Shining light on the transcriptome: Molecular regulatory networks leading to a fast-growth phenotype by continuous light in an environmentally sensitive teleost (Atherinopsidae).

Photoperiod can profoundly affect the physiology of teleost fish, including accelerated growth here defined as "fast growth phenotypes". However, molecular regulatory networks (MRNs) and biological processes being affected by continuous illumination and which allow some teleost species evident plasticity to thrive under this condition are not yet clear. Therefore, to provide a broad perspective of such mechanisms, Chirostoma estor fish were raised and sampled for growth under a simulated control (LD) 12 h Light: 12 h Dark or a continuous illumination (LL) 24 h Light: 0 h Dark since fertilization. The experiment lasted 12 weeks after hatching (wah), the time at which fish were sampled for growth, length, and whole-body cortisol levels. Additionally, 3 heads of fish from each treatment were used to perform a de novo transcriptome analysis using Next-Generation Sequencing. Fish in LL developed the fast growth phenotype with significant differences visible at 4 wah and gained 66% more mass by 12 wah than LD fish. Cortisol levels under LL were below basal levels at all times compared to fish in LD, suggesting circadian dysregulation effects. A strong effect of LL was observed in samples with a generalized down-regulation of genes except for Reactive Oxygen Species responses, genome stability, and growth biological processes. To our knowledge, this work is the first study using a transcriptomic approach to understand environmentally sensitive MRNs that mediate phenotypic plasticity in fish submitted to continuous illumination. This study gives new insights into the plasticity mechanisms of teleost fish under constant illumination.

The phytochemical 3,3'-diindolylmethane decreases expression of AR-controlled DNA damage repair genes through repressive chromatin modifications and is associated with DNA damage in prostate cancer cells.

Androgen receptor (AR) is a transcription factor involved in normal prostate physiology and prostate cancer (PCa) development. 3,3'-Diindolylmethane (DIM) is a promising phytochemical agent against PCa that affects AR activity and epigenetic regulators in PCa cells. However, whether DIM suppresses PCa via epigenetic regulation of AR target genes is unknown. We assessed epigenetic regulation of AR target genes in LNCaP PCa cells and showed that DIM treatment led to epigenetic suppression of AR target genes involved in DNA repair (PARP1, MRE11, DNA-PK). Decreased expression of these genes was accompanied by an increase in repressive chromatin marks, loss of AR occupancy and EZH2 recruitment to their regulatory regions. Decreased DNA repair gene expression was associated with an increase in DNA damage (γH2Ax) and up-regulation of genomic repeat elements LINE1 and α-satellite. Our results suggest that DIM suppresses AR-dependent gene transcription through epigenetic modulation, leading to DNA damage and genome instability in PCa cells.

Analysis of autophagic flux in response to sulforaphane in metastatic prostate cancer cells.

SCOPE: The phytochemical sulforaphane (SF) has been shown to decrease prostate cancer metastases in a genetic mouse model of prostate carcinogenesis, though the mechanism of action is not fully known. SF has been reported to stimulate autophagy, and modulation of autophagy has been proposed to influence SF cytotoxicity; however, no conclusions about autophagy can be drawn without assessing autophagic flux, which has not been characterized in prostate cancer cells following SF treatment. METHODS AND RESULTS: We conducted an investigation to assess the impact of SF on autophagic flux in two metastatic prostate cancer cell lines at a concentration shown to decrease metastasis in vivo. Autophagic flux was assessed by multiple autophagy related proteins and substrates. We found that SF can stimulate autophagic flux and cell death only at high concentrations, above what has been observed in vivo. CONCLUSION: These results suggest that SF does not directly stimulate autophagy or cell death in metastatic prostate cancer cells under physiologically relevant conditions, but instead supports the involvement of in vivo factors as important effectors of SF-mediated prostate cancer suppression.

Effects of sulforaphane and 3,3'-diindolylmethane on genome-wide promoter methylation in normal prostate epithelial cells and prostate cancer cells.

Epigenetic changes, including aberrant DNA methylation, result in altered gene expression and play an important role in carcinogenesis. Phytochemicals such as sulforaphane (SFN) and 3,3'-diindolylmethane (DIM) are promising chemopreventive agents for the treatment of prostate cancer. Both have been shown to induce re-expression of genes, including tumor suppressor genes silenced in cancer cells, via modulation of epigenetic marks including DNA methylation. However, it remained unclear the effects SFN and DIM on DNA methylation at a genomic scale. The goal of this study was to determine the genome-wide effects of SFN and DIM on promoter methylation in normal prostate epithelial cells and prostate cancer cells. Both SFN and DIM treatment decreased DNA methyltransferase expression in normal prostate epithelial cells (PrEC), and androgen-dependent (LnCAP) and androgen-independent (PC3) prostate cancer cells. The effects of SFN and DIM on promoter methylation profiles in normal PrEC, LnCAP and PC3 prostate cancer cells were determined using methyl-DNA immunoprecipitation followed by genome-wide DNA methylation array. We showed widespread changes in promoter methylation patterns, including both increased and decreased methylation, in all three prostate cell lines in response to SFN or DIM treatments. In particular, SFN and DIM altered promoter methylation in distinct sets of genes in PrEC, LnCAP, and PC3 cells, but shared similar gene targets within a single cell line. We further showed that SFN and DIM reversed many of the cancer-associated methylation alterations, including aberrantly methylated genes that are dysregulated or are highly involved in cancer progression. Overall, our data suggested that both SFN and DIM are epigenetic modulators that have broad and complex effects on DNA methylation profiles in both normal and cancerous prostate epithelial cells. Results from our study may provide new insights into the epigenetic mechanisms by which SFN and DIM exert their cancer chemopreventive effects.

TnaA, an SP-RING protein, interacts with Osa, a subunit of the chromatin remodeling complex BRAHMA and with the SUMOylation pathway in Drosophila melanogaster.

Tonalli A (TnaA) is a Drosophila melanogaster protein with an XSPRING domain. The XSPRING domain harbors an SP-RING zinc-finger, which is characteristic of proteins with SUMO E3 ligase activity. TnaA is required for homeotic gene expression and is presumably involved in the SUMOylation pathway. Here we analyzed some aspects of the TnaA location in embryo and larval stages and its genetic and biochemical interaction with SUMOylation pathway proteins. We describe that there are at least two TnaA proteins (TnaA130 and TnaA123) differentially expressed throughout development. We show that TnaA is chromatin-associated at discrete sites on polytene salivary gland chromosomes of third instar larvae and that tna mutant individuals do not survive to adulthood, with most dying as third instar larvae or pupae. The tna mutants that ultimately die as third instar larvae have an extended life span of at least 4 to 15 days as other SUMOylation pathway mutants. We show that TnaA physically interacts with the SUMO E2 conjugating enzyme Ubc9, and with the BRM complex subunit Osa. Furthermore, we show that tna and osa interact genetically with SUMOylation pathway components and individuals carrying mutations for these genes show a phenotype that can be the consequence of misexpression of developmental-related genes.

Open, repair and close again: chromatin dynamics and the response to UV-induced DNA damage.

Due to its link with human pathologies, including cancer, the mechanism of Nucleotide Excision Repair (NER) has been extensively studied. Most of the pathway and players have been defined using in vitro reconstitution experiments. However, in vivo, the NER machinery must deal with the presence of organized chromatin, which in some regions, such as heterochromatin, is highly condensed but still susceptible to DNA damage. A series of events involving different chromatin-remodeling factors and histone-modifying enzymes target chromatin regions that contain DNA lesions. CPDs change the structure of the nucleosome, allowing access to factors that can recognize the lesion. Next, DDB1-DDB2 protein complexes, which mono-ubiquitinate histones H2A, H3, and H4, recognize nucleosomes containing DNA lesions. The ubiquitinated nucleosome facilitates the recruitment of ATP-dependent chromatin-remodeling factors and the XPC-HR23B-Centrin 2 complex to the target region. Different ATP-dependent chromatin-remodeling factors, such as SWI/SNF and INO80, have been identified as having roles in the UV irradiation response prior to the action of the NER machinery. Subsequently, remodeling of the nucleosome allows enzymatic reactions by histone-modifying factors that may acetylate, methylate or demethylate specific histone residues. Intriguingly, some of these histone modifications are dependent on p53. These histone modifications and the remodeling of the nucleosome allow the entrance of TFIIH, XPC and other NER factors that remove the damaged strand; then, gap-filling DNA synthesis and ligation reactions are carried out after excision of the oligonucleotide with the lesion. Finally, after DNA repair, the initial chromatin structure has to be reestablished. Therefore, factors that modulate chromatin dynamics contribute to the NER mechanism, and they are significant in the future design of treatments for human pathologies related to genome instability and the appearance of drug-resistant tumors.

Drosophila p53 is required to increase the levels of the dKDM4B demethylase after UV-induced DNA damage to demethylate histone H3 lysine 9.

Chromatin undergoes a variety of changes in response to UV-induced DNA damage, including histone acetylation. In human and Drosophila cells, this response is affected by mutations in the tumor suppressor p53. In this work, we report that there is a global decrease in trimethylated Lys-9 in histone H3 (H3K9me3) in salivary gland cells in wild type flies in response to UV irradiation. In contrast, flies with mutations in the Dmp53 gene have reduced basal levels of H3K9me3, which are then increased after UV irradiation. The reduction of H3K9me3 in response to DNA damage occurs preferentially in heterochromatin. Our experiments demonstrate that UV irradiation enhances the levels of Lys-9 demethylase (dKDM4B) transcript and protein in wild type flies, but not in Dmp53 mutant flies. Dmp53 binds to a DNA element in the dKdm4B gene as a response to UV irradiation. Furthermore, heterozygous mutants for the dKdm4B gene are more sensitive to UV irradiation; they are deficient in the removal of cyclobutane-pyrimidine dimers, and the decrease of H3K9me3 levels following DNA damage is not observed in dKdm4B mutant flies. We propose that in response to UV irradiation, Dmp53 enhances the expression of the dKDM4B histone demethylase, which demethylates H3K9me3 preferentially in heterochromatin regions. This mechanism appears to be essential for the proper function of the nucleotide excision repair system.