Transcriptional activation by bidirectional RNA polymerase II elongation over a silent promoter

Transcriptional interference denotes negative cis effects between promoters. Here, we show that promoters can also interact positively. Bidirectional RNA polymerase II (Pol II) elongation over the silent human endogenous retrovirus ( HERV)-K18 promoter ( representative of 2.5 +/- 10(3) similar promoters genome-wide) activates transcription. In tandem constructs, an upstream promoter activates HERV-K18 transcription. This is abolished by inversion of the upstream promoter, or by insertion of a poly( A) signal between the promoters; transcription is restored by poly( A) signal mutants. TATA-box mutants in the upstream promoter reduce HERV-K18 transcription. Experiments with the same promoters in a convergent orientation produce similar effects. A small promoter deletion partially restores HERV-K18 activity, consistent with activation resulting from repressor repulsion by the elongating Pol II. Transcriptional elongation over this class of intragenic promoters will generate co-regulated sense - antisense transcripts, or, alternatively initiating transcripts, thus expanding the diversity and complexity of the human transcriptome

Assaying the regulatory potential of mammalian conserved non-coding sequences in human cells

The fraction of experimentally active conserved non-coding sequences within any given cell type is low, so classical assays are unlikely to expose their potential

A systematic enhancer screen using lentivector transgenesis identifies conserved and non-conserved functional elements at the olig1 and olig2 locus

Finding sequences that control expression of genes is central to understanding genome function. Previous studies have used evolutionary conservation as an indicator of regulatory potential. Here, we present a method for the unbiased in vivo screen of putative enhancers in large DNA regions, using the mouse as a model. We cloned a library of 142 overlapping fragments from a 200 kb-long murine BAC in a lentiviral vector expressing LacZ from a minimal promoter, and used the resulting vectors to infect fertilized murine oocytes. LacZ staining of E11 embryos obtained by first using the vectors in pools and then testing individual candidates led to the identification of 3 enhancers, only one of which shows significant evolutionary conservation. In situ hybridization and 3C/4C experiments suggest that this enhancer, which is active in the neural tube and posterior diencephalon, influences the expression of the Olig1 and/or Olig2 genes. This work provides a new approach for the large-scale in vivo screening of transcriptional regulatory sequences, and further demonstrates that evolutionary conservation alone seems too limiting a criterion for the identification of enhancers

Pleiotropic effects of trisomy and pharmacologic modulation on structural, functional, molecular, and genetic systems in a Down syndrome mouse model.

Down syndrome (DS) is characterized by skeletal and brain structural malformations, cognitive impairment, altered hippocampal metabolite concentration and gene expression imbalance. These alterations were usually investigated separately, and the potential rescuing effects of green tea extracts enriched in epigallocatechin-3-gallate (GTE-EGCG) provided disparate results due to different experimental conditions. We overcame these limitations by conducting the first longitudinal controlled experiment evaluating genotype and GTE-EGCG prenatal chronic treatment effects before and after treatment discontinuation. Our findings revealed that the Ts65Dn mouse model reflected the pleiotropic nature of DS, exhibiting brachycephalic skull, ventriculomegaly, neurodevelopmental delay, hyperactivity, and impaired memory robustness with altered hippocampal metabolite concentration and gene expression. GTE-EGCG treatment modulated most systems simultaneously but did not rescue DS phenotypes. On the contrary, the treatment exacerbated trisomic phenotypes including body weight, tibia microarchitecture, neurodevelopment, adult cognition, and metabolite concentration, not supporting the therapeutic use of GTE-EGCG as a prenatal chronic treatment. Our results highlight the importance of longitudinal experiments assessing the co-modulation of multiple systems throughout development when characterizing preclinical models in complex disorders and evaluating the pleiotropic effects and general safety of pharmacological treatments

Fine Tuning of Craniofacial Morphology by Distant-Acting Enhancers

The shape of the human face and skull is largely genetically determined, but the genetic drivers of craniofacial morphology remain poorly understood. Here we used a combination of epigenomic profiling, in vivo characterization of candidate enhancer sequences in transgenic mice, and targeted deletion experiments to examine the role of distant-acting enhancers in craniofacial development. We identified complex regulatory landscapes, consisting of enhancers that drive a remarkable spatial complexity of developmental expression patterns. Deletion of individual craniofacial enhancers from the mouse genome resulted in significant alterations of craniofacial shape, demonstrating their functional importance in defining face and skull morphology. These results demonstrate that enhancers play a pervasive role in mammalian craniofacial development and suggest that enhancer sequence variation contributes to human facial morphology

Disease-Causing 7.4 kb Cis-Regulatory Deletion Disrupting Conserved Non-Coding Sequences and Their Interaction with the FOXL2 Promotor: Implications for Mutation Screening

To date, the contribution of disrupted potentially cis-regulatory conserved non-coding sequences (CNCs) to human disease is most likely underestimated, as no systematic screens for putative deleterious variations in CNCs have been conducted. As a model for monogenic disease we studied the involvement of genetic changes of CNCs in the cis-regulatory domain of FOXL2 in blepharophimosis syndrome (BPES). Fifty-seven molecularly unsolved BPES patients underwent high-resolution copy number screening and targeted sequencing of CNCs. Apart from three larger distant deletions, a de novo deletion as small as 7.4 kb was found at 283 kb 5′ to FOXL2. The deletion appeared to be triggered by an H-DNA-induced double-stranded break (DSB). In addition, it disrupts a novel long non-coding RNA (ncRNA) PISRT1 and 8 CNCs. The regulatory potential of the deleted CNCs was substantiated by in vitro luciferase assays. Interestingly, Chromosome Conformation Capture (3C) of a 625 kb region surrounding FOXL2 in expressing cellular systems revealed physical interactions of three upstream fragments and the FOXL2 core promoter. Importantly, one of these contains the 7.4 kb deleted fragment. Overall, this study revealed the smallest distant deletion causing monogenic disease and impacts upon the concept of mutation screening in human disease and developmental disorders in particular

Evaluation of the regulatory potential of HSA21q conserved non-coding sequences (CNCs)

The comparative analysis of the human and mouse genomes has resulted in the identification of a large number of evolutionarily conserved sequences. The occurence of these so-called Conserved Non-Coding sequences (CNCs) has been further confirmed with the release of additional genomes. The strong evolutionary conservation of these sequences suggest they could contribute to the pool of yet non-identified functional sequences in the genome. This thesis has specifically focused on the functional analysis of a subset of HSA21 CNCs. The questions that were addressed are: i) what is the regulatory potentiel of CNCs in human cell lines, ii) what is the impact of phylogenetic conservation on their regulatory potential and iii) could CNCs contribute to human diseases?. Our study contributes to the functional annotation of CNCs by showing that the main role of mammalian CNCs might not be a regulatory one, however increasing the phylogenetic conservation favours a regulatory function