Mammalian TOR as a central modulator of the transcriptional response of keratinocyte stem cells to small temperature variations

Being in the layer at the interface of the body with the outside world, keratinocyte stem cells constantly adapt and respond to environmental cues such as temperature and oxygen. However, until now, experiments involving temperature have only examined cold and heat shocks. Here, our work illustrates that small variations of temperature (~1°C) are enough to affect colony size of keratinocyte stem cells and alter expression of some genes involved in various signalling pathways. The promoter regions of the temperature sensitive genes share a sequence located in some Alu short interspersed nuclear elements (Alu SINEs). This sequence termed TORRID contains several IFHL-like motifs (IFHL motif is related to ribogenesis in yeast) and interestingly a minimal TORRID sequence might be enough for a gene to acquire temperature sensitivity. Our findings demonstrated that the expression of temperature sensitive genes is regulated through the mammalian target of rapamycin (mTOR) pathway. The transcription factors ILF1 and RAP1, and histone deacetylase II (HDAC2) –human orthologs of yeast Fhl1p, Rap1 and Rpd3 respectively– associate with TORRID sequences. Small variations of temperature were enough to alter this association. Moreover, mTOR can translocate to the nucleus and associate with the TORRID sequences in a rapamycin and temperature dependent manner. Furthermore, Raptor and Rictor, members of the mTOR complexes 1 and 2 are also involved in this binding. Hence, our results reveal not only that TOR transcriptional activity is evolutionary conserved from yeast to human, but also that a small variation in temperature represents a new input to the mTOR pathway and fine-tune regulation of transcription through TORRID sequences. These results shed new light on the role of Alus in gene regulation.

Relationship between estrogen receptor alpha location and gene induction reveals the importance of downstream sites and cofactors

Mauro Delorenzi, Felix Naef

BACKGROUND: To understand cancer-related modifications to transcriptional programs requires detailed knowledge about the activation of signal-transduction pathways and gene expression programs. To investigate the mechanisms of target gene regulation by human estrogen receptor alpha (hERalpha), we combine extensive location and expression datasets with genomic sequence analysis. In particular, we study the influence of patterns of DNA occupancy by hERalpha on expression phenotypes. RESULTS: We find that strong ChIP-chip sites co-localize with strong hERalpha consensus sites and detect nucleotide bias near hERalpha sites. The localization of ChIP-chip sites relative to annotated genes shows that weak sites are enriched near transcription start sites, while stronger sites show no positional bias. Assessing the relationship between binding configurations and expression phenotypes, we find binding sites downstream of the transcription start site (TSS) to be equally good or better predictors of hERalpha-mediated expression as upstream sites. The study of FOX and SP1 cofactor sites near hERalpha ChIP sites shows that induced genes frequently have FOX or SP1 sites. Finally we integrate these multiple datasets to define a high confidence set of primary hERalpha target genes. CONCLUSION: Our results support the model of long-range interactions of hERalpha with the promoter-bound cofactor SP1 residing at the promoter of hERalpha target genes. FOX motifs co-occur with hERalpha motifs along responsive genes. Importantly we show that the spatial arrangement of sites near the start sites and within the full transcript is important in determining response to estrogen signaling.

BioMed Central2009

Assessing the role of Hes1 and identification of target genes in human and murine T-ALL

Silvia Wirth

The Notch signalling pathway is an ancient cell signalling mechanism that enables short-range communications between cells and controls a broad spectrum of cell fates and developmental processes. In the haematopoietic system Notch signalling has been linked to physiological, but also to pathological phenotypes of T cell development. Identification of numerous activating mutations within the NOTCH1 receptor in human T cell acute lymphoblastic leukaemia (T-ALL) patient samples demonstrated that aberrant activation of this signalling pathway is a frequent event in T-ALL. Active Notch signalling leads to the expression of a plethora of genes that might be drivers or passengers in tumorigenesis including Hes1, which encodes a transcriptional repressor. We have therefore studied Hes1 in the context of T-ALL in mouse models that mimic human disease and in human T-ALL cell lines with the aim to evaluate whether Hes1 affects disease development and disease maintenance and to characterize its function on a molecular level. The first part of this study is dedicated to the functional characterisation of Hes1 in vivo in T-ALL mouse models and in vitro in human T-ALL cell lines. Using a retroviral bone marrow transplant model of T-ALL in which progenitor cells are transduced with a retrovirus expressing constitutive active Notch1 intracellular domain (NICD) we could show that Hes1 is not required in established, late stage T-ALL cells, but that it promotes tumour progression in the early stages of the disease. We have also investigated the function of HES1 in human T-ALL and can show that the overexpression of dominant negative versions of Hes1 in two patient derived cell lines (T-ALL1, CUTLL-1) does not affect proliferation or apoptosis, emphasising that HES1 does not affect cell viability once cells are fully transformed to the leukemic state. In the second part of the study we set out to systematically delineate the gene regulatory networks down-stream of Hes1 by combining RNA-seq and ChIP-seq analysis. Comparing the gene expression of murine premalignant, early stage NICD induced CD4+CD8+ DP cells in the presence and absence of Hes1 by RNA-seq, we unexpectedly found a predominant upregulation of genes in cells expressing Hes1 indicating that Hes1 might rather act as a positive regulator of transcription than as a transcriptional repressor of distinct target genes. Hes1 might influence transcription through secondary or indirect effects as combinatorial analysis of RNA-seq and ChIP-seq led to the identification of only a small number of potential direct target genes that are transcriptionally regulated by Hes1. In summary, the role of Hes1 in promoting the early stages of the disease appears to be highly complex and is likely to involve both direct and indirect control of gene regulatory networks.

EPFL2014

Rhythmic modulation of transcriptional burst frequency in circadian gene promoters

Damien Lionel Nicolas

Isogenic cells sharing a common environment present a large degree of heterogeneity in gene expression, and stochasticity inherent to transcription substantially participates in this cell-to-cell variability. Notably, a majority of mammalian genes are transcribed during short periods termed transcription bursts followed by longer periods of transcriptional inactivity. Interestingly, genes display variability in the frequencies and sizes of their bursts, implying that different regulatory mechanisms actively participate in shaping gene-specific bursting signatures. However, the nature of these molecular mechanisms and their precise contribution to transcriptional bursting remains elusive. In this work, we used a short-lived luciferase reporter under the control of a Bmal1 circadian promoter stably inserted into the genome of NIH-3T3 cultured fibroblasts to quantify its transcriptional bursting along the circadian cycle and at three different reporter integration sites. By recording dynamic variations of the luminescence signal at the single-cell level and counting individual transcripts using smRNA-FISH at specific time-points, we could infer the transcriptional bursting parameters characteristic of these conditions using a telegraph (on-off) model of gene expression. We observed that while the integration site-specific differences in expression levels mainly arose from burst size dissimilarities, the burst frequency predominantly modulated the temporal variations in expression of Bmal1 over the circadian cycle. Thus, both parameters are uncoupled and can be independently modulated to regulate expression levels. By focusing on the molecular origins of bursting, we found that the rhythmic circadian modulation of burst frequencies depended on the presence of ROR responsive elements (ROREs) on Bmal1 promoter. These DNA motifs recruit the REV-ERBs repressors involved in the rhythmic regulation of the histone acetylation state at target promoters. Indeed, the H3K27ac profile in the Bmal1 promoter corresponded to that of its burst frequency. More generally, higher histone acetylation levels were observed during Bmal1 circadian peaks of expression, while H3K27ac signal did not vary between clones harboring different reporter integration sites. Similar properties were observed on other rhythmically expressed genes: despite variability in promoter motifs and expression phases, endogenous circadian genes displaying rhythmic variations in their promoter acetylation state also modulated their burst frequencies over the circadian period. By inferring the transcriptional bursting parameters of non-circadian genes using smRNA-FISH datasets, we also observed significant correlations between histone acetylation signal around promoters and the burst frequency. In conclusion, this study identified an association between the burst frequency and the histone acetylation state of promoters. While the molecular mechanisms behind this association remain elusive, it could be related to the facilitated binding of transcription regulators upon histone acetylation-mediated chromatin loosening. In this thesis we clarified how transcription of circadian genes is rhythmically modulated, and we further elucidated the link between molecular events and transcriptional bursting, with particular focus on histone acetylation.