Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human embryonic stem cells reveals conditioned media from SDEC supports proliferation on type I collagen

ABSTRACT: Conditioned media from a human embryonic germ cell-derived line called SDEC was found to be supportive of human embryonic stem cell growth in the absence of feeder layers on a simple type I collagen matrix. We performed gene expression studies comparing this line to non-supportive cell lines (WI-38 and Detroit 551) to try to identify gene targets responsible for this phenomenon. We used Affymetrix microarrays to identify genes that are differentially regulated in SDEC vs. non-supportive cell lines. The goal is to determine which genes maybe be contributing to human embryonic stem cell growth in the absence of a mouse fibroblast feeder layer. Experiment Overall Design: Total RNA samples were extracted from SDEC, WI-38, and Detroit 551 (D551) cell lines to compare gene expressions. Three biological replicates of each were analyzed via micorarray. Gene targets were identified by looking for highly differentially regulated genes in SDEC compared to the non-supportive lines. We concentrated on secreted proteins (which could potentially be secreted into the conditioned media) which we identified by functional annotations and literature research.

ORGANISM(S): Homo sapiens

SUBMITTER: Michael Betenbaugh

PROVIDER: E-GEOD-15400 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Higher-order chromosomal organization for transcription regulation is poorly understood in eukaryotes. Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIA-PET), we mapped long-range chromatin interactions associated with RNA polymerase II in human cells and uncovered widespread promoter-centered intragenic, extragenic, and intergenic interactions. These interactions further aggregated into higher-order clusters, wherein proximal and distal genes were engaged through promoter-promoter interactions. Most genes with promoter-promoter interactions were active and transcribed cooperatively, and some interacting promoters could influence each other implying combinatorial complexity of transcriptional controls. Comparative analyses of different cell lines showed that cell-specific chromatin interactions could provide structural frameworks for cell-specific transcription, and suggested significant enrichment of enhancer-promoter interactions for cell-specific functions. Furthermore, genetically-identified disease-associated noncoding elements were found to be spatially engaged with corresponding genes through long-range interactions. Overall, our study provides insights into transcription regulation by three-dimensional chromatin interactions for both housekeeping and cell-specific genes in human cells. RNA polymerase II (RNAPII) bound chromatin interactions were extracted with Chromatin Interaction Analysis with Paired-End Tag (ChIA-PET) sequencing, in order to study the transcription regulations with RNAPII-associated long-range chromatin interactions. Five cell lines, namely MCF7 (ATCC# HTB-22), K562 (ATCC# CCL-243), HCT116 (ATCC# CCL-247), HeLa (ATCC# CCL-2.2), and NB4 (Roussel and Lanotte, 2001) (provided by Dr. Sherman Weissman, Yale University), were grown under standard culture conditions and harvested at log phase. Harvested cells were cross-linked using 1% formaldehyde followed by neutralization with 0.2M glycine. Chromatin was isolated and subjected to ChIA-PET protocol as described in Fullwood et al (Fullwood et al: An oestrogen-receptor-alpha-bound human chromatin interactome. Nature 2009, 462(7269):58-64). The ChIA-PET sequence reads were processed and analyzed using ChIA-PET Tool (Li et al: ChIA-PET tool for comprehensive chromatin interaction analysis with paired-end tag sequencing. Genome Biol 2010, 11(2):R22).

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Transcription profiling of human embryonic stem cells reveals conditioned media from SDEC supports proliferation on type I collagen

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