Skip to main content Skip to navigation

The mechanism of a tissue-specific regulation of gene expression by a ubiquitously expressed transcription factor

Primary Supervisor: Professor Constanze Bonifer, Institute of Cancer and Genomic Sciences

Secondary supervisor: Salam Assi

PhD project title: The mechanism of a tissue-specific regulation of gene expression by a ubiquitously expressed transcription factor

University of Registration: University of Birmingham

Project outline:

Mammalian development is regulated by the interplay of transcription factors with the epigenetic regulatory machinery. A large number of tissue-specific transcription factors have been described in whose absence a specific cell lineage is not, or less efficiently formed. However, many widely expressed genes are regulated by factors that are ubiquitously expressed and which cooperate with tissue-specific factors to activate differential gene expression programmes and thus drive development. An interesting observation is that knock-out of such genes can have surprisingly tissue-specific effects. This is also true for one of the most enigmatic of all generic transcription factors (TFs), the Sp-family of factors, with the best studies one being Sp1. This TF binds to almost every promoter in the mammalian genome, but its precise role in regulating specific genes is still not understood in spite of decades of research.

Both Sp1 and the closely related family member Sp3 are ubiquitously expressed and recognise the same DNA binding motifs (1). Sp1 DNA-binding is indispensable for normal mammalian embryo development, since mice expressing a truncated version of Sp1 lacking the DNA binding domain (DBD) (Sp1ΔDBD/ΔDBD) demonstrate multiple heterogeneous phenotypic abnormalities and die in utero (2). Interestingly, it was not possible to identify the specific role of Sp1 in a specific pathway by conditional deletion, as no defects were observed when the gene was deleted later in development, indicating that the developmental defects in Sp1ΔDBD/ΔDBD mice were cumulative. Sp3-/- mice with a complete gene knock-out survive until birth but die shortly after due to respiratory failure (3). From these experiments it was clear that while there may be some functional redundancy, tightly regulated levels of both factors are required throughout normal embryonic development.

To examine the mechanism of how a ubiquitously expressed TF regulates tissue-specific gene expression at the global level, and to bypass the problems with embryonic lethality in mice we used the in vitro differentiation of mouse embryonic stem cells (ESC) into blood cells as a tractable model to shed light on the role of Sp factors in developmental gene expression (4). We showed that the complete knock-out of Sp1 is incompatible with differentiation (5). Sp1ΔDBD/ΔDBD cells are unable to terminally differentiate and this phenotype was associated with a progressive deregulation in gene expression (5). We showed that while Sp3 can compensate for Sp1 binding at some target genes it cannot support hematopoietic specification alone. Most importantly, we showed by single cell RNA sequencing that the differentiation trajectory leading to hematopoietic differentiation is severely disturbed in Sp1ΔDBD/ΔDBD cells. Cells execute the order of cell fate decisions correctly, but not as a cohort and with delayed kinetics. These results mean that Sp-factors cooperate with tissue specific TFs to ensure that every gene is switched on in the right cell type and at the right time. Moreover, it was shown that a specific mutation of Sp1 has the opposite effect of driving excessive levels of transcription that lead to disease highlighting again the important role of this factor in gene expression control (6). However, the molecular mechanism of this cooperation is entirely unclear. One potential mechanism is that Sp1/Sp3 factors are required to set up a correct promoter structure.

This studentship will test this hypothesis by addressing the question of how these factors perform this function. We will study (i) how promoters carrying mutant Sp factors cooperate with distal gene regulatory elements binding tissue-specific TFs in nuclear space, (ii) whether Sp1 factors are important for RNA-Polymerase II binding, (iii) which part of these proteins is essential for cooperation and finally, (iv) at the single cell level, whether mutant proteins are capable of associating with a spatially confined nuclear transcription compartment.

References:

  1. O'Connor L, Gilmour J, Bonifer C. The Role of the Ubiquitously Expressed Transcription Factor Sp1 in Tissue-specific Transcriptional Regulation and in Disease. Yale J Biol Med. 2016 Dec 23;89(4):513-525.
  2. Marin M, Karis A, Visser P, Grosveld F, Philipsen S. Transcription factor Sp1 is essential for early embryonic development but dispensable for cell growth and differentiation. Cell. 1997 May 16;89(4):619-28.
  3. Bouwman P, Göllner H, Elsässer HP, Eckhoff G, Karis A, Grosveld F, Philipsen S, Suske G. Transcription factor Sp3 is essential for post-natal survival and late tooth development. EMBO J. 2000 Feb 15;19(4):655-61. doi: 10.1093/emboj/19.4.655.
  4. Goode DK, Obier N, Vijayabaskar MS, Lie-A-Ling M, Lilly AJ, Hannah R, Lichtinger M, Batta K, Florkowska M, Patel R, Challinor M, Wallace K, Gilmour J, Assi SA, Cauchy P, Hoogenkamp M, Westhead DR, Lacaud G, Kouskoff V, Göttgens B, Bonifer C. Dynamic Gene Regulatory Networks Drive Hematopoietic Specification and Differentiation. Dev Cell. 2016 Mar 7;36(5):572-87
  5. Gilmour J, O'Connor L, Middleton CP, Keane P, Gillemans N, Cazier JB, Philipsen S, Bonifer C. Robust hematopoietic specification requires the ubiquitous Sp1 and Sp3 transcription factors. Epigenetics Chromatin. 2019 Jun 4;12(1):33.
  6. Tummala H, Walne A.J, Bewicke-Copley F, Ellison A, Pontikos N, Bridger M.G., Rio-Machin A, Sidhu J.K, Wang J, Hasle H, Fitzgibbon J, Vulliamy T, Dokal I . A frameshift variant in specificity protein 1 triggers superactivation of Sp1-mediated transcription in familial bone marrow failure. Proc Natl Acad Sci U S A . 2020 Jul 21;117(29):17151-17155.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Stem Cells

    Techniques that will be undertaken during the project:

    • The project will involve:
    • Growing mouse embryonic stem cells, differentiation of these cells into blood cells,
    • Genome editing to generate Sp1 mutants, chromatin immunoprecipitation
    • Experiments, ATAC-Seq, single-gene and global gene expression analysis both at bulk and at single cell level, promoter-enhancer cooperation assays, and advanced imaging analysis. The student will also be expected to train in bioinformatics and in the analysis and integration of genome-wide next generation sequencing data from different sources

    Contact: Professor Constanze Bonifer, University of Birmingham