Interphase | EPFL Graph Search

L’interphase est la période du cycle cellulaire qui est caractérisée par un accroissement du volume cellulaire, la cellule transcrit ses gènes et les chromosomes sont dupliqués. Elle peut être subdivisée en plusieurs phases :

la phase G (de l’anglais growth ou gap), au cours de laquelle la cellule croît et effectue les fonctions pour lesquelles elle est programmée génétiquement : biosynthèse des protéines C'est notamment durant cette phase que les jeunes cellules filles fraîchement divisées grandissent jusqu'à atteindre leur taille finale ;
la phase S (de l'anglais synthesis, à cause de la synthèse de nouvelles molécules d’ADN), au cours de laquelle le matériel chromosomique est doublé par réplication de l'ADN. C'est ce qu'on appelle la duplication des chromosomes ;
la phase G, où la cellule se prépare à se diviser en deux cellules filles. À l'issue de cette phase, chaque chromosome est parfaitement identique à son homologue sur le plan morphologique et du point de vue des gèn

Proteins Binding to and Regulating Human Telomeric Transcripts

Katarzyna Sikora

Telomere are the protective terminal structures of linear chromosomes. The repetitive TTAGGG sequences are bound by the dedicated shelterin complex, as well as by histones carrying modifications characteristic of silent chromatin. Like the centromeric heterochromatin, telomeres are transcribed, resulting in a heterogenous population of RNAs, containing the unique subtelomeric as well as the common telomeric sequences. The telomeric transcripts, TERRAs, are capped, and a fraction of them is polyadenylated. The presence of the poly(A) tail confers increased stability to the RNA, otherwise relatively short-lived. HnRNPA1 is an abundant, mostly nuclear protein, committed to multiple steps of mRNA biogenesis and trafficking.In vitro, hnRNPA1 shows a striking affinity for UUAGGG repeats. It associates with telomeres, and contributes to positive regulation of their length. In this work, we performed a screen for factors binding to UUAGGG repeats in vitro. We obtained a list of proteins specifically or preferentially associating with a telomeric bait oligonucleotide. We found hnRNPA1 among the most abundant proteins selected on UUAGGG repeats. Based on prelimary results in our lab, hnRNPA1 associates with endogenous TERRA. We set out to further characterize this interaction. We observed preferential binding of hnRNPA1 to nonadenylated telomeric transcripts, which was preserved in the cytoplasmic and nucleoplasmic cell fractions. Despite this bias, a knockdown of hnRNPA1 failed to disturb the ratio between poly(A)- and poly(A)+ TERRA. Our laboratory has previously observed disappearance of TERRA foci in interphase cells depleted for hnRNPA1. We asked, whether this effect could be explained by a change in subcellular localization of the telomeric RNA. In our hands, however, hnRNPA1 knockdown did not affect TERRA fractionation behaviour. We conclude that hnRNPA1 association with TERRA is affected by the presence of the poly(A) tail, and possibly, proteins binding to it. Based on the results obtained in cells knocked down for hnRNPA1, we infer that this protein is not linked to TERRA biogenesis nor to its cellular localization.

EPFL2011

Mitotic chromosome binding predicts transcription factor properties in interphase

Mahé Cécile Raccaud

Mammalian transcription factors (TFs) differ broadly in their nuclear mobility and sequence-specific/non-specific DNA binding affinity. How these properties affect the ability of TFs to occupy their specific binding sites in the genome and modify the epigenetic landscape to regulate transcription is unclear. TFs also differ in their ability to associate with mitotic chromosomes, which has been shown to be mediated largely by non-specific binding. The TFs identified so far for their ability to bind mitotic chromatin are known cell fate regulators and often have been shown to have pioneering activity (i.e. they are able to bind and open closed chromatin regions). However, the number of TFs tested for mitotic chromosome binding (MCB) and the knowledge about properties allowing TFs to bind to mitotic chromatin are limited. Thus, we performed a large-scale quantification MCB by TFs. Out of 502 quantified TFs, we observed that 22% of TFs were enriched on mitotic chromosomes, 24% were depleted and 54% were neither enriched nor depleted, and these behaviors were largely cell-type non-specific. As TF-DNA interactions are mediated by their DNA-binding domains, we investigated their influence on MCB. However, despite the fact that TFs bearing certain types of DNA-binding domains were more likely to be enriched or depleted from mitotic chromosomes, it was not sufficient to explain differential MCB. Amino acid sequence-based analysis using machine learning showed that electrostatic properties impact the propensity of TFs to bind mitotic chromosomes. This confirms that co-localization of TFs with mitotic chromosome is affected by non-specific electrostatic protein-DNA interactions. Therefore, we next investigated whether the MCB reflected properties of TFs in interphase. We observed a clear correlation of MCB with DNA co-localization in interphase fibroblasts and with mobility in both interphase and mitosis. As mobility is influenced by both TF diffusion and binding to DNA, we excluded that differences in diffusion, caused by TF sizes, could explain the variability in mobility rates, and tested whether TFs differed in their association with specific DNA sites. Single molecule imaging of DNA binding in live cells showed that differences in MCB were correlated with differences in relative TF on-rates, but not with TFs residence times. To test whether this affected target site occupancy, genome-wide mapping of TF binding was performed, showing that TFs associating to mitotic chromosomes also have a higher interphase genome occupancy and greater ability to find their specific sites in the genome. Lastly, we investigated the correlation between the abilities to bind mitotic chromatin and to modify chromatin accessibility and observed that the broader impact of TFs with a high MCB on chromatin accessibility can be explained by their high number of bound target sites, and not by a higher potency to alter chromatin accessibility. Altogether, our data suggests that TFs differ broadly in their non-specific DNA binding properties, which regulate their search efficiency and thereby their occupancy of specific sites in the genome. We suggest that measuring mitotic chromosome association of TFs by fluorescence microscopy can be used as a metric of non-specific DNA binding.

EPFL2019

Eukaryotic transcription factor binding kinetics

Andrea Callegari

Transcription represents a key regulatory step in gene expression and transcription factors (TFs) are the key molecular players of this process. TFs inhibit or promote the assembly of the transcriptional machinery by binding DNA and several co-factors. Whereas biochemical studies suggested stable TF-DNA interactions (timescales of hours), fluorescence microscopy showed that TFs bind and dissociate from DNA within few seconds. However, the biological implications of such a dynamic behaviour are still elusive. TF-DNA binding kinetics have been mainly measured in interphase cells, i.e. when active transcription occurs. Though recent findings suggest that even when transcription is silenced in mitosis, some TFs bind to chromatin, a phenomenon known as âmitotic bookmarkingâ. This mechanism was proposed to regulate phenotypic maintenance by ensuring faithful gene reactivation at the mitosis-G1 transition. At present, though, evidence on whether and how binding kinetics determine TFsâ mitotic chromosome binding is missing. In this work, we aimed to determine the role of TFs-DNA binding kinetics in two functional aspects: (i) transcriptional activation of genes involved in the inflammatory response; (ii) differential mitotic chromatin binding of TFs regulating reprogramming and differentiation. We investigated the first aspect by measuring the binding kinetics and transcriptional activity of p65-NFï«B and mutants to its DNA-binding domain (DBD) and transactivation domains (TADs). By performing single-molecule tracking (SMT) of fluorescent p65 molecules and extrapolating p65 transcriptional activation potentials from RNAseq, we demonstrated that p65 binding time (t_b) generally correlates to its capability to induce transcriptional activation. Unexpectedly, deletion of p65 transactivation domains (TADs) did not significantly reduce p65 t_b (~3-6 s) if compared to wt-p65 (t_b~4 s), although it strongly inhibited p65-mediated transcriptional activation. We also showed ~1000-fold reduction of DNA-binding affinity (Y36A/E39D mutant) retained ~3% of long-lasting binding events (t_b~12 ð ) linked to no-activity. This prompted us to speculatively suggest that p65 may bind DNA indirectly. In a second part, we completed the kinetic characterization of Sox2 and Oct4, two TFs of the differentiation/reprogramming network. Preliminary findings showed that Sox2 and Oct4 display different mitotic chromosome binding and comparable dissociation rates from mitotic chromatin. Thus, we tested whether their distinct retention on mitotic chromosomes could arise from different association rates. To test this hypothesis, we performed SMT of Sox2- or Oct4-Halo in ES living cells to compute their bound fraction during different phases of their cell cycle. Sox2 and Oct4 were equally retained on interphase cells and did not show significant differences even during mitosis, suggesting that neither dissociation nor association rates at specific binding sites determine the differential retentions of the two TFs. Our findings indicate that (i) p65 binding kinetics predict functional outcomes as long as protein-protein interactions are preserved; (ii) such interactions do not stabilize p65-DNA binding but critically define binding events as transcriptionally productive; (iii) differential retention of Sox2 and Oct4 on mitotic chromatin is not due to differences in their binding kinetics at specific sites, suggesting a contribution from TFs-DNA non-specific interactions.

EPFL2016