Transcription factor effects on chromatin organisation and gene regulation

The intranuclear DNA of all eukaryotic cells is packed into chromatin, generating a repressive environment for the genome. Remodelling of the local chromatin structure is a vital process that allows access to the obscured DNA sequence and may induce the expression of specific genes. To be able to find the appropriate gene in the vast amount of DNA the cell relies on sequence specific transcription factors, which then trigger gene induction. Correct control of gene expression is of fundamental importance for cell function. In this thesis the effect of transcription factor DNA binding on chromatin organisation and transcription was examined in vivo. The MMTV LTR (mouse mammary tumour virus long terminal repeat) was used as a model promoter, and oocytes from Xenopus laevis as cellular system.

The ubiquitous transcription factors NF1 (Nuclear Factor 1) and Oct1 (octamer binding factor 1) were found to cooperate in binding and enhanced basal transcription at the non-hormone induced MMTV LTR, which thus is accessible to these factors. Oct1, but not NF1, was found to increase both the basal and the hormone induced transcription. Together NF1 and Oct1 greatly enhanced the MMTV transcription in a synergistic manner. The basis for this cooperativity was an NF1 and Oct1 presetting of the MMTV LTR specific nucleosomal array. This preset state was functionally relevant, as it enabled a more rapid and stronger hormone response. This indicates a fundamental role of the ubiquitous factors NF1 and Oct1 in setting up a chromatin structure poised for transcription.

Cooperative binding of GR (glucocorticoid receptor), NF1 and Oct1 upon hormone activation was also revealed. This was explained by a common binding platform at the enhanceosome. A direct effect of NF1 binding on enhanceosome stability was detected.

Novel transcription factor sites for FoxA (Forkhead box A) was found in the MMTV LTR. An upstream FoxA site was found to inhibit hormone dependent MMTV transcription. This was likely due to a structural/sterical effect mediated through FoxA1 DNA bending. In the promoter proximal area, a double FoxA site was found to activate basal transcription through FoxA1 activation domains. Binding of FoxA1 occurred independently at each site, and in the absence of hormone activated GR. At the chromatin level, FoxA1 binding resulted in a perturbed chromatin organisation, containing an accessible C-nucleosome. Thus, FoxA1 was able to bind the chromatinised MMTV LTR and create an open structure, i.e. act as a pioneer transcription factor. In addition, the FoxA1 activation domains stabilised an altered organisation of the MMTV enhanceosome.

Co-expression of NF1, Oct1 and GR with FoxA1 resulted in a displacement of FoxA1 from the upstream inhibitory site. In concordance the FoxA1 mediated transcriptional inhibition on the hormone activated MMTV LTR was nullified. This could possibly be explained by secondary effects stemming from NF1 and Oct1 influence on the MMTV LTR nucleosomal array. NF1 and Oct1 increased stability for the active MMTV nucleosomal array might be incompatible with FoxA1 binding at the upstream site. Similarly, in a recent report FoxA1 was found to be displaced from several promoters upon ER (estrogen receptor) induction in MCF7 cells. Together this indicates an important role of FoxA1 in nuclear receptor induced transcription.

We have been able to study both the cooperative and competitive nature of interactions within an enhanceosome. The differences revealed here emphasise the importance of context effects on transcription factors.

List of scientific papers

I. Belikov S, Astrand C, Holmqvist PH, Wrange O (2004). Chromatin-mediated restriction of nuclear factor 1/CTF binding in a repressed and hormone-activated promoter in vivo. Mol Cell Biol. 24(7): 3036-47.
https://pubmed.ncbi.nlm.nih.gov/15024090

II. Belikov S, Holmqvist PH, Astrand C, Wrange O (2004). Nuclear factor 1 and octamer transcription factor 1 binding preset the chromatin structure of the mouse mammary tumor virus promoter for hormone induction. J Biol Chem. 279(48): 49857-67. Epub 2004 Sep 20
https://pubmed.ncbi.nlm.nih.gov/15381691

III. Holmqvist PH, Belikov S, Zaret KS, Wrange O (2005). FoxA1 binding to the MMTV LTR modulates chromatin structure and transcription. Exp Cell Res. 304(2): 593-603. Epub 2004 Dec 30
https://pubmed.ncbi.nlm.nih.gov/15748903

IV. Holmqvist PH, Belikov S, Wrange O (2005). Functional analysis of FoxA1 interactions at the MMTV LTR. [Manuscript]