RESUMO
Nuclear factor-kappa B (NF-κB) transcription factors coordinate gene expression in response to a broad array of cellular signals. In vertebrates, there are five NF-κB proteins (c-Rel, RelA/p65, RelB, p50, and p52) that can form various dimeric combinations exhibiting both common and dimer-specific DNA-binding specificity. In this chapter, we describe the use of the nuclear extract protein-binding microarray (nextPBM), a high-throughput method to characterize the DNA binding of transcription factors present in cell nuclear extracts. NextPBMs allow for sensitive analysis of the DNA binding of NF-κB dimers and their interactions with cell-specific cofactors.
Assuntos
Análise Serial de Proteínas , Animais , DNA/genética , DNA/metabolismo , NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , Extratos Vegetais , Ligação Proteica , Transdução de Sinais , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
The sea urchin larval skeleton offers a simple model for formation of developmental patterns. The calcium carbonate skeleton is secreted by primary mesenchyme cells (PMCs) in response to largely unknown patterning cues expressed by the ectoderm. To discover novel ectodermal cues, we performed an unbiased RNA-Seq-based screen and functionally tested candidates; we thereby identified several novel skeletal patterning cues. Among these, we show that SLC26a2/7 is a ventrally expressed sulfate transporter that promotes a ventral accumulation of sulfated proteoglycans, which is required for ventral PMC positioning and skeletal patterning. We show that the effects of SLC perturbation are mimicked by manipulation of either external sulfate levels or proteoglycan sulfation. These results identify novel skeletal patterning genes and demonstrate that ventral proteoglycan sulfation serves as a positional cue for sea urchin skeletal patterning.