Differential DNA methylation and CTCF binding between the ESR1 promoter a of MCF-7 and MDA-MB-231 breast cancer cells.

BACKGROUND: ESR1 is expressed by 60-70% of breast tumours. it's a good prognosis factor and the target of hormone therapy. Optimization of ESR1 reactivation therapy is currently ongoing. Here we probe if the transcription factor CTCF plays a role in the differential expression of ESR1 in the breast cancer cell lines MCF-7 (ESR1+) and MDA-MB-231 (ESR1-). METHODS AND RESULTS: Knockdown of CTCF in MCF-7 resulted in decreased ESR1 gene expression. CTCF binds to the promoter of ESR1 in MCF-7 but not in MDA-MB-231 cells. CTCF ESR1 binding sites are unmethylated in MCF7 but methylated in MDA-MB-231 cells. CONCLUSION: ESR1 expression in MCF7 cells is dependent on CTCF expression. CTCF can bind to specific regions of the promotor of ESR1 gene in MCF-7 cells but not in MDA-MB-231 cells, this correlates with the methylation status of these regions and could be involved in the transcriptional regulation of ESR1.

TGF-ß/SMAD canonical pathway induces the expression of transcriptional cofactor TAZ in liver cancer cells.

The TGF-ß and Hippo pathways are critical for liver size control, regeneration, and cancer progression. The transcriptional cofactor TAZ, also named WWTR1, is a downstream effector of Hippo pathway and plays a key role in the maintenance of liver physiological functions. However, the up-regulation of TAZ expression has been associated with liver cancer progression. Recent evidence shows crosstalk of TGF-ß and Hippo pathways, since TGF-ß modulates TAZ expression through different mechanisms in a cellular context-dependent manner but supposedly independent of SMADs. Here, we evaluate the molecular interplay between TGF-ß pathway and TAZ expression and observe that TGF-ß induces TAZ expression through SMAD canonical pathway in liver cancer HepG2 cells. Therefore, TAZ cofactor is a primary target of TGF-ß/SMAD-signaling, one of the pathways altered in liver cancer.

DNMT3B overexpression downregulates genes with CpG islands, common motifs, and transcription factor binding sites that interact with DNMT3B.

DNA methylation is a key epigenetic modification to regulate gene expression in mammalian cells. Abnormal DNA methylation in gene promoters is common across human cancer types. DNMT3B is the main de novo methyltransferase enhanced in several primary tumors. How de novo methylation is established in genes related to cancer is poorly understood. CpG islands (CGIs), common sequences, and transcription factors (TFs) that interact with DNMT3B have been associated with abnormal de novo methylation. We initially identified cis elements associated with DNA methylation to investigate the contribution of DNMT3B overexpression to the deregulation of its possible target genes in an epithelial cell model. In a set of downregulated genes (n = 146) from HaCaT cells with DNMT3B overexpression, we found CGI, common sequences, and TFs Binding Sites that interact with DNMT3B (we called them P-down-3B). PPL1, VAV3, IRF1, and BRAF are P-down-3B genes that are downregulated and increased their methylation in DNMT3B presence. Together these findings suggest that methylated promoters aberrantly have some cis elements that could conduce de novo methylation by DNMT3B.

Interplay Between the Histone Variant H2A.Z and the Epigenome in Pancreatic Cancer.

BACKGROUND: The oncogenic process is orchestrated by a complex network of chromatin remodeling elements that shape the cancer epigenome. Histone variant H2A.Z regulates DNA control elements such as promoters and enhancers in different types of cancer; however, the interplay between H2A.Z and the pancreatic cancer epigenome is unknown. OBJECTIVE: This study analyzed the role of H2A.Z in different DNA regulatory elements. METHODS: We performed Chromatin Immunoprecipitation Sequencing assays (ChiP-seq) with total H2A.Z and acetylated H2A.Z (acH2A.Z) antibodies and analyzed published data from ChIP-seq, RNA-seq, bromouridine labeling-UV and sequencing (BruUV-seq), Hi-C and ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) in the pancreatic cancer cell line PANC-1. RESULTS: The results indicate that total H2A.Z facilitates the recruitment of RNA polymerase II and transcription factors at promoters and enhancers allowing the expression of pro-oncogenic genes. Interestingly, we demonstrated that H2A.Z is enriched in super-enhancers (SEs) contributing to the transcriptional activation of key genes implicated in tumor development. Importantly, we established that H2A.Z contributes to the three-dimensional (3D) genome organization of pancreatic cancer and that it is a component of the Topological Associated Domains (TADs) boundaries in PANC-1 and that total H2A.Z and acH2A.Z are associated with A and B compartments, respectively. CONCLUSIONS: H2A.Z participates in the biology and development of pancreatic cancer by generating a pro-oncogenic transcriptome through its posttranslational modifications, interactions with different partners, and regulatory elements, contributing to the oncogenic 3D genome organization. These data allow us to understand the molecular mechanisms that promote an oncogenic transcriptome in pancreatic cancer mediated by H2A.Z.

Cancer Epigenetics: An Overview.

Cancer is a complex disease caused by genetic and epigenetic alterations in the control of cell division. Findings from the field of cancer genomics and epigenomics have increased our understanding of the origin and evolution of tumorigenic processes, greatly advancing our knowledge of the molecular etiology of cancer. Consequently, any contemporary view of cancer research must consider tumorigenesis as a cellular phenomenon that is a result of the interplay between genetic and epigenetic mutations and their interaction with environmental factors, including our microbiome, that influences cellular metabolism and proliferation rates. The integration and better knowledge of these processes will help us to improve diagnosis, prognosis, and future genetic and epigenetic therapies. Here, I present an overview of the epigenetic processes that are affected in cancer and how they contribute to the onset and progression of the disease. Finally, I discuss how the development of sophisticated experimental approaches and computational tools, including novel ways to exploit large data sets, could contribute to the better understanding and treatment of cancer.

Emerging Functions of lncRNA Loci beyond the Transcript Itself.

Thousands of long noncoding RNAs (lncRNAs) are actively transcribed in mammalian genomes. This class of RNAs has important regulatory functions in a broad range of cellular processes and diseases. Numerous lncRNAs have been demonstrated to mediate gene regulation through RNA-based mechanisms. Simultaneously, non-functional lncRNA transcripts derived from the activity of lncRNA loci have been identified, which underpin the notion that a considerable fraction of lncRNA loci exert regulatory functions through mechanisms associated with the production or the activity of lncRNA loci beyond the synthesized transcripts. We particularly distinguish two main RNA-independent components associated with regulatory effects; the act of transcription and the activity of DNA regulatory elements. We describe the experimental approaches to distinguish and understand the functional mechanisms derived from lncRNA loci. These scenarios reveal emerging mechanisms important to understanding the lncRNA implications in genome biology.

Development and Testing of a Low-Cost Inactivation Buffer That Allows for Direct SARS-CoV-2 Detection in Saliva.

Massive testing is a cornerstone in efforts to effectively track infections and stop COVID-19 transmission, including places with good vaccination coverage. However, SARS-CoV-2 testing by RT-qPCR requires specialized personnel, protection equipment, commercial kits, and dedicated facilities, which represent significant challenges for massive testing in resource-limited settings. It is therefore important to develop testing protocols that are inexpensive, fast, and sufficiently sensitive. Here, we optimized the composition of a buffer (PKTP), containing a protease, a detergent, and an RNase inhibitor, which is compatible with the RT-qPCR chemistry, allowing for direct SARS-CoV-2 detection from saliva without extracting RNA. PKTP is compatible with heat inactivation, reducing the biohazard risk of handling samples. We assessed the PKTP buffer performance in comparison to the RNA-extraction-based protocol of the US Centers for Disease Control and Prevention in saliva samples from 70 COVID-19 patients finding a good sensitivity (85.7% for the N1 and 87.1% for the N2 target) and correlations (R = 0.77, p < 0.001 for N1, and R = 0.78, p < 0.001 for N2). We also propose an auto-collection protocol for saliva samples and a multiplex reaction to minimize the PCR reaction number per patient and further reduce costs and processing time of several samples, while maintaining diagnostic standards in favor of massive testing.

Genome accessibility dynamics in response to phosphate limitation is controlled by the PHR1 family of transcription factors in Arabidopsis.

As phosphorus is one of the most limiting nutrients in many natural and agricultural ecosystems, plants have evolved strategies that cope with its scarcity. Genetic approaches have facilitated the identification of several molecular elements that regulate the phosphate (Pi) starvation response (PSR) of plants, including the master regulator of the transcriptional response to phosphate starvation PHOSPHATE STARVATION RESPONSE1 (PHR1). However, the chromatin modifications underlying the plant transcriptional response to phosphate scarcity remain largely unknown. Here, we present a detailed analysis of changes in chromatin accessibility during phosphate starvation in Arabidopsis thaliana root cells. Root cells undergo a genome-wide remodeling of chromatin accessibility in response to Pi starvation that is often associated with changes in the transcription of neighboring genes. Analysis of chromatin accessibility in the phr1 phl2 double mutant revealed that the transcription factors PHR1 and PHL2 play a key role in remodeling chromatin accessibility in response to Pi limitation. We also discovered that PHR1 and PHL2 play an important role in determining chromatin accessibility and the associated transcription of many genes under optimal Pi conditions, including genes involved in the PSR. We propose that a set of transcription factors directly activated by PHR1 in Pi-starved root cells trigger a second wave of epigenetic changes required for the transcriptional activation of the complete set of low-Pi-responsive genes.

Vegfa promoter gene hypermethylation at HIF1α binding site is an early contributor to CKD progression after renal ischemia.

Chronic hypoxia is a major contributor to Chronic Kidney Disease (CKD) after Acute Kidney Injury (AKI). However, the temporal relation between the acute insult and maladaptive renal response to hypoxia remains unclear. In this study, we analyzed the time-course of renal hemodynamics, oxidative stress, inflammation, and fibrosis, as well as epigenetic modifications, with focus on HIF1α/VEGF signaling, in the AKI to CKD transition. Sham-operated, right nephrectomy (UNx), and UNx plus renal ischemia (IR + UNx) groups of rats were included and studied at 1, 2, 3, or 4 months. The IR + UNx group developed CKD characterized by progressive proteinuria, renal dysfunction, tubular proliferation, and fibrosis. At first month post-ischemia, there was a twofold significant increase in oxidative stress and reduction in global DNA methylation that was maintained throughout the study. Hif1α and Vegfa expression were depressed in the first and second-months post-ischemia, and then Hif1α but not Vegfa expression was recovered. Interestingly, hypermethylation of the Vegfa promoter gene at the HIF1α binding site was found, since early stages of the CKD progression. Our findings suggest that renal hypoperfusion, inefficient hypoxic response, increased oxidative stress, DNA hypomethylation, and, Vegfa promoter gene hypermethylation at HIF1α binding site, are early determinants of AKI-to-CKD transition.

Generation of Functional Genetic Study Models in Zebrafish Using CRISPR-Cas9.

CRISPR-Cas9 is a method for genome editing that can be used efficiently for in vivo applications; the basic implementation of this method is used to generate genome site-directed sequence eliminations. Here we describe a protocol for genome editing using CRISPR-Cas9 in zebrafish (Danio rerio) one-cell embryos.

An Intronic Alu Element Attenuates the Transcription of a Long Non-coding RNA in Human Cell Lines.

Alu elements are primate-specific repeats and represent the most abundant type of transposable elements (TE) in the human genome. Genome-wide analysis of the enrichment of histone post-translational modifications suggests that human Alu sequences could function as transcriptional enhancers; however, no functional experiments have evaluated the role of Alu sequences in the control of transcription in situ. The present study analyses the regulatory activity of a human Alu sequence from the AluSx family located in the second intron of the long intergenic non-coding RNA Linc00441, found in divergent orientation to the RB1 gene. We observed that the Alu sequence acts as an enhancer element based on reporter gene assays while CRISPR-Cas9 deletions of the Alu sequence in K562 cells resulted in a marked transcriptional upregulation of Linc00441 and a decrease in proliferation. Our results suggest that an intragenic Alu sequence with enhancer activity can act as a transcriptional attenuator of its host lincRNA.

Novel tephritid-specific features revealed from cytological and transcriptomic analysis of Anastrepha ludens embryonic development.

Anastrepha ludens is a major pest of fruits including citrus and mangoes in Mexico and Central America with major economic and social impacts. Despite its importance, our knowledge on its embryonic development is scarce. Here, we report the first cytological study of embryonic development in A. ludens and provide a transcriptional landscape during key embryonic stages. We established 17 stages of A. ludens embryogenesis that closely resemble the morphological events observed in Drosophila. In addition to the extended duration of embryonic development, we observed notable differences including yolk extrusion at both poles of the embryo, distinct nuclear division waves in the syncytial blastoderm and a heterochronic change during the involution of the head. Characterization of the transcriptional dynamics during syncytial blastoderm, cellular blastoderm and gastrulation, showed that approximately 9000 different transcripts are present at each stage. Even though we identified most of the transcripts with a role during embryonic development present in Drosophila, including sex determination genes, a number of transcripts were absent not only in A. ludens but in other tephritids such as Ceratitis capitata and Bactrocera dorsalis. Intriguingly, some A. ludens embryo transcripts encode proteins present in other organisms but not in other flies. Furthermore, we developed an RNA in situ hybridization protocol that allowed us to obtain the expression patterns of genes whose functions are important in establishing the embryonic body pattern. Our results revealed novel tephritid-specific features during A. ludens embryonic development and open new avenues for strategies aiming to control this important pest.

Genome-wide 5-hydroxymethylcytosine (5hmC) emerges at early stage of in vitro differentiation of a putative hepatocyte progenitor.

A basic question linked to differential patterns of gene expression is how cells reach different fates despite using the same DNA template. Since 5-hydroxymethylcytosine (5hmC) emerged as an intermediate metabolite in active DNA demethylation, there have been increasing efforts to elucidate its function as a stable modification of the genome, including a role in establishing such tissue-specific patterns of expression. Recently we described TET1-mediated enrichment of 5hmC on the promoter region of the master regulator of hepatocyte identity, HNF4A, which precedes differentiation of liver adult progenitor cells in vitro. Here, we studied the genome-wide distribution of 5hmC at early in vitro differentiation of human hepatocyte-like cells. We found a global increase in 5hmC as well as a drop in 5-methylcytosine after one week of in vitro differentiation from bipotent progenitors, at a time when the liver transcript program is already established. 5hmC was overall higher at the bodies of overexpressed genes. Furthermore, by modifying the metabolic environment, an adenosine derivative prevents 5hmC enrichment and impairs the acquisition of hepatic identity markers. These results suggest that 5hmC could be a marker of cell identity, as well as a useful biomarker in conditions associated with cell de-differentiation such as liver malignancies.

In situ dissection of domain boundaries affect genome topology and gene transcription in Drosophila.

Chromosomes are organized into high-frequency chromatin interaction domains called topologically associating domains (TADs), which are separated from each other by domain boundaries. The molecular mechanisms responsible for TAD formation are not yet fully understood. In Drosophila, it has been proposed that transcription is fundamental for TAD organization while the participation of genetic sequences bound by architectural proteins (APs) remains controversial. Here, we investigate the contribution of domain boundaries to TAD organization and the regulation of gene expression at the Notch gene locus in Drosophila. We find that deletion of domain boundaries results in TAD fusion and long-range topological defects that are accompanied by loss of APs and RNA Pol II chromatin binding as well as defects in transcription. Together, our results provide compelling evidence of the contribution of discrete genetic sequences bound by APs and RNA Pol II in the partition of the genome into TADs and in the regulation of gene expression in Drosophila.

Enhancer RNAs: Insights Into Their Biological Role.

Enhancers play a central role in the transcriptional regulation of metazoans. Almost a decade ago, the discovery of their pervasive transcription into noncoding RNAs, termed enhancer RNAs (eRNAs), opened a whole new field of study. The presence of eRNAs correlates with enhancer activity; however, whether they act as functional molecules remains controversial. Here we review direct experimental evidence supporting a functional role of eRNAs in transcription and provide a general pipeline that could help in the design of experimental approaches to investigate the function of eRNAs. We propose that induction of transcriptional activity at enhancers promotes an increase in its activity by an RNA-mediated titration of regulatory proteins that can impact different processes like chromatin accessibility or chromatin looping. In a few cases, transcripts originating from enhancers have acquired specific molecular functions to regulate gene expression. We speculate that these transcripts are either nonannotated long noncoding RNAs (lncRNAs) or are evolving toward functional lncRNAs. Further work will be needed to comprehend better the biological activity of these transcripts.

CTCF knockout reveals an essential role for this protein during the zebrafish development.

Chromatin regulation and organization are essential processes that regulate gene activity. The CCCTC-binding factor (CTCF) is a protein with different and important molecular functions related with chromatin dynamics. It is conserved since invertebrates to vertebrates, posing it as a factor with an important role in the physiology. In this work, we aimed to understand the distribution and functional relevance of CTCF during the embryonic development of the zebrafish (Danio rerio). We generated a zebrafish specific anti-Ctcf antibody, and found this protein to be ubiquitous, through different stages and tissues. We used the CRISPR-Cas9 system to induce molecular alterations in the locus. This resulted in early lethality. We delayed the lethality performing knockdown morpholino experiments, and found an aberrant embryo morphology involving malformations in structures through all the length of the embryo. These phenotypes were rescued with human CTCF mRNA injections, showing the specificity of the morpholinos and a partial functional conservation between the fish and the human proteins. Lastly, we found that the pro-apoptotic genes p53 and bbc3/PUMA are deregulated in the ctcf morpholino-injected embryos. In conclusion, CTCF is a ubiquitous factor during the zebrafish development, which regulates the correct formation of different structures of the embryo, and its deregulation impacts on essential cell survival genes. Overall, this work provides a basis to look for the particular functions of CTCF in the different developing tissues and organs of the zebrafish.

Developing in 3D: the role of CTCF in cell differentiation.

CTCF is a highly conserved zinc-finger DNA-binding protein that mediates interactions between distant sequences in the genome. As a consequence, CTCF regulates enhancer-promoter interactions and contributes to the three-dimensional organization of the genome. Recent studies indicate that CTCF is developmentally regulated, suggesting that it plays a role in cell type-specific genome organization. Here, we review these studies and discuss how CTCF functions during the development of various cell and tissue types, ranging from embryonic stem cells and gametes, to neural, muscle and cardiac cells. We propose that the lineage-specific control of CTCF levels, and its partnership with lineage-specific transcription factors, allows for the control of cell type-specific gene expression via chromatin looping.

Genome Editing During Development Using the CRISPR-Cas Technology.

Over the years, the study of gene function during development involved the implementation of sophisticated transgenic strategies to visualize how organisms change during their lifetime. These strategies are diverse and extremely useful and allowed the discovery of some of the fundamental mechanisms governing organism's development. Such strategies can be time-consuming, in some cases expensive, and require complex infrastructure. With the advent of the genome editing CRISPR-Cas9 RNA-guided DNA endonuclease system a tremendous progress has been achieved in manipulating diverse organisms and cell types. In recent years this system has contributed importantly to the design of novel experimental strategies to further understand developmental processes, to generate genetically modified animal models, and develop disease models. Here we highlight examples in which the genome editing CRISPR-Cas9 system has been employed to understand the mechanisms controlling embryonic development and disease.

Epigenetic Effects of an Adenosine Derivative in a Wistar Rat Model of Liver Cirrhosis.

The pathological characteristic of cirrhosis is scarring which results in a structurally distorted and dysfunctional liver. Previously, we demonstrated that Col1a1 and Pparg genes are deregulated in CCl4 -induced cirrhosis but their normal expression levels are recovered upon treatment with IFC-305, an adenosine derivative. We observed that adenosine was able to modulate S-adenosylmethionine-dependent trans-methylation reactions, and recently, we found that IFC-305 modulates HDAC3 expression. Here, we investigated whether epigenetic mechanisms, involving DNA methylation processes and histone acetylation, could explain the re-establishment of gene expression mediated by IFC-305 in cirrhosis. Therefore, Wistar rats were CCl4 treated and a sub-group received IFC-305 to reverse fibrosis. Global changes in DNA methylation, 5-hydroxymethylation, and histone H4 acetylation were observed after treatment with IFC-305. In particular, during cirrhosis, the Pparg gene promoter is depleted of histone H4 acetylation, whereas IFC-305 administration restores normal histone acetylation levels which correlates with an increase of Pparg transcript and protein levels. In contrast, the promoter of Col1a1 gene is hypomethylated during cirrhosis but gains DNA methylation upon treatment with IFC-305 which correlates with a reduction of Col1a1 transcript and protein levels. Our results suggest a model in which cirrhosis results in a general loss of permissive chromatin histone marks which triggers the repression of the Pparg gene and the upregulation of the Col1a1 gene. Treatment with IFC-305 restores epigenetic modifications globally and specifically at the promoters of Pparg and Col1a1 genes. These results reveal one of the mechanisms of action of IFC-305 and suggest a possible therapeutic function in cirrhosis. J. Cell. Biochem. 119: 401-413, 2018. © 2017 Wiley Periodicals, Inc.

Erratum to: "Monoallelic germline methylation and sequence variant in the promoter of the RB1 gene: a possible constitutive epimutation in hereditary retinoblastoma".

[This corrects the article DOI: 10.1186/s13148-015-0167-0.].