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Xenopus laevis

ABSTRACT: RNAseq and ChIPseq profiling of Foxn4 and Foxj1 in multiciliated cells

PROVIDER: PRJNA351216 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR4473800.fastq.gz	Fastqsanger.gz
	SRR4473810.fastq.gz	Fastqsanger.gz
	SRR4473801.fastq.gz	Fastqsanger.gz
	SRR4473811.fastq.gz	Fastqsanger.gz
	SRR4478521.fastq.gz	Fastqsanger.gz

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Xenopus laevis

Project description:RNAseq profiling of multiciliated cells

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Histone modification and transcription factor ChIPseq of Xenopus laevis epithelial progenitors

Project description:To determine the positions of promoters and enhancers in developing Xenopus laevis epithelial progenitors, we performed ChIPseq on the histone modifications H3K4me3 and H3K27ac. We also performed ChIPseq on the transcription factors foxj1 (in the presence or absence of rfx2), myb (in the presence or absence of multicilin), and rad21. Some embryos were harvested as wild-types; in other experiments, we injected embryos with mRNAs encoding FLAG-foxj1 (with and without rfx2 morpholino) or GFP-myb (with and without an inducible form of multicilin (mcidas-HGR)). We then isolated epithelial progenitors surgically and, when injected with multicilin, induced at mid-stage 11. We then harvested chromatin at 9 hours after induction (roughly stage 18) and performed ChIPseq using antibodies against endogenous targets (H3K4me3, H3K27ac, rad21) or protein tags (FLAG, GFP). We then sequenced these libraries, aligned the reads to the X. laevis genome (version 9.1) with bwa mem and called peaks with HOMER, using input as background.

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Xenopus laevis

Project description:Histone modification and transcription factor ChIPseq of Xenopus laevis epithelial progenitors

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RNAseq analysis of Xenopus laevis wildtype and emi2 mutant multiciliated cells during and post differentiation

Project description:Multiciliated cells (MCCs) form when progenitors massively expand centriole number, yielding the hundreds of basal bodies required to extend multiple motile cilia. This organelle biogenesis is promoted transcriptionally by Multicilin acting in a complex with the E2F transcription factors, which activates the expression of genes known to be involved required to form centrioles during the cell cycle, but also requires a cell cycle state required for the centrosome cycle. Multicilin also activates the expression of Emi2 (fbxo43) an inhibitor of the APC/C ubiquitin ligase. In Emi2 mutants, basal body formation during MCCl differentiation is blocked. RNAseq analysis is used to show that gene expression associated with MCC differentiation is upregulated normally in Emi2 mutants, but surprisingly is not downregulated when MCC differentiation is complete. Emi2 is required to promote the centrosomal cycle during MCC differentiation, but then also acts to turn off gene expression associated with centriole biogenesis in differentiated cells.

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Multicilin drives centriole biogenesis via E2f proteins

Project description:Biochemistry suggests e2f4 forms a complex with the coiled-coiled protein multicilin (MCIDAS), a protein that is necessary and sufficient to specify multiciliated cells in vertebrates. Here, we performed RNAseq on Xenopus laevis ectoderm in the presence of multicilin alone or multicilin and a dominant-negative e2f4 construct. We also performed ChIPseq on e2f4 in the presence or absence of multicilin. Taken together, these data demonstrate how multicilin affects e2f4 genomic targets and their downstream transcription. RNAseq: misexpression of multicilin-HGR +/- dominant-negative e2f4 messenger RNAs in X. laevis animal caps, multicilin induced with dexamethasone at mid-stage 11 and harvested at 3 timepoints (3, 6, and 9 hours after induction, roughly corresponding to stages 13, 16, and 18) with 3 biological replicates. ChIPseq: misexpression of e2f4-GFP +/- multicilin-HGR messenger RNAs in X. laevis animal caps, multicilin induced at mid-stage 11 and harvested at one timepoint (6 hours after induction, roughly corresponding to stage 16), immunoprecipitated with anti-GFP and sequenced; 2 biological replicates. Background was input prior to IP.

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