Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

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

Nature volume 514, pages 257–261 (2014)Cite this article

Subjects

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Co-localization of Pit1 and matrin-3 on enhancers.
Figure 2: Pit1 association with LIS resistant nuclear component is β-catenin- and SATB1- dependent.
Figure 3: β-catenin and SATB1 influence transcription of genes regulated by Pit1 enhancers.
Figure 4: Naturally occurring R271W mutation in human Pit1 affects ability of Pit1 to interact with matrin-3 rich network.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

Microarray data are deposited in GEO under accession number GSE58009.

References

  1. McGinnis, W., Levine, M. S., Hafen, E., Kuroiwa, A. & Gehring, W. J. A conserved DNA sequence in homoeotic genes of the Drosophila Antennapedia and bithorax complexes. Nature 308, 428–433 (1984)

    Article  ADS  CAS  Google Scholar 

  2. Scott, M. P. & Weiner, A. J. Structural relationships among genes that control development: sequence homology between the Antennapedia, Ultrabithorax, and fushi tarazu loci of Drosophila. Proc. Natl Acad. Sci. USA 81, 4115–4119 (1984)

    Article  ADS  CAS  Google Scholar 

  3. Affolter, M., Schier, A. & Gehring, W. J. Homeodomain proteins and the regulation of gene expression. Curr. Opin. Cell Biol. 2, 485–495 (1990)

    Article  CAS  Google Scholar 

  4. Prince, K. L., Walvoord, E. C. & Rhodes, S. J. The role of homeodomain transcription factors in heritable pituitary disease. Nature Rev. Endocrinol. 7, 727–737 (2011)

    Article  CAS  Google Scholar 

  5. Heintzman, N. D. & Ren, B. Finding distal regulatory elements in the human genome. Curr. Opin. Genet. Dev. 19, 541–549 (2009)

    Article  CAS  Google Scholar 

  6. Zeitz, M. J., Malyavantham, K. S., Seifert, B. & Berezney, R. Matrin 3: chromosomal distribution and protein interactions. J. Cell. Biochem. 108, 125–133 (2009)

    Article  CAS  Google Scholar 

  7. Nickerson, J. Experimental observations of a nuclear matrix. J. Cell Sci. 114, 463–474 (2001)

    CAS  PubMed  Google Scholar 

  8. Kohwi-Shigematsu, T. et al. Genome organizing function of SATB1 in tumor progression. Semin. Cancer Biol. 23, 72–79 (2013)

    Article  CAS  Google Scholar 

  9. Radovick, S. et al. A mutation in the POU-homeodomain of Pit-1 responsible for combined pituitary hormone deficiency. Science 257, 1115–1118 (1992)

    Article  ADS  CAS  Google Scholar 

  10. Zhu, X., Wang, J., Ju, B. G. & Rosenfeld, M. G. Signaling and epigenetic regulation of pituitary development. Curr. Opin. Cell Biol. 19, 605–611 (2007)

    Article  CAS  Google Scholar 

  11. Ingraham, H. A. et al. A tissue-specific transcription factor containing a homeodomain specifies a pituitary phenotype. Cell 55, 519–529 (1988)

    Article  CAS  Google Scholar 

  12. Nakayasu, H. & Berezney, R. Nuclear matrins: identification of the major nuclear matrix proteins. Proc. Natl Acad. Sci. USA 88, 10312–10316 (1991)

    Article  ADS  CAS  Google Scholar 

  13. Romig, H., Fackelmayer, F. O., Renz, A., Ramsperger, U. & Richter, A. Characterization of SAF-A, a novel nuclear DNA binding protein from HeLa cells with high affinity for nuclear matrix/scaffold attachment DNA elements. EMBO J. 11, 3431–3440 (1992)

    Article  CAS  Google Scholar 

  14. Olson, L. E. et al. Homeodomain-mediated beta-catenin-dependent switching events dictate cell-lineage determination. Cell 125, 593–605 (2006)

    Article  CAS  Google Scholar 

  15. Su, L. K., Vogelstein, B. & Kinzler, K. W. Association of the APC tumor suppressor protein with catenins. Science 262, 1734–1737 (1993)

    Article  ADS  CAS  Google Scholar 

  16. Graham, T. A., Weaver, C., Mao, F., Kimelman, D. & Xu, W. Crystal structure of a beta-catenin/Tcf complex. Cell 103, 885–896 (2000)

    Article  CAS  Google Scholar 

  17. Notani, D. et al. Global regulator SATB1 recruits beta-catenin and regulates T(H)2 differentiation in Wnt-dependent manner. PLoS Biol. 8, e1000296 (2010)

    Article  Google Scholar 

  18. Dickinson, L. A., Dickinson, C. D. & Kohwi-Shigematsu, T. An atypical homeodomain in SATB1 promotes specific recognition of the key structural element in a matrix attachment region. J. Biol. Chem. 272, 11463–11470 (1997)

    Article  CAS  Google Scholar 

  19. Cai, S., Han, H. J. & Kohwi-Shigematsu, T. Tissue-specific nuclear architecture and gene expression regulated by SATB1. Nature Genet. 34, 42–51 (2003)

    Article  CAS  Google Scholar 

  20. Mancini, M. G., Liu, B., Sharp, Z. D. & Mancini, M. A. Subnuclear partitioning and functional regulation of the Pit-1 transcription factor. J. Cell. Biochem. 72, 322–338 (1999)

    Article  CAS  Google Scholar 

  21. Mirkovitch, J., Mirault, M. E. & Laemmli, U. K. Organization of the higher-order chromatin loop: specific DNA attachment sites on nuclear scaffold. Cell 39, 223–232 (1984)

    Article  CAS  Google Scholar 

  22. Li, W. et al. Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation. Nature 498, 516–520 (2013)

    Article  ADS  CAS  Google Scholar 

  23. Kipp, M. et al. SAF-Box, a conserved protein domain that specifically recognizes scaffold attachment region DNA. Mol. Cell. Biol. 20, 7480–7489 (2000)

    Article  CAS  Google Scholar 

  24. Aravind, L. & Koonin, E. V. SAP – a putative DNA-binding motif involved in chromosomal organization. Trends Biochem. Sci. 25, 112–114 (2000)

    Article  CAS  Google Scholar 

  25. Reeves, R. Molecular biology of HMGA proteins: hubs of nuclear function. Gene 277, 63–81 (2001)

    Article  CAS  Google Scholar 

  26. Xu, L. et al. Signal-specific co-activator domain requirements for Pit-1 activation. Nature 395, 301–306 (1998)

    Article  ADS  CAS  Google Scholar 

  27. Li, S. et al. Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain gene pit-1. Nature 347, 528–533 (1990)

    Article  ADS  CAS  Google Scholar 

  28. Gibcus, J. H. & Dekker, J. The hierarchy of the 3D genome. Mol. Cell 49, 773–782 (2013)

    Article  CAS  Google Scholar 

  29. Bickmore, W. A. & van Steensel, B. Genome architecture: domain organization of interphase chromosomes. Cell 152, 1270–1284 (2013)

    Article  CAS  Google Scholar 

  30. S’iakste N. I & S’iakste T. G Transcription factors and the nuclear matrix [in Russian]. Mol. Biol. (Mosk.) 35, 739–749 (2001)

    Google Scholar 

  31. Wang, D. et al. Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA. Nature 474, 390–394 (2011)

    Article  CAS  Google Scholar 

  32. Core, L. J., Waterfall, J. J. & Lis, J. T. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322, 1845–1848 (2008)

    Article  ADS  CAS  Google Scholar 

  33. Gasser, S. M. . Vassetzky, Y. S. in Chromatin: A Practical Approach (ed. Gould, H. ) pp. 111–124 (Oxford Univ. Press, 1998)

    Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

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

    Dorota Skowronska-Krawczyk, Qi Ma, Michal Schwartz, Kathleen Scully, Wenbo Li, Zhijie Liu, Havilah Taylor, Jessica Tollkuhn, Kenneth A. Ohgi, Dimple Notani & Michael G. Rosenfeld

  2. The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, 5290002, Israel

    Michal Schwartz

  3. Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA

    Yoshinori Kohwi & Terumi Kohwi-Shigematsu

Authors
  1. Dorota Skowronska-Krawczyk
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qi Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michal Schwartz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kathleen Scully
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wenbo Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Zhijie Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Havilah Taylor
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jessica Tollkuhn
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Kenneth A. Ohgi
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Dimple Notani
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Yoshinori Kohwi
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Terumi Kohwi-Shigematsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Michael G. Rosenfeld
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.S.-K. and M.G.R. conceived the project. D.S.-K. performed the majority of the experiments; Q.M. performed the bioinformatic analyses; M.S. contributed three dimensional immuno-FISH. W.L. contributed GRO-Seqs; K.A.O. assisted in deep-sequencing library preparations and sequencing; additional experiments/methods were contributed by K.S., J.T., Z.L. and D.N.; H.T. and K.S. assisted in animal-based experiments. Y.K. and T.K.-S. contributed critical insights and reagents. D.S.-K., K.S. and M.G.R. wrote the manuscript. All authors discussed the results and commented on or edited the manuscript.

Corresponding authors

Correspondence to Dorota Skowronska-Krawczyk or Michael G. Rosenfeld.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Matrin-3 co-localizes with enhancers in GC cells.

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.

Extended Data Figure 2 Co-localization and interaction of Pit-1 with β-catenin and Satb1.

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.

Extended Data Figure 3 β-catenin and SATB1 have transcriptional effects on Pit1 targets.

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).

Supplementary information

Supplementary Table 1

This file contains Supplementary Table 1. (XLSX 1456 kb)

Supplementary Table 2

This file contains Supplementary Table 2. (XLSX 19 kb)

Supplementary Table 3

This file contains Supplementary Table 3. (XLSX 75 kb)

Supplementary Table 4

This file contains Supplementary Table 4. (XLSX 52 kb)

Supplementary Table 5

This file contains Supplementary Table 5. (XLSX 22 kb)

Supplementary Table 6

This file contains Supplementary Table 6. (XLSX 19 kb)

Supplementary Table 7

This file contains Supplementary Table 7. (XLSX 151 kb)

Supplementary Table 8

This file contains Supplementary Table 8. (XLSX 45 kb)

Supplementary Table 9

This file contains Supplementary Table 9. (XLSX 11 kb)

Supplementary Table 10

This file contains Supplementary Table 10. (XLSX 13 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature13573

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing