Transcription Termination Factor (TTF1), a multifunctional regulator of ribosomal RNA gene activity and cell growth
Dany S. Sibai1,2, Michel G. Tremblay1, Frédéric Lessard1, Marianne-Sabourine Félix1, Thomas Moss 1,2 1 Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre, Laval University, Quebec, Canada; 2 Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Laval University, Quebec, Canada.
Transcription Termination Factor I (TTF1) is a Myb-family sequence specific factor whose unique N-terminal negative regulatory domain (NRD) inhibits DNA binding. TTF1 is the ortholog of Reb1p, one of two essential Nucleosome Free Regions (NFRs) inducers in yeast. Mouse and human TTF1 are believed to be responsible for termination of 47S pre-ribosomal RNA (47S rRNA) transcription and for imposing a unidirectional mode of DNA replication across the ribosomal RNA gene (rDNA) repeats. TTF1 has also been implicated in rDNA silencing via a lncRNA, chromatin remodelling and DNA CpG methylation (meCpG) as well as in mediating p14/19-ARF tumour suppressor activity. Moreover, studies have demonstrated a potential role for TTF1 in nucleosome positioning and transcriptional activation. However, to date these potential functions have not been directly tested in vivo.
We have now generated mouse cell lines conditionally expressing TTF1. Since loss of TTF1 was expected to cause inviability, an epitope tagged, doxycycline inducible ttf1 cDNA was first introduced into the NIH3T3 mouse cell line and then CRISPR/Cas9 was used to induce homozygous deletions in the endogenous ttf1 alleles. The resulting cell lines have been used to determine the role TTF1 plays in cell growth, regulation of lncRNA synthesis and rDNA silencing, and how these functions are inter-related. To do this we have applied a range of techniques including RNA metabolic labeling, psoralen accessibility crosslinking, lncRNA mapping, chromatin immunoprecipitation (ChIP-qPCR and DChIP-Seq) and CpG methylation mapping. We show that TTF1 protein levels determine the cell proliferation rate by regulating ribosome production and total cellular RNA levels rather than CpG methylation and rDNA silencing. The data further show that the lncRNA is indeed generated from the enhancer-associated “Spacer” promoter and suggest an alternative scenario for lncRNA function in which the rDNA activity is regulated by promoter occlusion.