-catenin or SATB1 were downregulated (Fig. S3h). Finally, we confirmed that lack of Pit1 protein inhibited association of selected Pit1-dependent enhancers with matrin-3 (Fig. 3d, Fig. S3f). Together, these data suggest that functional interaction between Pit1-dependent regulatory elements and matrin-3 requires association of the DNA-bound Pit1 with β-catenin and SATB1. Consistent with this suggestion, lack of matrin3 had a negative effect on the expression of target genes (Fig. S3i).


Required enhancer-matrin-3 network interactions for a homeodomain transcription program.

Skowronska-Krawczyk D, Ma Q, Schwartz M, Scully K, Li W, Liu Z, Taylor H, Tollkuhn J, Ohgi KA, Notani D, Kohwi Y, Kohwi-Shigematsu T, Rosenfeld MG - Nature (2014)

β-catenin and SATB1 influence transcription of genes regulated by Pit1 enhancersa, Genes containing Pit1 enhancers (991) are significantly downregulated upon knockdown of β-catenin and Satb1. RPKM – reads per kilobase per million b, GRO-seq tag distribution on both strands of Pit1 bound enhancers before and after treatment of GC cells with β-catenin and Satb1 siRNAs. c, ChIP-qPCR analysis of Satb1 association with Pit1 enhancers upon either Pit1 or β-catenin knockdown. Experiments were repeated 3 times, and p-values calculated using student’s two tailed t-test. (+/− SD; *p<0.05, **p<0.01, ***p<0.001) d, ChIP-qPCR analysis of matrin-3 association with Pit1 enhancers upon Pit1 knockdown. Experiments repeated 2–4 times, p-values calculated using student’s two tailed t-test. (+/− SD; *p<0.05, **p<0.01, ***p<0.001)
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Figure 3: β-catenin and SATB1 influence transcription of genes regulated by Pit1 enhancersa, Genes containing Pit1 enhancers (991) are significantly downregulated upon knockdown of β-catenin and Satb1. RPKM – reads per kilobase per million b, GRO-seq tag distribution on both strands of Pit1 bound enhancers before and after treatment of GC cells with β-catenin and Satb1 siRNAs. c, ChIP-qPCR analysis of Satb1 association with Pit1 enhancers upon either Pit1 or β-catenin knockdown. Experiments were repeated 3 times, and p-values calculated using student’s two tailed t-test. (+/− SD; *p<0.05, **p<0.01, ***p<0.001) d, ChIP-qPCR analysis of matrin-3 association with Pit1 enhancers upon Pit1 knockdown. Experiments repeated 2–4 times, p-values calculated using student’s two tailed t-test. (+/− SD; *p<0.05, **p<0.01, ***p<0.001)
Mentions: Global run-on (GRO-seq) analysis using GC cells before and after simultaneous knockdown of both β-catenin and Satb1 revealed that in total, 1350 coding gene nascent transcripts were downregulated and 916 genes were upregulated under these double-knockdown conditions (Fig. S3a). Out of the 1350 genes positively regulated by both β-catenin and SATB1 (Supplementary Table 8), 991 had Pit1 bound enhancer elements within 200kbp of their start sites (Fig. S3a), and all 991 were significantly downregulated (Fig. 3a). Consistent with their functional importance22 the expression of the eRNAs associated with these 991 Pit1-bound enhancers was significantly affected in the simultaneous knockdown of β-catenin and Satb1 (Fig. 3b). ChIP-seq analysis revealed that Satb1 and β-catenin proteins were located at the center of Pit1-positive enhancers (Fig. S3b–d), strongly supporting the co-binding model of these three proteins. Based on this analysis, we selected several highly down-regulated genes that contain Pit1 enhancers (Fig. S3c) and observed that knockdown of either of the two proteins significantly diminished target gene expression (Fig. S3g). Additionally, knockdown of either Pit1 or β-catenin affected Satb1 association with enhancer elements, as assessed by ChIP-qPCR (Fig. 3c, Fig. S3e). Association of matrin-3 with Pit-1 dependent enhancers was significantly reduced when either β-catenin or SATB1 were downregulated (Fig. S3h). Finally, we confirmed that lack of Pit1 protein inhibited association of selected Pit1-dependent enhancers with matrin-3 (Fig. 3d, Fig. S3f). Together, these data suggest that functional interaction between Pit1-dependent regulatory elements and matrin-3 requires association of the DNA-bound Pit1 with β-catenin and SATB1. Consistent with this suggestion, lack of matrin3 had a negative effect on the expression of target genes (Fig. S3i).

Bottom Line: Here investigation of a developmentally required POU-homeodomain transcription factor, Pit1 (also known as Pou1f1), has revealed that, unexpectedly, binding of Pit1-occupied enhancers to a nuclear matrin-3-rich network/architecture is a key event in effective activation of the Pit1-regulated enhancer/coding gene transcriptional program.The matrin-3 network-tethered R271W Pit1 mutant, but not the untethered protein, restores Pit1-dependent activation of the enhancers and recruitment of co-activators, exemplified by p300, causing both enhancer RNA transcription and target gene activation.These studies have thus revealed an unanticipated homeodomain factor/β-catenin/Satb1-dependent localization of target gene regulatory enhancer regions to a subnuclear architectural structure that serves as an underlying mechanism by which an enhancer-bound homeodomain factor effectively activates developmental gene transcriptional programs.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
Homeodomain proteins, described 30 years ago, exert essential roles in development as regulators of target gene expression; however, the molecular mechanisms underlying transcriptional activity of homeodomain factors remain poorly understood. Here investigation of a developmentally required POU-homeodomain transcription factor, Pit1 (also known as Pou1f1), has revealed that, unexpectedly, binding of Pit1-occupied enhancers to a nuclear matrin-3-rich network/architecture is a key event in effective activation of the Pit1-regulated enhancer/coding gene transcriptional program. Pit1 association with Satb1 (ref. 8) and β-catenin is required for this tethering event. A naturally occurring, dominant negative, point mutation in human PIT1(R271W), causing combined pituitary hormone deficiency, results in loss of Pit1 association with β-catenin and Satb1 and therefore the matrin-3-rich network, blocking Pit1-dependent enhancer/coding target gene activation. This defective activation can be rescued by artificial tethering of the mutant R271W Pit1 protein to the matrin-3 network, bypassing the pre-requisite association with β-catenin and Satb1 otherwise required. The matrin-3 network-tethered R271W Pit1 mutant, but not the untethered protein, restores Pit1-dependent activation of the enhancers and recruitment of co-activators, exemplified by p300, causing both enhancer RNA transcription and target gene activation. These studies have thus revealed an unanticipated homeodomain factor/β-catenin/Satb1-dependent localization of target gene regulatory enhancer regions to a subnuclear architectural structure that serves as an underlying mechanism by which an enhancer-bound homeodomain factor effectively activates developmental gene transcriptional programs.

Show MeSH
Related in: MedlinePlus