LRF Promotes Indirectly Advantageous Chromatin Conformation via BGLT3-lncRNA Expression and Switch from Fetal to Adult Hemoglobin.

The hemoglobin switch from fetal (HbF) to adult (HbA) has been studied intensively as an essential model for gene expression regulation, but also as a beneficial therapeutic approach for ß-hemoglobinopathies, towards the objective of reactivating HbF. The transcription factor LRF (Leukemia/lymphoma-related), encoded from the ZBTB7A gene has been implicated in fetal hemoglobin silencing, though has a wide range of functions that have not been fully clarified. We thus established the LRF/ZBTB7A-overexpressing and ZBTB7A-knockdown K562 (human erythroleukemia cell line) clones to assess fetal vs. adult hemoglobin production pre- and post-induction. Transgenic K562 clones were further developed and studied under the influence of epigenetic chromatin regulators, such as DNA methyl transferase 3 (DNMT3) and Histone Deacetylase 1 (HDAC1), to evaluate LRF's potential disturbance upon the aberrant epigenetic background and provide valuable information of the preferable epigenetic frame, in which LRF unfolds its action on the ß-type globin's expression. The ChIP-seq analysis demonstrated that LRF binds to γ-globin genes (HBG2/1) and apparently associates BCL11A for their silencing, but also during erythropoiesis induction, LRF binds the BGLT3 gene, promoting BGLT3-lncRNA production through the γ-δ intergenic region of ß-type globin's locus, triggering the transcriptional events from γ- to ß-globin switch. Our findings are supported by an up-to-date looping model, which highlights chromatin alterations during erythropoiesis at late stages of gestation, to establish an "open" chromatin conformation across the γ-δ intergenic region and accomplish ß-globin expression and hemoglobin switch.

"Crosstalk between non-coding RNAs and transcription factor LRF in non-small cell lung cancer".

Epigenetic approaches in direct correlation with assessment of critical genetic mutations in non-small cell lung cancer (NSCLC) are currently very intensive, as the epigenetic components underlying NSCLC development and progression have attained high recognition. In this level of research, established human NSCLC cell lines as well as experimental animals are widely used to detect novel biomarkers and pharmacological targets to treat NSCLC. The epigenetic background holds a great potential for the identification of epi-biomarkers for treatment response however, it is highly complex and requires precise definition as these phenomena are variable between NSCLC subtypes and systems origin. We engaged an in-depth characterization of non-coding (nc)RNAs prevalent in human KRAS-mutant NSCLC cell lines A549 and H460 and mouse KRAS-mutant NSCLC tissue by Next Generation Sequencing (NGS) and quantitative Real Time PCRs (qPCRs). Also, the transcription factor (TF) LRF, a known epigenetic silencer, was examined as a modulator of non-coding RNAs expression. Finally, interacting networks underlying epigenetic variations in NSCLC subtypes were created. Data derived from our study highlights the divergent epigenetic profiles of NSCLC of human and mouse origin, as well as the significant contribution of 12qf1: 109,709,060-109,747,960 mouse chromosomal region to micro-RNA upregulated species. Furthermore, the novel epigenetic miR-148b-3p/lncPVT1/ZBTB7A axis was identified, which differentiates human cell line of lung adenocarcinoma from large cell lung carcinoma, two characteristic NSCLC subtypes. The detailed recording of epigenetic events in NSCLC and combinational studies including networking between ncRNAs and TFs validate the identification of significant epigenetic features, prevailing in NSCLC subtypes and among experimental models. Our results enrich knowledge in the field and empower research on the epigenetic prognostic biomarkers of the disease progression, NSCLC subtypes discrimination and advancement to patient-tailored treatments.

Fetal hemoglobin induction in azacytidine responders enlightens methylation patterns related to blast clearance in higher-risk MDS and CMML.

BACKGROUND: As new treatment options for patients with higher-risk myelodysplastic syndromes are emerging, identification of prognostic markers for hypomethylating agent (HMA) treatment and understanding mechanisms of their delayed and short-term responses are essential. Early fetal hemoglobin (HbF) induction has been suggested as a prognostic indicator for decitabine-treated patients. Although epigenetic mechanisms are assumed, responding patients' epigenomes have not been thoroughly examined. We aimed to clarify HbF kinetics and prognostic value for azacytidine treated patients, as well as the epigenetic landscape that might influence HbF re-expression and its clinical relevance. RESULTS: Serial HbF measurements by high-performance liquid chromatography (n = 20) showed induction of HbF only among responders (p = 0.030). Moreover, HbF increase immediately after the first azacytidine cycle demonstrated prognostic value for progression-free survival (PFS) (p = 0.032, HR = 0.19, CI 0.24-1.63). Changes in methylation patterns were revealed with methylated DNA genome-wide sequencing analysis (n = 7) for FOG-1, RCOR-1, ZBTB7A and genes of the NuRD-complex components. Targeted pyrosequencing methodology (n = 28) revealed a strong inverse correlation between the degree of γ-globin gene (HBG2) promoter methylation and baseline HbF levels (p = 0.003, rs = - 0.663). A potential epigenetic mechanism of HbF re-expression in azacytidine responders was enlightened by targeted methylation analysis, through hypomethylation of site -53 of HBG2 promoter (p = 0.039, rs = - 0.504), which corresponds to MBD2-NuRD binding site, and to hypermethylation of the CpG326 island of ZBTB7A (p = 0.05, rs = 0.482), a known HbF repressor. These changes were associated to blast cell clearance (pHBG2 = 0.011, rs = 0.480/pZBTB7A = 0.026, rs = 0.427) and showed prognostic value for PFS (pZBTB7A = 0.037, HR = 1.14, CI 0.34-3.8). CONCLUSIONS: Early HbF induction is featured as an accessible prognostic indicator for HMA treatment and the proposed potential epigenetic mechanism of HbF re-expression in azacytidine responders includes hypomethylation of the γ-globin gene promoter region and hypermethylation of the CpG326 island of ZBTB7A. The association of these methylation patterns with blast clearance and their prognostic value for PFS paves the way to discuss in-depth azacytidine epigenetic mechanism of action.

Transcriptional repression of lncRNA and miRNA subsets mediated by LRF during erythropoiesis.

Non-coding RNA (ncRNA) species, mainly long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been currently imputed for lesser or greater involvement in human erythropoiesis. These RNA subsets operate within a complex circuit with other epigenetic components and transcription factors (TF) affecting chromatin remodeling during cell differentiation. Lymphoma/leukemia-related (LRF) TF exerts higher occupancy on DNA CpG rich sites and is implicated in several differentiation cell pathways and erythropoiesis among them and also directs the epigenetic regulation of hemoglobin transversion from fetal (HbF) to adult (HbA) form by intervening in the γ-globin gene repression. We intended to investigate LRF activity in the evolving landscape of cells' commitment to the erythroid lineage and specifically during HbF to HbA transversion, to qualify this TF as potential repressor of lncRNAs and miRNAs. Transgenic human erythroleukemia cells, overexpressing LRF and further induced to erythropoiesis, were subjected to expression analysis in high LRF occupancy genetic loci-producing lncRNAs. LRF abundance in genetic loci transcribing for studied lncRNAs was determined by ChIP-Seq data analysis. qPCRs were performed to examine lncRNA expression status. Differentially expressed miRNA pre- and post-erythropoiesis induction were assessed by next-generation sequencing (NGS), and their promoter regions were charted. Expression levels of lncRNAs were correlated with DNA methylation status of flanked CpG islands, and contingent co-regulation of hosted miRNAs was considered. LRF-binding sites were overrepresented in LRF overexpressing cell clones during erythropoiesis induction and exerted a significant suppressive effect towards lncRNAs and miRNA collections. Based on present data interpretation, LRF's multiplied binding capacity across genome is suggested to be transient and associated with higher levels of DNA methylation. KEY MESSAGES: During erythropoiesis, LRF displays extensive occupancy across genetic loci. LRF significantly represses subsets of lncRNAs and miRNAs during erythropoiesis. Promoter region CpG islands' methylation levels affect lncRNA expression. MiRNAs embedded within lncRNA loci show differential regulation of expression.

Medicines for Obesity: Appraisal of Clinical Studies with Grading of Recommendations, Assessment, Development, and Evaluation Tool.

We evaluated the quality of evidence from phase III/IV clinical trials of drugs against obesity using the principles of Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) tool. Our systematic review evaluates the quality of clinical evidence from existing clinical trials and not the pharmacological efficacy of anti-obesity therapies. A literature search using select keywords in separate was performed in PubMed and ClinicalTrials.gov databases for phase III/IV clinical trials during the last ten years. Our findings indicate that the quality of existing clinical evidence from anti-obesity trials generally ranges from low to moderate. Most trials suffered from publication bias. Less frequently, trials suffered from the risk of bias mainly due to lack of blindness in the treatment. Our work indicates that additional higher-quality clinical trials are needed to gain more confidence in the estimate of the effect of currently used anti-obesity medicines, to allow more informed clinical decisions, thus reducing the risk of implementing potentially ineffective or even harmful therapeutic strategies.