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RNA-seq on dual-FACS sorted Drosophila Intestinal stem cell and Enteroblast, and effect of SPEN loss of function on gene expression

ABSTRACT: Precise regulation of stem cell self-renewal and differentiation properties is essential for tissue homeostasis. Using the adult Drosophila intestine to study molecular mechanisms controlling stem cell properties, we identify the gene split-ends (spen) in a genetic screen as a novel regulator of intestinal stem cell fate. Spen family genes encode conserved RNA recognition motif-containing proteins that are reported to have roles in RNA splicing and transcriptional regulation. We demonstrate that spen loss of function in intestinal stem cells results in an abnormal increase in the number of stem cell-like cells and that Spen acts to control early commitment events of the stem cells. Using two-color cell sorting of stem cells and their daughters, we characterize spen-dependent changes in RNA abundance and exon usage, and find potential key regulators downstream of spen. Our work identifies spen as an important regulator of adult stem cells in the Drosophila intestine, provides new insight to Spen-family protein functions, and may also shed light on Spen’s mode of action in other developmental contexts.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE84367 | GEO | 2017/07/20

SECONDARY ACCESSION(S): PRJNA329039

REPOSITORIES: GEO

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SPEN is a molecular integrator that bridges Xist with transcriptional and epigenetic control of X-chromosome inactivation

Project description:Xist represents a paradigm for long non-coding RNA function in epigenetic regulation, although how it mediates X-chromosome inactivation (XCI) remains largely unexplained. Multiple Xist-RNA binding proteins have recently been identified, including SPEN/SHARP, whose knockdown has been associated with deficient XCI at multiple loci. Here we demonstrate that SPEN is a key orchestrator of XCI in vivo and unravel its mechanism of action. We show that SPEN is essential for initiating gene silencing on the X chromosome in preimplantation mouse embryos and embryonic stem cells. On the other hand, SPEN is dispensable for maintenance of XCI in neural progenitor cells, although it significantly dampens expression of genes that escape from XCI. During initiation of XCI, we show by live-cell imaging and CUT&RUN approaches that SPEN is immediately recruited to the X chromosome upon Xist up-regulation, where it is targeted to enhancers and promoters of actively transcribed genes. SPEN rapidly disengages from chromatin once silencing is accomplished, implying a need for active transcription to tether it to chromatin. We define SPEN’s SPOC (SPEN paralog and ortholog C-terminal) domain as a major effector of SPEN’s gene silencing function, and show that artificial tethering of SPOC to Xist RNA is sufficient to mediate X-linked gene silencing. We identify SPOC’s protein partners which include NCOR/SMRT, the m6A RNA methylation machinery, the NuRD complex, RNA polymerase II and factors involved in regulation of transcription initiation and elongation. We propose that SPEN acts as a molecular integrator for initiation of XCI, bridging Xist RNA with the transcription machinery as well as nucleosome remodelers and histone deacetylases, at active enhancers and promoters.

2021-04-19 | PXD015699 | Pride

Study of SPEN knockdown in MCF10A cells

Project description:In this experiment, we knocked down the expression of Split Ends (SPEN), a gene that we found was involved in the regulation of primary cilia formation, in MCF10A cells, to evaluate its effects on transcription. Split Ends (SPEN) is a transcriptional coregulator that have formerly identified as a tumour suppressor gene in ER-positive breast cancers. We expect the knockdown of SPEN in MCF10A cells to be accompanied with the down-regulation of genes involved in ciliogenesis as we observed that SPEN knockdown decreases primary cilia formation in those cells. We chose MCF10A cells because they harbour lots of primary cilia and therefore represent a good model to study the effect of SPEN on primary cilia formation.

2017-08-15 | E-MTAB-4974 | biostudies-arrayexpress

Study of SPEN overexpression in T47D cells. (2)

Project description:We investigated the functions/pathways affected by SPEN in breast cancer by global expression profiling in a cell model, where the human breast cancer cell line, T47D, were transfected with an expression vector coding for SPEN.

2015-10-21 | E-MTAB-2182 | biostudies-arrayexpress

A SPEN Complex Serves as a Scaffold to Coordinate Multiple Epigenetic Regulatory Mechanisms in Breast Cancer Stem Cells

Project description:There is substantial evidence that many cancers, including human breast cancer are hierarchically organized and driven by a cellular population that displays stem cell properties. These stem-like cells (CSC’s) mediate tumor metastasis and by virtue of their relative therapeutic resistance mediate tumor recurrence. Both normal and malignant stem cells are regulated through epigenetic mechanisms which involve DNA and histone modifications as well as noncoding RNAs. However, the mechanisms responsible for coordinating these multiple levels of epigenetic regulation in CSC’s remain elusive. Here, we identify a SPEN-Xist complex in breast CSC’s cells which, by virtue of inclusion of multiple epigenetic regulatory proteins, provides a scaffold for coordinating multiple levels of epigenetic regulation in these cells. Genetic knockdown of SPEN or XIST significantly reduces the CSC population as accessed by ALDH expression, sphere formation or tumor initiating capacity in xerograph models. Gene expression analysts suggests that the SPEN- XIST complex modulates multiple CSC regulatory pathways via the coordination of epigenetic modulatory proteins. This work has fundamental implications for understanding the epigenetic regulation of cancer stem cells as well as for developing strategies to target these cells

2020-01-25 | GSE144234 | GEO

Study of SPEN knockdown in MCF-7 cells. (2)

Project description:We investigated the functions/pathways affected by SPEN knockdown in breast cancer by global expression profiling in a cell model, where the human breast cancer cell line, MCF-7, were transfected with an shRNA targeting SPEN mRNA.

2015-10-21 | E-MTAB-2183 | biostudies-arrayexpress

Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation [irCLIP-seq]

Project description:We show that Spen, an Xist binding repressor protein essential for XCI, binds to ancient retroviral RNA transcribed genome-wide, performing a surveillance role to recruit chromatin silencing machinery to these parasitic loci. Spen inactivation leads to de-repression of endogenous retroviral (ERV) elements in embryonic stem cells, with gain of chromatin accessibility, active histone modifications, and ERV RNA transcription. Spen binds directly to ERV RNAs that are highly similar to the A-repeat of Xist, a region critical for Xist-mediated gene silencing. ERV RNA and Xist A-repeat bind the RRM3 domain of Spen in a competitive manner and insertion of an ERV into an A-repeat deficient Xist rescues binding of Xist RNA to Spen.

2020-05-16 | GSE131376 | GEO

Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation [ATAC-Seq]

2020-05-16 | GSE131374 | GEO

Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation [RNA-Seq]

2020-05-16 | GSE131373 | GEO

Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation [ChIP-Seq]

2020-05-16 | GSE131412 | GEO

Effect of estradiol and tamoxifen on SPEN-expressing and control T47D cells.

Project description:Split Ends (SPEN) is a transcriptional coregulator that have formerly identified as a tumour suppressor gene in ER-positive breast cancers. However, ER-positive breast cancers are diagnosed at similar frequencies in pre- and post-menopausal women who show significantly different circulating hormone levels. This therefore raises the possibility that SPEN functions under hormone-depleted settings may contrast with its roles in the presence of hormones. We therefore attempted to explore the cellular functions regulated by SPEN under hormone-depleted settings using a previously established model with T47D cells stably transfected with a control vector (non-target) or SPEN-expressing vector. In particular, we attempted to investigate the hormone-independent transcriptional program regulated by SPEN in breast cancer. To achieve this, we have treated previously established T47D cells stably transfected with a control vector (non-target) or SPEN-expressing vector. These cells were allowed to grow in hormone-depleted conditions for 4 days. To minimize external biases introduced by hormone depletion or any transcriptional contribution from the estrogen receptor (ER), we also performed gene expression profiling analyses on the same cells but stimulated with an estrogen receptor (ER) agonist (Estradiol) or antagonist (Tamoxifen).

2017-08-15 | E-MTAB-4975 | biostudies-arrayexpress

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