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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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Pit1 association with LIS resistant nuclear component is β-catenin- and SATB1- dependenta, BLRP-β-catenin immunoprecipitated from cytoplasmic or nuclear fractions of GC cells. Co-puryifying factors identified by mass spectrometry. b, Co-IP-Western analysis confirmed Pit1:β-catenin interaction and revealed interaction with SATB1. c, GST-pulldown showing β-catenin armadillo repeat 8 (within region previously shown to interact with Prop1 and Lef1) is required for interaction with Pit1. d, Nascent GH transcripts levels after siRNA knockdown of β-catenin and Pit1 analyzed by RT-qPCR. Experiments were repeated 2 times, and p-values calculated using student’s two tailed t-test. (+/− SD; **p<0.01) e, Co-IP of HA-tagged Pit1 protein in 293T cells before and after β-catenin and Satb1 knockdown showing Pit1 interacts simultaneously with both proteins. f, LIS nuclear extraction before and after β-catenin or/and Satb1 knockdown shows that both proteins are needed for Pit1 retention in LIS resistant fraction. g, Co-IP of Pit1 protein in GC cells before and after simultaneous knockdown of β-catenin and Satb1 shows interaction of Pit1 with matrin-3 is dependent on both. h, Examples of immuno-FISH experiments showing GH locus (green) colocalizing with matrin3 (red) with higher frequency in control culture conditions than after siRNA β-catenin and siRNA Satb1 treatment in GC cells. In Pit1 positive, non-GH-expressing MMQ cells, significantly fewer GH loci are associated with matrin3. The chart represents the count of percentage of signals exhibiting co-localization in control vs. siβ-catenin and siSATB1 conditions compared to MMQ cells; n≥200, +/− SD, ***p<0.001
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Figure 2: Pit1 association with LIS resistant nuclear component is β-catenin- and SATB1- dependenta, BLRP-β-catenin immunoprecipitated from cytoplasmic or nuclear fractions of GC cells. Co-puryifying factors identified by mass spectrometry. b, Co-IP-Western analysis confirmed Pit1:β-catenin interaction and revealed interaction with SATB1. c, GST-pulldown showing β-catenin armadillo repeat 8 (within region previously shown to interact with Prop1 and Lef1) is required for interaction with Pit1. d, Nascent GH transcripts levels after siRNA knockdown of β-catenin and Pit1 analyzed by RT-qPCR. Experiments were repeated 2 times, and p-values calculated using student’s two tailed t-test. (+/− SD; **p<0.01) e, Co-IP of HA-tagged Pit1 protein in 293T cells before and after β-catenin and Satb1 knockdown showing Pit1 interacts simultaneously with both proteins. f, LIS nuclear extraction before and after β-catenin or/and Satb1 knockdown shows that both proteins are needed for Pit1 retention in LIS resistant fraction. g, Co-IP of Pit1 protein in GC cells before and after simultaneous knockdown of β-catenin and Satb1 shows interaction of Pit1 with matrin-3 is dependent on both. h, Examples of immuno-FISH experiments showing GH locus (green) colocalizing with matrin3 (red) with higher frequency in control culture conditions than after siRNA β-catenin and siRNA Satb1 treatment in GC cells. In Pit1 positive, non-GH-expressing MMQ cells, significantly fewer GH loci are associated with matrin3. The chart represents the count of percentage of signals exhibiting co-localization in control vs. siβ-catenin and siSATB1 conditions compared to MMQ cells; n≥200, +/− SD, ***p<0.001

Mentions: Because the pituitary specific homeodomain protein, Prop1, interacts with β-catenin to activate target gene expression14, we investigated β-catenin interacting partners in differentiated pituitary cells by expressing β-catenin fused to the biotin ligase recognition peptide (BLRP), along with biotinylating enzyme BirA, in GC cells. Biotinylated β-catenin was present in both the cytoplasmic and the nuclear compartments (Fig. 2a, Fig. S2a). Streptavidin pulldown, followed by mass spectrometry, revealed that while cytoplasmic β-catenin interacting factors included α-catenin, as previously described15, Pit1 peptides predominated in the nuclear β-catenin fraction (Fig. 2a, Supplementary Table 4–7). This putative Pit1:β-catenin interaction was confirmed between endogenous proteins by co-IP/Western blot experiments using nuclear extracts from GC pituitary cells (Fig. 2b). Using GST-pulldown assays, armadillo repeat 8 of β-catenin proved sufficient to mediate this interaction, exactly within a region that has been previously described to interact with Prop114 as well as Lef/Tcf16 (Fig 2c, Fig S2b).


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)

Pit1 association with LIS resistant nuclear component is β-catenin- and SATB1- dependenta, BLRP-β-catenin immunoprecipitated from cytoplasmic or nuclear fractions of GC cells. Co-puryifying factors identified by mass spectrometry. b, Co-IP-Western analysis confirmed Pit1:β-catenin interaction and revealed interaction with SATB1. c, GST-pulldown showing β-catenin armadillo repeat 8 (within region previously shown to interact with Prop1 and Lef1) is required for interaction with Pit1. d, Nascent GH transcripts levels after siRNA knockdown of β-catenin and Pit1 analyzed by RT-qPCR. Experiments were repeated 2 times, and p-values calculated using student’s two tailed t-test. (+/− SD; **p<0.01) e, Co-IP of HA-tagged Pit1 protein in 293T cells before and after β-catenin and Satb1 knockdown showing Pit1 interacts simultaneously with both proteins. f, LIS nuclear extraction before and after β-catenin or/and Satb1 knockdown shows that both proteins are needed for Pit1 retention in LIS resistant fraction. g, Co-IP of Pit1 protein in GC cells before and after simultaneous knockdown of β-catenin and Satb1 shows interaction of Pit1 with matrin-3 is dependent on both. h, Examples of immuno-FISH experiments showing GH locus (green) colocalizing with matrin3 (red) with higher frequency in control culture conditions than after siRNA β-catenin and siRNA Satb1 treatment in GC cells. In Pit1 positive, non-GH-expressing MMQ cells, significantly fewer GH loci are associated with matrin3. The chart represents the count of percentage of signals exhibiting co-localization in control vs. siβ-catenin and siSATB1 conditions compared to MMQ cells; n≥200, +/− SD, ***p<0.001
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4358797&req=5

Figure 2: Pit1 association with LIS resistant nuclear component is β-catenin- and SATB1- dependenta, BLRP-β-catenin immunoprecipitated from cytoplasmic or nuclear fractions of GC cells. Co-puryifying factors identified by mass spectrometry. b, Co-IP-Western analysis confirmed Pit1:β-catenin interaction and revealed interaction with SATB1. c, GST-pulldown showing β-catenin armadillo repeat 8 (within region previously shown to interact with Prop1 and Lef1) is required for interaction with Pit1. d, Nascent GH transcripts levels after siRNA knockdown of β-catenin and Pit1 analyzed by RT-qPCR. Experiments were repeated 2 times, and p-values calculated using student’s two tailed t-test. (+/− SD; **p<0.01) e, Co-IP of HA-tagged Pit1 protein in 293T cells before and after β-catenin and Satb1 knockdown showing Pit1 interacts simultaneously with both proteins. f, LIS nuclear extraction before and after β-catenin or/and Satb1 knockdown shows that both proteins are needed for Pit1 retention in LIS resistant fraction. g, Co-IP of Pit1 protein in GC cells before and after simultaneous knockdown of β-catenin and Satb1 shows interaction of Pit1 with matrin-3 is dependent on both. h, Examples of immuno-FISH experiments showing GH locus (green) colocalizing with matrin3 (red) with higher frequency in control culture conditions than after siRNA β-catenin and siRNA Satb1 treatment in GC cells. In Pit1 positive, non-GH-expressing MMQ cells, significantly fewer GH loci are associated with matrin3. The chart represents the count of percentage of signals exhibiting co-localization in control vs. siβ-catenin and siSATB1 conditions compared to MMQ cells; n≥200, +/− SD, ***p<0.001
Mentions: Because the pituitary specific homeodomain protein, Prop1, interacts with β-catenin to activate target gene expression14, we investigated β-catenin interacting partners in differentiated pituitary cells by expressing β-catenin fused to the biotin ligase recognition peptide (BLRP), along with biotinylating enzyme BirA, in GC cells. Biotinylated β-catenin was present in both the cytoplasmic and the nuclear compartments (Fig. 2a, Fig. S2a). Streptavidin pulldown, followed by mass spectrometry, revealed that while cytoplasmic β-catenin interacting factors included α-catenin, as previously described15, Pit1 peptides predominated in the nuclear β-catenin fraction (Fig. 2a, Supplementary Table 4–7). This putative Pit1:β-catenin interaction was confirmed between endogenous proteins by co-IP/Western blot experiments using nuclear extracts from GC pituitary cells (Fig. 2b). Using GST-pulldown assays, armadillo repeat 8 of β-catenin proved sufficient to mediate this interaction, exactly within a region that has been previously described to interact with Prop114 as well as Lef/Tcf16 (Fig 2c, Fig S2b).

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