Homeodomain proteins, described 30 years ago1,2, exert essential roles in development as regulators of target gene expression3,4; 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 enhancers5 to a nuclear matrin-3-rich network/architecture6,7 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 deficiency9, 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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We thank M. Ghassemian for assistance with mass spectrometry; C. Nelson for cell culture assistance; J. Hightower for assistance with figures and manuscript preparation; T. Suter for help with images analysis. We acknowledge J. Santini and the UCSD Neuroscience Microscopy Shared Facility (Grant P30 NS047101) for imaging. These studies were supported by grants NS034934, DK039949, DK018477, HL065445, CA173903 to M.G.R. from NIH. D.S.-K. was supported by EMBO Long Term Fellowship, The Swiss National Science Foundation and The San Diego Foundation. M.G.R. is an Investigator with HHMI.
The authors declare no competing financial interests.
Extended data figures and tables
a, Validation of Pit1 antibody specificity by qPCR analysis of ChIP signals on two known targets; the GH and NeuroD4 enhancers shows lack of Pit1 signal after Pit1 knockdown. Experiment repeated 2 times, P values were calculated using Student’s two tailed t-test (± s.d.; ***P < 0.001). b, Western-blot confirming Pit1 knockdown efficiency in samples used to assess Pit1 antibody specificity. c, Co-immunoprecipitation of HA-tagged, overexpressed Pit1 protein from 293T cells confirms interaction of Pit1 protein with endogenous matrin-3 and hnRNP-U proteins (nt, non-transfected). d, Reciprocal co-immunoprecipitation of matrin-3 followed by western blot analysis confirmed interactions of matrin-3 with Pit1. e, Super-resolution image (×100, OMX DeltaVision) of immunostaining with anti-matrin-3 antibody in GC cells reveals “structure-like” matrin-3 network. f, ChIP-seq analysis reveals that ∼50% of matrin-3 DNA binding sites co-localize with the enhancer mark, H3K4me2, in GC cells. g, Meta-analysis plot of ChIP-seq data shows that Pit1 bound enhancers co-localize with matrin-3, H3K4me1, H3K4me2 and H3K27Ac, but not with a mark of silent chromatin, H3K27me3. x-axis is the distance from the Pit1 peak in base pairs. h, Western blot confirming matrin-3 knockdown efficiency in GC cells. i, Validation of matrin-3 antibody specificity by qPCR analysis of ChIP signal on known Pit1 targets shows lack of matrin-3 signal after matrin-3 knockdown. Experiment repeated 2 times, P values were calculated using Student’s two tailed t-test (± s.d.; **P < 0.01, ***P < 0.001). j, Single optical sections of immuno-FISH experiment in GC cells. From left: matrin-3, GH-FISH, DAPI, merge; each staining shown separately in black and white, and merged as matrin-3 antibody staining in red and the GH locus labelled with a DNA probe in green.
a, Western blot showing that biotinylated BLRP-β-catenin is present in the nuclear and cytoplasmic fractions of GC cells. Biotinylated BLRP-β-catenin is visualized with streptavidin-horse radish peroxidase. b, Bacterially expressed GST–β-catenin fusion proteins used to map interaction with Pit-1 in GC cell extracts. c, Western blot validation of siRNA knockdown of β-catenin and Pit-1 proteins in GC cell samples. d, RT-qPCR analysis of Satb1 mRNA expression in embryonic and adult mouse pituitary glands showing increased expression that parallels differentiation of Pit1-dependent cell types, n = 3, ± s.d. e, Immunohistochemical staining of SATB1 and GH in the anterior lobe of an adult mouse pituitary showing co-localization of signals at the cellular level. f, Western blot showing co-immunoprecipitation of SATB1 with HA-tagged Pit1 in 293T cells. g, RT-qPCR analysis of GH nascent transcript levels in E16.5 mouse pituitaries harbouring a Prop1-CRE conditional deletion of Satb1, ± s.d., *P < 0.05, WT = 4, cKO = 12. h, Schematic representation of the LIS-extraction procedure used to isolate matrin-3-rich network. i, Pit-1 co-localizes with Satb1 and matrin-3 in the LIS-resistant insoluble fraction whereas β-catenin is present in both LIS-extracted and LIS-resistant insoluble fractions in GC cells. j, Western blot confirming β-catenin and Satb1 knockdown efficiency in GC cells. k, siRNA knockdown of β-catenin and Satb1 in GC cells does not affect either the subcellular distribution or the level of Pit1 protein. l, After siRNA knockdown of β-catenin and Satb1 followed by LIS extraction, Pit-1 is detectable in the “looped-out DNA” fraction. m, Immuno-FISH in GC cells before and after siRNA knockdown of β-catenin and Satb1 and in MMQ cells. Single optical section of each experiment is presented. In merge: Matrin3 antibody immunostaining in red and GH loci labelled with a fluorescent green DNA probe.
a, Most β-catenin/Satb1-activated target genes are associated with Pit-1-bound enhancers in GC cells. b, Example picture of ChIP-seq and GRO-seq analysis of a Pit-1/Satb1 target gene, the NeuroD4 locus. c, Location of Pit1-dependent enhancers (in yellow) relative to the transcription start sites of selected target genes. d, Heat-map plot of ChIP-seq tag distribution on Pit1 enhancers showing enrichment of Satb1 and β-catenin signal in centre of enhancer. e, ChIP-qPCR analysis of Satb1 association with Pit1 enhancers upon either Pit1 or β-catenin knockdown. Experiments repeated 3 times, P values calculated using Student’s two tailed t-test (± s.d.; *P < 0.05, **P < 0.01, ***P < 0.001). f, 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 (± s.d.; *P < 0.05, **P < 0.01, ***P < 0.001). g, qPCR analysis showing significant change in expression of selected Pit1 target genes upon either Pit1 or Satb1 knockdown (± s.d.; ***P < 0.001). h, ChIP-qPCR analysis of matrin-3 association with Pit1-dependent enhancers following siRNA knockdown of β-catenin or Satb1. Experiment repeated 2–3 times, P values were calculated using Student’s two tailed t-test (± s.d.; *P < 0.05, **P < 0.01, ***P < 0.001). i, RT-qPCR analysis of relative expression of target gene mRNA following siRNA knockdown of matrin-3.
Extended Data Figure 4 Pit-1 R271W mutant protein binds cognate DNA sites but does not associate with the nuclear LIS-resistant insoluble fraction.
a, RT-qPCR analysis of mRNA expression of Pit1-dependent target genes in GC cells. Both overexpressed, wild-type and R271W, Pit1 proteins compete for binding to recognition sites with endogenous Pit1. Experiment repeated 2 times, P values were calculated using Student’s two tailed t-test (± s.d.; *P < 0.05). b, Heat map of ChIP-seq data showing that overexpressed HA-Pit1 WT and HA-Pit1 R271W bind to the same enhancer DNA recognition sites as endogenous Pit1 protein. c, Scattered dot plot of genome-wide peaks consistency analysis between the wild-type and R271W Pit1 to Pit1 enhancers with Pearson correlation coefficient shows a strong resemblance of the data. d, ChIP-seq analysis comparing normalized tag density on GC samples containing overexpressed wild-type and R271W Pit1 protein. x-axis represents the number of base pairs from the centre of the Pit1 peak. e, Both wild-type and R271W Pit1 protein express at similar levels and partition in the nuclear fraction. f, Fusion of the SAF matrix-association domain results in fractionation of R271W-SAF Pit1 protein in LIS-resistant insoluble fraction of GC cells. g, Western blot showing equivalent expression of all HA-tagged Pit1 constructs in GC cells. Asterisk, product of degradation of overexpressed Pit1 protein. h, i, ChIP-qPCR (h) and RT-qPCR (i) analysis of effect of overexpression of different forms of Pit1 protein on p300 association and enhancer RNA (eRNA) expression, respectively, in the absence of endogenous Pit1. Experiments have been repeated 2–4 times, P values were calculated using Student’s two tailed t-test (± s.d.; **P < 0.01, ***P < 0.001).
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Skowronska-Krawczyk, D., Ma, Q., Schwartz, M. et al. Required enhancer–matrin-3 network interactions for a homeodomain transcription program. Nature 514, 257–261 (2014). https://doi.org/10.1038/nature13573
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