Chromatin endogenous cleavage (ChEC) uses fusion of a protein of interest to micrococcal nuclease (MNase) to target calcium-dependent cleavage to specific genomic loci in vivo. Here we report the combination of ChEC with high-throughput sequencing (ChEC-seq) to map budding yeast transcription factor (TF) binding. Temporal analysis of ChEC-seq data reveals two classes of sites for TFs, one displaying rapid cleavage at sites with robust consensus motifs and the second showing slow cleavage at largely unique sites with low-scoring motifs. Sites with high-scoring motifs also display asymmetric cleavage, indicating that ChEC-seq provides information on the directionality of TF-DNA interactions. Strikingly, similar DNA shape patterns are observed regardless of motif strength, indicating that the kinetics of ChEC-seq discriminates DNA recognition through sequence and/or shape. We propose that time-resolved ChEC-seq detects both high-affinity interactions of TFs with consensus motifs and sites preferentially sampled by TFs during diffusion and sliding.
SUMMARY
Interactions of transcription factors (TFs) with DNA comprise a complex interplay between base-specific amino acid contacts and readout of DNA structure. Recent studies highlighted the complementarity of DNA sequence and shape in modeling TF binding in vitro. Here, we provide a comprehensive evaluation of in vivo datasets to assess the predictive power obtained by augmenting various DNA sequence-based models of TF binding sites (TFBSs) with DNA shape features (helix twist, minor groove width, propeller twist, and roll). Results from 400 human ChIP-seq datasets for 76 TFs show that combining DNA shape features with position specific scoring matrix (PSSM) scores improves TFBS predictions. Improvement was also observed using TF flexible models and a machine-learning approach using a binary encoding of nucleotides in lieu of PSSMs. Incorporating DNA shape information is most beneficial for E2F and MADS-domain TF families. Our findings indicate that incorporating DNA sequence and shape information benefits the modeling of TF binding under complex in vivo conditions.
SUMMARY
The functionality of stem cells declines during aging thereby contributing to aging-associated impairments in tissue regeneration and function1. Alterations in developmental pathways have been associated with declines in stem cell function during aging2–6 but the nature of this process remains poorly defined. Hox genes are key regulators of stem cells and tissue patterning during embryogenesis with an unknown role in aging7,8. This study identifies an altered epigenetic stress response in muscle stem cells (also known as satellite cells = SCs) of aged compared to young mice. This includes aberrant global and site-specific induction of active chromatin marks in activated SCs from aged mice resulting in the specific induction of Hoxa9 among all Hox genes. Hoxa9 in turn activates several developmental pathways and represents a decisive factor separating gene expression of SCs from aged compared to young mice. This includes most of the currently known inhibitors of SC function in aging muscle such as Wnt-, TGFß-, JAK/STAT- and senescence signaling2–4,6. Inhibition of aberrant chromatin activation or deletion of Hoxa9 suffices to improve SC function and muscle regeneration in aged mice, while overexpression of Hoxa9 mimics aging-associated defects in SCs from young mice, which can be rescued by inhibition of Hoxa9-targeted developmental pathways. Together, these data delineate an altered epigenetic stress response in activated SCs from aged mice, which limits SC function and muscle regeneration by Hoxa9-dependent activation of developmental pathways.
Chromatin endogenous cleavage (ChEC) uses fusion of a protein of interest to micrococcal nuclease (MNase) to target calcium-dependent cleavage to specific genomic loci in vivo. Here we report the combination of ChEC with high-throughput sequencing (ChEC-seq) to map budding yeast transcription factor (TF) binding. Temporal analysis of ChEC-seq data reveals two classes of sites for TFs, one displaying rapid cleavage at sites with robust consensus motifs and the second showing slow cleavage at largely unique sites with low-scoring motifs. Sites with high-scoring motifs also display asymmetric cleavage, indicating that ChEC-seq provides information on the directionality of TF-DNA interactions. Strikingly, similar DNA shape patterns are observed regardless of motif strength, indicating that the kinetics of ChEC-seq discriminates DNA recognition through sequence and/or shape. We propose that time-resolved ChEC-seq detects both high-affinity interactions of TFs with consensus motifs and sites preferentially sampled by TFs during diffusion and sliding.
Pancreatic cancer is a drug resistant hypovascular tumor. Although there are many studies on the mechanism of chemoresistance in pancreatic cancers, studies on the relationship between ABCG2 and chemoresistance during hypoxia of pancreatic cancer are rare. Hypoxia-inducible factor-1 (HIF-1a) is a master regulator of the transcriptional response to oxygen deprivation in cancer cells. The aim of this study was to examine the role of ABCG 2 and HIF-1a in mediating chemoresistance during hypoxia in pancreatic cancer. In this study, we detected the expression levels of ABCG 2 , ERK/phosphorylated-ERK (p-ERK) and HIF-1a by immunohistochemistry in fresh pancreatic cancer and paracarcinoma tissues obtained from 25 patients. The mechanism by which p-ERK1/2 and HIF-1a affect ABCG 2 s expression was analyzed in the hypoxic cultured human pancreatic cancer cell line Capan-2. ABCG2-mediatedregulation of gemcitabine response under hypoxic conditions in pancreatic cancer cells was observed. It was found that ABCG 2 , ERK/p-ERK and HIF-1a were overexpressed in cancer tissues. ABCG2, HIF-1a and p-ERK levels were demonstrated to be high during hypoxic conditions in pancreatic cancer cells. Hypoxia induced phosphorylation of ERK1/2 to activate HIF-1a and contribute the ABCG 2 expression and mediated gemcitabine chemoresistance in pancreatic cancer cells. Hypoxic conditions induced HIF-1a binding to target gene sequences in the ABCG 2 promoter, resulting in increased transcription in pancreatic cancer cells. We demonstrated that hypoxia-induced chemoresistance is due to the regulation of ABCG 2 through the activation of ERK1/2/HIF-1a. ABCG 2 could serve as a predictor of gemcitabine response and, potentially, as a chemotherapeutic target in pancreatic cancer. Inhibition of ERK1/2 and HIF-1acould result in increased gemcitabine sensitization in pancreatic cancer with highly expressed ABCG 2 cell member protein.
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