Search

Friend of GATA1 (FOG-1) is an essential transcriptional co-factor of the master erythroid transcription factor GATA1. The knockout of the Zfpm1 gene, coding for FOG-1, results in early embryonic lethality due to anemia in mice, similar to the embryonic lethal phenotype of the Gata1 gene knockout. However, a detailed molecular analysis of the Zfpm1 knockout phenotype in erythropoiesis is presently incomplete. To this end, we used CRISPR/Cas9 to knockout Zfpm1 in mouse erythroleukemic (MEL) cells. Phenotypic characterization of DMSO-induced terminal erythroid differentiation showed that the Zfpm1 knockout MEL cells did not progress past the proerythroblast stage of differentiation. Expression profiling of the Zfpm1 knockout MEL cells by RNAseq showed a lack of upregulation of erythroid-related gene expression profiles. Bioinformatic analysis highlighted cholesterol transport as a pathway affected in the Zfpm1 knockout cells. Moreover, we show that the cholesterol transporters Abca1 and Ldlr fail to be repressed during erythroid differentiation in Zfpm1 knockout cells, resulting in higher intracellular lipid levels and higher membrane fluidity. We also show that in FOG-1 knockout cells, the nuclear levels of SREBP2, a key transcriptional regulator of cholesterol biosynthesis and transport, are markedly increased. On the basis of these findings we propose that FOG-1 (and, potentially, GATA1) regulate cholesterol homeostasis during erythroid differentiation directly through the downregulation of cholesterol transport genes and indirectly, through the repression of the SREBP2 transcriptional activator of cholesterol homeostasis. Taken together, our work provides a molecular basis for understanding FOG-1 functions in eryhropoiesis and reveals a novel role for FOG-1 in cholesterol transport., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Roussis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Three-dimensional (3D) genome folding has a fundamental role in the regulation of developmental genes by facilitating or constraining chromatin interactions between cis-regulatory elements (CREs). Polycomb response elements (PREs) are a specific kind of CRE involved in the memory of transcriptional states in Drosophila melanogaster. PREs act as nucleation sites for Polycomb group (PcG) proteins, which deposit the repressive histone mark H3K27me3, leading to the formation of a class of topologically associating domain (TAD) called a Polycomb domain. PREs can establish looping contacts that stabilize the gene repression of key developmental genes during development. However, the mechanism by which PRE loops fine-tune gene expression is unknown. Using clustered regularly interspaced short palindromic repeats and Cas9 genome engineering, we specifically perturbed PRE contacts or enhancer function and used complementary approaches including 4C-seq, Hi-C and Hi-M to analyze how chromatin architecture perturbation affects gene expression. Our results suggest that the PRE loop at the dac gene locus acts as a constitutive 3D chromatin scaffold during Drosophila development that forms independently of gene expression states and has a versatile function; it restricts enhancer-promoter communication and contributes to enhancer specificity., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Gene silencing by heterochromatin plays a crucial role in cell identity. Here, we characterize the localization, the biogenesis, and the function of an atypical heterochromatin, which is simultaneously enriched in the typical H3K9me3 mark and in H3K36me3, a histone mark usually associated with gene expression. We identified thousands of dual regions in mouse embryonic stem (ES) cells that rely on the histone methyltransferases SET domain bifurcated 1 (SETDB1) and nuclear set domain (NSD)-containing proteins to generate H3K9me3 and H3K36me3, respectively. Upon SETDB1 removal, dual domains lose both marks, gain signatures of active enhancers, and come into contact with upregulated genes, suggesting that it might be an important pathway by which genes are controlled by heterochromatin. In differentiated tissues, a subset of these dual domains is destabilized and becomes enriched in active enhancer marks, providing a mechanistic insight into the involvement of heterochromatin in the maintenance of cell identity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The genome folds into a hierarchy of three-dimensional structures within the nucleus. At the sub-megabase scale, chromosomes form topologically associating domains (TADs) 1-4 . However, how TADs fold in single cells is elusive. Here, we reveal TAD features inaccessible to cell population analysis by using super-resolution microscopy. TAD structures and physical insulation associated with their borders are variable between individual cells, yet chromatin intermingling is enriched within TADs compared to adjacent TADs in most cells. The spatial segregation of TADs is further exacerbated during cell differentiation. Favored interactions within TADs are regulated by cohesin and CTCF through distinct mechanisms: cohesin generates chromatin contacts and intermingling while CTCF prevents inter-TAD contacts. Furthermore, TADs are subdivided into discrete nanodomains, which persist in cells depleted of CTCF or cohesin, whereas disruption of nucleosome contacts alters their structural organization. Altogether, these results provide a physical basis for the folding of individual chromosomes at the nanoscale.

To understand the role of the extensive senescence-associated 3D genome reorganization, we generated genome-wide chromatin interaction maps, epigenome, replication-timing, whole-genome bisulfite sequencing, and gene expression profiles from cells entering replicative senescence (RS) or upon oncogene-induced senescence (OIS). We identify senescence-associated heterochromatin domains (SAHDs). Differential intra- versus inter-SAHD interactions lead to the formation of senescence-associated heterochromatin foci (SAHFs) in OIS but not in RS. This OIS-specific configuration brings active genes located in genomic regions adjacent to SAHDs in close spatial proximity and favors their expression. We also identify DNMT1 as a factor that induces SAHFs by promoting HMGA2 expression. Upon DNMT1 depletion, OIS cells transition to a 3D genome conformation akin to that of cells in replicative senescence. These data show how multi-omics and imaging can identify critical features of RS and OIS and discover determinants of acute senescence and SAHF formation., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Polycomb repressive complexes 1 and 2 have been historically described as transcriptional repressors, but recent reports suggest that PRC1 might also support activation, although the underlying mechanisms remain elusive. Here, we show that stage-specific PRC1 binding at a subset of active promoters and enhancers during Drosophila development coincides with the formation of three-dimensional (3D) loops, an increase in expression during development and repression in PRC1 mutants. Dissection of the dachshund locus indicates that PRC1-anchored loops are versatile architectural platforms that persist when surrounding genes are transcriptionally active and fine-tune their expression. The analysis of RING1B binding profiles and 3D contacts during neural differentiation in mice suggests that this role is conserved in mammals., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Eukaryotic chromosomes are organized in multiple scales, from nucleosomes to chromosome territories. Recently, genome-wide methods identified an intermediate level of chromosome organization, topologically associating domains (TADs), that play key roles in transcriptional regulation. However, these methods cannot directly examine the interplay between transcriptional activation and chromosome architecture while maintaining spatial information. Here we present a multiplexed, sequential imaging approach (Hi-M) that permits simultaneous detection of chromosome organization and transcription in single nuclei. This allowed us to unveil the changes in 3D chromatin organization occurring upon transcriptional activation and homologous chromosome unpairing during awakening of the zygotic genome in intact Drosophila embryos. Excitingly, the ability of Hi-M to explore the multi-scale chromosome architecture with spatial resolution at different stages of development or during the cell cycle will be key to understanding the mechanisms and consequences of the 4D organization of the genome., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Interphase chromatin is organized into topologically associating domains (TADs). Within TADs, chromatin looping interactions are formed between DNA regulatory elements, but their functional importance for the establishment of the 3D genome organization and gene regulation during development is unclear. Using high-resolution Hi-C experiments, we analyze higher order 3D chromatin organization during Drosophila embryogenesis and identify active and repressive chromatin loops that are established with different kinetics and depend on distinct factors: Zelda-dependent active loops are formed before the midblastula transition between transcribed genes over long distances. Repressive loops within polycomb domains are formed after the midblastula transition between polycomb response elements by the action of GAGA factor and polycomb proteins. Perturbation of PRE function by CRISPR/Cas9 genome engineering affects polycomb domain formation and destabilizes polycomb-mediated silencing. Preventing loop formation without removal of polycomb components also decreases silencing efficiency, suggesting that chromatin architecture can play instructive roles in gene regulation during development. VIDEO ABSTRACT., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Deciphering the rules of genome folding in the cell nucleus is essential to understand its functions. Recent chromosome conformation capture (Hi-C) studies have revealed that the genome is partitioned into topologically associating domains (TADs), which demarcate functional epigenetic domains defined by combinations of specific chromatin marks. However, whether TADs are true physical units in each cell nucleus or whether they reflect statistical frequencies of measured interactions within cell populations is unclear. Using a combination of Hi-C, three-dimensional (3D) fluorescent in situ hybridization, super-resolution microscopy, and polymer modeling, we provide an integrative view of chromatin folding in Drosophila . We observed that repressed TADs form a succession of discrete nanocompartments, interspersed by less condensed active regions. Single-cell analysis revealed a consistent TAD-based physical compartmentalization of the chromatin fiber, with some degree of heterogeneity in intra-TAD conformations and in cis and trans inter-TAD contact events. These results indicate that TADs are fundamental 3D genome units that engage in dynamic higher-order inter-TAD connections. This domain-based architecture is likely to play a major role in regulatory transactions during DNA-dependent processes.

Chromosome conformation capture technologies have revealed important insights into genome folding. Yet, how spatial genome architecture is related to gene expression and cell fate remains unclear. We comprehensively mapped 3D chromatin organization during mouse neural differentiation in vitro and in vivo, generating the highest-resolution Hi-C maps available to date. We found that transcription is correlated with chromatin insulation and long-range interactions, but dCas9-mediated activation is insufficient for creating TAD boundaries de novo. Additionally, we discovered long-range contacts between gene bodies of exon-rich, active genes in all cell types. During neural differentiation, contacts between active TADs become less pronounced while inactive TADs interact more strongly. An extensive Polycomb network in stem cells is disrupted, while dynamic interactions between neural transcription factors appear in vivo. Finally, cell type-specific enhancer-promoter contacts are established concomitant to gene expression. This work shows that multiple factors influence the dynamics of chromatin interactions in development., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Inborn defects in DNA repair are associated with complex developmental disorders whose causal mechanisms are poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the nucleotide excision repair (NER) structure-specific endonuclease ERCC1-XPF complex interacts with the insulator binding protein CTCF, the cohesin subunits SMC1A and SMC3 and with MBD2; the factors co-localize with ATRX at the promoters and control regions (ICRs) of imprinted genes during postnatal hepatic development. Loss of Ercc1 or exposure to MMC triggers the localization of CTCF to heterochromatin, the dissociation of the CTCF-cohesin complex and ATRX from promoters and ICRs, altered histone marks and the aberrant developmental expression of imprinted genes without altering DNA methylation. We propose that ERCC1-XPF cooperates with CTCF and cohesin to facilitate the developmental silencing of imprinted genes and that persistent DNA damage triggers chromatin changes that affect gene expression programs associated with NER disorders.

GATA-1 and PU.1 are two important hematopoietic transcription factors that mutually inhibit each other in progenitor cells to guide entrance into the erythroid or myeloid lineage, respectively. PU.1 controls its own expression during myelopoiesis by binding to the distal URE enhancer, whose deletion leads to acute myeloid leukemia (AML). We herein present evidence that GATA-1 binds to the PU.1 gene and inhibits its expression in human AML-erythroleukemias (EL). Furthermore, GATA-1 together with DNA methyl Transferase I (DNMT1) mediate repression of the PU.1 gene through the URE. Repression of the PU.1 gene involves both DNA methylation at the URE and its histone H3 lysine-K9 methylation and deacetylation as well as the H3K27 methylation at additional DNA elements and the promoter. The GATA-1-mediated inhibition of PU.1 gene transcription in human AML-EL mediated through the URE represents important mechanism that contributes to PU.1 downregulation and leukemogenesis that is sensitive to DNA demethylation therapy.

The clear connection between ribosome biogenesis dysfunction and specific hematopoiesis-related disorders prompted us to examine the role of critical lineage-specific transcription factors in the transcriptional regulation of ribosomal protein (RP) genes during terminal erythroid differentiation. By applying EMSA and ChIP methodologies in mouse erythroleukemia cells we show that GATA1 and PU.1 bind in vitro and in vivo the proximal promoter region of the RPS19 gene which is frequently mutated in Diamond-Blackfan Anemia. Moreover, ChIPseq data analysis also demonstrates that several RP genes are enriched as potential GATA1 and PU.1 gene targets in mouse and human erythroid cells, with GATA1 binding showing an association with higher ribosomal protein gene expression levels during terminal erythroid differentiation in human and mouse. Our results suggest that RP gene expression and hence balanced ribosome biosynthesis may be specifically and selectively regulated by lineage specific transcription factors during hematopoiesis, a finding which may be clinically relevant to ribosomopathies.

Concentrations of Cd, Cu, Cr, Ni, Zn, Fe and metallothioneins (MTs) were measured in the gonads of Paracentrotus lividus from Amvrakikos gulf (Ionian Sea, Greece). Three natural populations were selected; two of them, growing inside the gulf (Agios Thomas and Koronisia), presented higher density and smaller body size than the population living in a coastal area just outside the gulf (Mytikas). Metal and MT levels were not elevated, with the exception of Zn, showing high values related to the reproduction stage of the sea urchins. Significant geographical variations were recorded in the concentrations of Cu, Zn, Cd, Cr and MTs. The highest mean and maximum values of Cu, Zn and MTs were recorded in Agios Thomas while Cd and Cr were higher in Mytikas population. Copper, Zn, Fe and MT concentrations were negatively correlated to the sea urchin body size, while a positive concentration-size relationship was observed for Cd. Although all studied populations grow in a low metal level marine environment, urchins with smaller body size living in a food limited marine environment showed higher gonadosomatic index, metal concentrations and MT levels in their gonads (Agios Thomas and Koronisia) than larger specimens growing in a food unlimited area (Mytikas).

The ubiquitously expressed transcription factor TFII-I exerts both positive and negative effects on transcription. Using biotinylation tagging technology and high-throughput sequencing, we determined sites of chromatin interactions for TFII-I in the human erythroleukemia cell line K562. This analysis revealed that TFII-I binds upstream of the transcription start site of expressed genes, both upstream and downstream of the transcription start site of repressed genes, and downstream of RNA polymerase II peaks at the ATF3 and other stress responsive genes. At the ATF3 gene, TFII-I binds immediately downstream of a Pol II peak located 5 kb upstream of exon 1. Induction of ATF3 expression increases transcription throughout the ATF3 gene locus which requires TFII-I and correlates with increased association of Pol II and Elongin A. Pull-down assays demonstrated that TFII-I interacts with Elongin A. Partial depletion of TFII-I expression caused a reduction in the association of Elongin A with and transcription of the DNMT1 and EFR3A genes without a decrease in Pol II recruitment. The data reveal different interaction patterns of TFII-I at active, repressed, or inducible genes, identify novel TFII-I interacting proteins, implicate TFII-I in the regulation of transcription elongation and provide insight into the role of TFII-I during the response to cellular stress., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We report the genomic occupancy profiles of the key hematopoietic transcription factor GATA-1 in pro-erythroblasts and mature erythroid cells fractionated from day E12.5 mouse fetal liver cells. Integration of GATA-1 occupancy profiles with available genome-wide transcription factor and epigenetic profiles assayed in fetal liver cells enabled as to evaluate GATA-1 involvement in modulating local chromatin structure of target genes during erythroid differentiation. Our results suggest that GATA-1 associates preferentially with changes of specific epigenetic modifications, such as H4K16, H3K27 acetylation and H3K4 di-methylation. Furthermore, we used random forest (RF) non-linear regression to predict changes in the expression levels of GATA-1 target genes based on the genomic features available for pro-erythroblasts and mature fetal liver-derived erythroid cells. Remarkably, our prediction model explained a high proportion of 62% of variation in gene expression. Hierarchical clustering of the proximity values calculated by the RF model produced a clear separation of upregulated versus downregulated genes and a further separation of downregulated genes in two distinct groups. Thus, our study of GATA-1 genome-wide occupancy profiles in mouse primary erythroid cells and their integration with global epigenetic marks reveals three clusters of GATA-1 gene targets that are associated with specific epigenetic signatures and functional characteristics.

MicroRNAs (miRNAs) are a class of small regulatory genes regulating gene expression by targeting messenger RNA. Though computational methods for miRNA target prediction are the prevailing means to analyze their function, they still miss a large fraction of the targeted genes and additionally predict a large number of false positives. Here we introduce a novel algorithm called DIANA-microT-ANN which combines multiple novel target site features through an artificial neural network (ANN) and is trained using recently published high-throughput data measuring the change of protein levels after miRNA overexpression, providing positive and negative targeting examples. The features characterizing each miRNA recognition element include binding structure, conservation level, and a specific profile of structural accessibility. The ANN is trained to integrate the features of each recognition element along the 3'untranslated region into a targeting score, reproducing the relative repression fold change of the protein. Tested on two different sets the algorithm outperforms other widely used algorithms and also predicts a significant number of unique and reliable targets not predicted by the other methods. For 542 human miRNAs DIANA-microT-ANN predicts 120000 targets not provided by TargetScan 5.0. The algorithm is freely available at http://microrna.gr/microT-ANN.

Background: High-throughput gene expression experiments are widely used to identify the role of genes involved in biological conditions of interest. MicroRNAs (miRNA) are regulatory molecules that have been functionally associated with several developmental programs and their deregulation with diverse diseases including cancer., Methodology/principal Findings: Although miRNA expression levels may not be routinely measured in high-throughput experiments, a possible involvement of miRNAs in the deregulation of gene expression can be computationally predicted and quantified through analysis of overrepresented motifs in the deregulated genes 3' untranslated region (3'UTR) sequences. Here, we introduce a user-friendly web-server, DIANA-mirExTra (www.microrna.gr/mirextra) that allows the comparison of frequencies of miRNA associated motifs between sets of genes that can lead to the identification of miRNAs responsible for the deregulation of large numbers of genes. To this end, we have investigated different approaches and measures, and have practically implemented them on experimental data., Conclusions/significance: On several datasets of miRNA overexpression and repression experiments, our proposed approaches have successfully identified the deregulated miRNA. Beyond the prediction of miRNAs responsible for the deregulation of transcripts, the web-server provides extensive links to DIANA-mirPath, a functional analysis tool incorporating miRNA targets in biological pathways. Additionally, in case information about miRNA expression changes is provided, the results can be filtered to display the analysis for miRNAs of interest only.

Unlabelled: MicroRNAs (miRNAs) are a class of short endogenously expressed RNA molecules that regulate gene expression by binding directly to the messenger RNA of protein coding genes. They have been found to confer a novel layer of genetic regulation in a wide range of biological processes. Computational miRNA target prediction remains one of the key means used to decipher the role of miRNAs in development and disease. Here we introduce the basic idea behind the experimental identification of miRNA targets and present some of the most widely used computational miRNA target identification programs. The review includes an assessment of the prediction quality of these programs and their combinations., Supplementary Information: Supplementary data are available at Bioinformatics online.

Background: MicroRNAs are small endogenously expressed non-coding RNA molecules that regulate target gene expression through translation repression or messenger RNA degradation. MicroRNA regulation is performed through pairing of the microRNA to sites in the messenger RNA of protein coding genes. Since experimental identification of miRNA target genes poses difficulties, computational microRNA target prediction is one of the key means in deciphering the role of microRNAs in development and disease., Results: DIANA-microT 3.0 is an algorithm for microRNA target prediction which is based on several parameters calculated individually for each microRNA and combines conserved and non-conserved microRNA recognition elements into a final prediction score, which correlates with protein production fold change. Specifically, for each predicted interaction the program reports a signal to noise ratio and a precision score which can be used as an indication of the false positive rate of the prediction., Conclusion: Recently, several computational target prediction programs were benchmarked based on a set of microRNA target genes identified by the pSILAC method. In this assessment DIANA-microT 3.0 was found to achieve the highest precision among the most widely used microRNA target prediction programs reaching approximately 66%. The DIANA-microT 3.0 prediction results are available online in a user friendly web server at http://www.microrna.gr/microT.

Summary: DIANA-mirPath is a web-based computational tool developed to identify molecular pathways potentially altered by the expression of single or multiple microRNAs. The software performs an enrichment analysis of multiple microRNA target genes comparing each set of microRNA targets to all known KEGG pathways. The combinatorial effect of co-expressed microRNAs in the modulation of a given pathway is taken into account by the simultaneous analysis of multiple microRNAs. The graphical output of the program provides an overview of the parts of the pathway modulated by microRNAs, facilitating the interpretation and presentation of the analysis results., Availability: The software is available at http://microrna.gr/mirpath and is free for all users with no login or download requirement.

Computational microRNA (miRNA) target prediction is one of the key means for deciphering the role of miRNAs in development and disease. Here, we present the DIANA-microT web server as the user interface to the DIANA-microT 3.0 miRNA target prediction algorithm. The web server provides extensive information for predicted miRNA:target gene interactions with a user-friendly interface, providing extensive connectivity to online biological resources. Target gene and miRNA functions may be elucidated through automated bibliographic searches and functional information is accessible through Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The web server offers links to nomenclature, sequence and protein databases, and users are facilitated by being able to search for targeted genes using different nomenclatures or functional features, such as the genes possible involvement in biological pathways. The target prediction algorithm supports parameters calculated individually for each miRNA:target gene interaction and provides a signal-to-noise ratio and a precision score that helps in the evaluation of the significance of the predicted results. Using a set of miRNA targets recently identified through the pSILAC method, the performance of several computational target prediction programs was assessed. DIANA-microT 3.0 achieved there with 66% the highest ratio of correctly predicted targets over all predicted targets. The DIANA-microT web server is freely available at www.microrna.gr/microT.

A method is described that allows an accurate mapping of 3' ends of RNAs. In this method a labeled DNA probe, containing the presumed 3' end of the RNA under analysis is allowed to anneals to the RNA itself. Mung-bean nuclease is then used to digest single strands of both RNA and DNA. Electrophoretic fractionation of "protected" undigested, labeled DNA is than performed using a sequence reaction of a known DNA as length marker. This procedure was applied to the analysis of both a polyA RNA (Interleukin 10 mRNA) and non polyA RNAs (sea urchin 18S and 26S rRNAs). This method might be potentially relevant for the evaluation of the role of posttrascriptional control of IL-10 in the pathogenesis of the immune and inflammatory mediated diseases associated to ageing. This might allow to develop new strategies to approach to the diagnosis and therapy of age related diseases.

TarBase5.0 is a database which houses a manually curated collection of experimentally supported microRNA (miRNA) targets in several animal species of central scientific interest, plants and viruses. MiRNAs are small non-coding RNA molecules that exhibit an inhibitory effect on gene expression, interfering with the stability and translational efficiency of the targeted mature messenger RNAs. Even though several computational programs exist to predict miRNA targets, there is a need for a comprehensive collection and description of miRNA targets with experimental support. Here we introduce a substantially extended version of this resource. The current version includes more than 1300 experimentally supported targets. Each target site is described by the miRNA that binds it, the gene in which it occurs, the nature of the experiments that were conducted to test it, the sufficiency of the site to induce translational repression and/or cleavage, and the paper from which all these data were extracted. Additionally, the database is functionally linked to several other relevant and useful databases such as Ensembl, Hugo, UCSC and SwissProt. The TarBase5.0 database can be queried or downloaded from http://microrna.gr/tarbase.