Nucleosome regulatory dynamics in response to TGFß.

Nucleosomes play important roles in a cell beyond their basal functionality in chromatin compaction. Their placement affects all steps in transcriptional regulation, from transcription factor (TF) binding to messenger ribonucleic acid (mRNA) synthesis. Careful profiling of their locations and dynamics in response to stimuli is important to further our understanding of transcriptional regulation by the state of chromatin. We measured nucleosome occupancy in human hepatic cells before and after treatment with transforming growth factor beta 1 (TGFß1), using massively parallel sequencing. With a newly developed method, SuMMIt, for precise positioning of nucleosomes we inferred dynamics of the nucleosomal landscape. Distinct nucleosome positioning has previously been described at transcription start site and flanking TF binding sites. We found that the average pattern is present at very few sites and, in case of TF binding, the double peak surrounding the sites is just an artifact of averaging over many loci. We systematically searched for depleted nucleosomes in stimulated cells compared to unstimulated cells and identified 24 318 loci. Depending on genomic annotation, 44-78% of them were over-represented in binding motifs for TFs. Changes in binding affinity were verified for HNF4α by qPCR. Strikingly many of these loci were associated with expression changes, as measured by RNA sequencing.

p53 regulates epithelial-mesenchymal transition induced by transforming growth factor ß.

Epithelial plasticity characterizes embryonic development and diseases such as cancer. Epithelial-mesenchymal transition (EMT) is a reversible and guided process of plasticity whereby embryonic or adult epithelia acquire mesenchymal properties. Multiple signaling pathways control EMT, and the transforming growth factor ß (TGFß) pathway plays a central role as its inducer. Here, we analyzed the role of the tumor suppressor protein p53 in TGFß-induced EMT in a well-established mammary epithelial cell model. We found that diploid NMuMG mammary cells bi-allelically express a wild type and a missense mutant (R277C) form of p53. Global reduction of both forms of p53 led to an enhanced EMT response to TGFß. Conversely, stabilization of wild type p53 using the compound nutlin had a negative impact on EMT. After silencing both p53 forms, rescue experiments using either wild type or R277C mutant p53 revealed that wild type p53 inhibited, whereas the R277C mutant did not significantly affect, the TGFß-driven EMT response. Under serum-free culture conditions, silencing of total p53 levels led to higher numbers of mammospheres characterized by larger size. Rescue of the silenced endogenous p53 with R277C mutant p53, in contrast, suppressed both size and numbers of the mammospheres. This work proposes that wild type p53 controls the efficiency by which mammary epithelial cells undergo EMT in response to TGFß.

PU.1 and bacterial metabolites regulate the human gene CAMP encoding antimicrobial peptide LL-37 in colon epithelial cells.

Mammalian antimicrobial peptides contribute to the protective barrier against microbes at epithelial surfaces. This study focuses on the promoter of the human CAMP gene, encoding the antimicrobial peptide LL-37, and induction of the gene in the colonic epithelial cell line HT-29. CAMP promoter segments were inserted in front of a luciferase reporter in order to identify regulatory regions. A transcription promoting region was identified and the transcription factor PU.1 of the Ets family was recruited to this region as shown by ChIP analysis. This ties PU.1 to the regulation of human innate epithelial defences for the first time. In addition, the conserved second intron was found to exert a transcription enhancing effect in cooperation with the 3' end of the proximal promoter, and the importance of two upstream AUG codons was examined. Moreover, we here demonstrate that lithocholic acid enhances CAMP transcription, and does so additively with butyrate. Thus, a crosstalk between bacteria and host epithelia of the gut could be partially mediated via these two bacterial products to obtain gut homeostasis.

The antimicrobial peptide rCRAMP is present in the central nervous system of the rat.

The brain is protected against invading pathogens by the blood-brain barrier, and also by its own innate defence system consisting of microglia and neurons in a coordinated network. Antimicrobial peptides are a part of the innate immune system at epithelial surfaces, and may also have important functions in the brain. Recently, we characterized the rat homologue of the human cathelicidin LL-37, designated rCRAMP. Here we present several lines of evidence for this peptide being expressed in rat CNS. (1) A peptide/protein extract of rat brain is active against bacteria in a salt-dependent manner. (2) Western blot analysis demonstrates the presence of rCRAMP in rat brain extract. (3) rCRAMP peptide and mRNA are present mainly in specific CNS regions (olfactory bulb, cerebellum, medulla oblongata and spinal cord). (4) rCRAMP-like immunoreactivity is detected in olfactory bulb, cerebellum and spinal cord by immunohistochemistry. (5) Moreover, the transcript of rCRAMP is detected in primary cultures from hippocampus, striatum, cerebellum and spinal cord, as shown with RT-PCR and Southern blot analyses. In addition, the rCRAMP peptide exhibits in vitro activity against the neuropathogenic bacterium Neisseria meningitidis. Taken together, these data suggest that the cathelicidin rCRAMP may play a role in the innate immunity of the CNS.