Dataset Information

Insights into Bidirectional Gene Expression Control Using the Canonical GAL1/GAL10 Promoter.

ABSTRACT: Despite advances made in understanding the effects of promoter structure on transcriptional activity, limited knowledge exists regarding the role played by chromatin architecture in transcription. Previous work hypothesized that transcription from the bidirectional GAL1/GAL10 promoter is controlled through looping of its UAS region around a nonstandard nucleosome. Here, by editing the GAL1/GAL10 promoter at high resolution, we provide insights into bidirectional expression control. We demonstrate that the first and fourth Gal4 binding sites within the UAS do not functionally contribute to promoter activation. Instead, these sites, along with nearby regulatory regions, contribute to the directional regulation of gene expression. Furthermore, Gal4 binding to the third binding site is critical for gene expression, while binding to the other three sites is not sufficient for transcriptional activation. Because the GAL1/GAL10 UAS can activate gene expression in many eukaryotes, the regulatory mechanism presented is expected to operate broadly across the eukaryotic clade.

SUBMITTER: Elison GL

PROVIDER: S-EPMC6263159 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Insights into Bidirectional Gene Expression Control Using the Canonical GAL1/GAL10 Promoter.

Elison Gregory L GL Xue Yuan Y Song Ruijie R Acar Murat M

Cell reports 20181001 3

Despite advances made in understanding the effects of promoter structure on transcriptional activity, limited knowledge exists regarding the role played by chromatin architecture in transcription. Previous work hypothesized that transcription from the bidirectional GAL1/GAL10 promoter is controlled through looping of its UAS region around a nonstandard nucleosome. Here, by editing the GAL1/GAL10 promoter at high resolution, we provide insights into bidirectional expression control. We demonstrat ...[more]

PMID: 30332652

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Project description:OBJECTIVE:Functional investigation of novel gene/protein targets associated with adipocyte differentiation or function heavily relies on efficient and accessible tools to manipulate gene expression in adipocytes in vitro. Recent advances in gene-editing technologies such as CRISPR-Cas9 have not only eased gene editing but also greatly facilitated modulation of gene expression without altering the genome. Here, we aimed to develop and validate a competent in vitro adipocyte model of controllable functionality as well as multiplexed gene manipulation in adipocytes, using the CRISPRa "SAM" system and siRNAs to simultaneously overexpress and silence selected genes in the same cell populations. METHODS:We introduced a stable expression of dCas9-VP64 and MS2-P65, the core components of the CRIPSRa SAM system, in mesenchymal C3H/10T1/2 cells through viral delivery and used guide RNAs targeting Pparγ2, Prdm16, Zfp423, or Ucp1 to control the expression of key genes involved in adipocyte differentiation and function. We additionally co-transfected mature adipocytes with sgRNA plasmids and siRNA to simultaneously up-regulate and silence selected genes. Quantitative gene expression, oxygen consumption, fluorescence-activated cell sorting and immunocytochemistry served as validation proxies in pre- or mature adipocytes. RESULTS:CRISPRa SAM-mediated up-regulation of a key adipogenic gene, Pparγ2, was successfully achieved using selected sgRNAs targeting the Pparγ2 promoter region (i.e. up to 104 fold); this induction was long lasting and sufficient to promote adipogenesis. Furthermore, co-activation of Pparγ2 with either Prdm16 or Zfp423 transcripts drove distinct thermogenic gene expression patterns associated with increased or decreased oxygen consumption, respectively, mimicking typical characteristics of brite/beige or white cell lineages. Lastly, we demonstrated that up-regulation of endogenous genes in mature adipocytes was also easily and efficiently achieved using CRISPRa SAM, here exemplified by targeted Ucp1 overexpression (up to 4 × 103 fold), and that it was compatible with concomitant gene silencing using siRNA, allowing for bidirectional manipulation of gene expression in the same cell populations. CONCLUSIONS:We demonstrate that the CRISPRa SAM system can be easily adopted and used to efficiently manipulate gene expression in pre- and mature adipocytes in vitro. Moreover, we describe a novel methodological approach combining the activation of endogenous genes and siRNA-mediated gene silencing, thus providing a powerful tool to functionally decipher genetic factors controlling adipogenesis and adipocyte functions.

Dataset Information

Insights into Bidirectional Gene Expression Control Using the Canonical GAL1/GAL10 Promoter.

Publications

Insights into Bidirectional Gene Expression Control Using the Canonical GAL1/GAL10 Promoter.

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