Nouria Hernandez performed her thesis research on mRNA splicing with Dr. Walter Keller at the University of Heidelberg in Germany and received her PhD in 1983. She did her postdoctoral studies with Dr. Alan M. Weiner at Yale University in New Haven, Connecticut, USA, working on 3’ end formation of the U1 small nuclear RNA. She then joined Cold Spring Harbor Laboratory at Cold Spring Harbor, New York, in 1986 as an Assistant Professor. She became a Cold Spring Harbor Laboratory Professor in 1993 and joined the Howard Hughes Medical Institute as an Associate Investigator in 1994. She became a full Howard Hughes Medical Institute Investigator in 1999. In 2005, she joined the faculty of the UNIL as a Professor and as the Director of the Center for Integrative Genomics (CIG).
Regulation of human RNA polymerase II and III transcription, small nuclear RNA genes, CK2, cell cycle, chromatin.
Mechanisms of basal and regulated RNA polymerase II and III transcription of ncRNA genes in mammalian cells
The task of transcribing the human genome is shared among three main RNA polymerases (RNAPs) known as RNAP-I, -II, and -III, as well as a newly identified single polypeptide RNAP-IV. RNAP-I transcribes the repeated 45S transcription unit, which gives rise to the 28S, 18S, and 5.8S ribosomal RNAs. RNAP-II transcribes the mRNA genes encoding proteins as well as most small nuclear RNA (snRNA) and microRNA genes. Thus, in contrast to RNAP-I, RNAP-II recognizes a large variety of promoter structures, reflecting the intricate regulation of its target genes in processes such as cell growth, proliferation, differentiation, and responses to various stresses. spRNAP-IV is thought to transcribe a few hundred mRNA-encoding genes. RNAPIII transcribes a collection of short genes encoding RNAs that are essential for cellular metabolism as well as some regulatory RNAs such as microRNAs. We are interested in mechanisms of transcription regulation of genes producing transcripts that do not code for proteins, so-called non-coding RNA (ncRNA) genes. In particular we study the mechanisms that govern transcription of RNAP-II snRNA genes as well as transcription of RNAP-III genes, which according to current knowledge all give rise to ncRNAs. Both classes of genes are relatively understudied compared to classical RNAP-II mRNA-encoding genes, yet their regulation is of great importance for cell metabolism. Our recent focus has been in
- The determination of the RNAP-III transcriptome in the human genome,
- The mechanisms of transcription activation of RNAP-III snRNA promoters, and
- The unexpected role of a subunit of the snRNA activating protein complex (SNAPc ), a transcription factor binding to the RNAP-II and RNAP-III core snRNA promoters.
Targets for basal transcription factors used by RNAP-II snRNA promoters and RNAP-III promoters
In collaboration with Dr. H. Stunnenberg, Radboud University, we set out to characterize targets for SNAPc and RNAP-III in the human genome. We used an anti-TBP antibody we developed many years ago to select TBP-binding fragments and create a DNA array, which was then probed with DNA from chromatin immunoprecipitations performed with antibodies directed against various transcription factors including:
- The RNAP-III transcription factors Brf1 and Bdp1;
- RNAP-III itself; and iii) the five SNAPc subunits. The results showed nearly perfect colocalization of Brf1, Bdp1, and RNAP-III on RNAPIII promoters, and good colocalization of the various SNAPc subunits on both RNAP-II and RNAP-III snRNA promoters. We are now extending this work to the entire genome using the ChIP-Seq methodology.
Activation of RNAP-III type 3 promoters and RNAP-II snRNA promoters
Our studies on the mechanisms of transcription activation have been centered on the role of the zinc finger protein Staf in activating transcription from the RNAP-III U6 promoter. We found that Staf can bind to preassembled chromatin templates and activate transcription in vitro, suggesting that it recruits activities that modify the chromatin. Indeed, purification of Staf-associated proteins and their identification by multi-dimensional protein identification technology (Mud-PIT) revealed a number of proteins linked to chromatin remodeling and histone modification, among them the chromodomain-helicase-DNA binding protein 8 (CHD8). We showed that CHD8 binds to histone H3 di- and tri- methylated on lysine 4, resides on the human U6 promoter as well as on the mRNA IRF3 promoter in vivo, and is involved in efficient transcription from both these promoters. This suggests that RNAP-III transcription requires chromatin remodeling and uses some of the same factors used for chromatin remodeling at RNAP-II promoters.
An unexpected function for a subunit of SNAPc
The unexpected role of a SNAPc subunit was discovered during a routine examination of SNAPc localization. We found that the SNAP45 subunit localizes to centrosomes during parts of mitosis, as well as to the spindle midzone during anaphase and the mid-body during telophase. Consistent with localization to these mitotic structures, both down- and up-regulation of SNAP45 led to a G2/M arrest with cells displaying abnormal mitotic structures. In contrast, down-regulation of SNAP190, another SNAPc subunit, led to an accumulation of cells with a G0/G1 DNA content. Thus, SNAP45 seems to play two roles in the cell, one as a subunit of the transcription factor SNAPc, and another as a factor required for proper mitotic progression.
Bonhoure, N., Byrnes, A., Moir, R.D., Hodroj, W., Preitner, F., Praz, V., Marcelin, G., Chua. S.C. Jr., Martinez-Lopez, N., Singh, R., Moullan, N., Auwerx, J., Willemin, G., Shah, H., Hartil, K., Vaitheesvaran, B., Kurland, I., *Hernandez, N., *Willis, I.M. (2015). Loss of the RNA polymerase III repressor MAF1 confers obesity resistance. Genes Dev. 29, 934-47. URL
Orioli, A., Praz, V., Lhôte, P., Hernandez, N. (2016). Human MAF1 targets and represses active RNA polymerase III genes by preventing recruitment rather than inducing long-term transcriptional arrest. Genome Res. 26, 624-635. URL
Mange, F., Praz, V., Migliavacca, E., Willis, I.M., Schütz, F., Hernandez, N., and the CycliX Consortium (2017). Diurnal regulation of RNA polymerase III transcription is under the control of both the feeding-fasting response and the circadian clock. Genome Res. 27, 973-984. URL
*: co-corresponding authors
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