Plasmodium spp. parasites from the human host to the mosquito vector depends on circulating gametocytes in the peripheral blood of the vertebrate host. Once ingested by the mosquito during blood meals, these sexual forms undergo a series of radical morphological and metabolic changes to survive and progress from the gut to the salivary glands, where they will be waiting to be injected into the vertebrate host. The design of effective transmission-blocking strategies requires a thorough understanding of all the mechanisms that drive the development of gametocytes, gametes, sexual reproduction, and subsequent differentiation within the mosquito. The drastic changes in Plasmodium falciparum shape and function throughout its life cycle rely on the tight regulation of stage-specific gene expression. This review outlines the mechanisms involved in Plasmodium falciparum sexual stage development in both the human and mosquito vector, and zygote to oocyst differentiation. Functional studies unravel mechanisms employed by P. falciparum to orchestrate the expression of stage-specific functional products required to succeed in its complex life cycle, thus providing us with potential targets for developing new therapeutics. These mechanisms are based on studies conducted with various Plasmodium species, including predominantly P. falciparum and the rodent malaria parasites P. berghei. However, the great potential of epigenetics, genomics, transcriptomics, proteomics, and functional genetic studies to improve the understanding of malaria as a disease remains partly untapped because of limitations in studies using human malaria parasites and field isolates.Malaria elimination may never succeed without the implementation of transmission-blocking strategies. The transmission of  Plasmodium and is transmitted through the bite of a mosquito vector of the genus Anopheles. In humans, malaria is caused by six Plasmodium species, including Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale curtisi, Plasmodium ovale wallikeri, and the zoonotic parasite Plasmodium knowlesi. Of the human malaria parasites, P. falciparum causes the most significant morbidity and mortality. In 2021, clinical malaria affected 247,000 million individuals worldwide, leading to 619,000 deaths, with the highest burden observed in sub-Saharan Africa [Malaria is a mosquito-borne infection caused by an apicomplexan parasite of the genus Plasmodium transmission from the human host to the mosquito vector and the parasite stages within the mosquito are promising targets for successfully interrupting the parasite life cycle and reducing the malaria burden in endemic areas [With the renewed interest in malaria elimination and eradication, it has been recognized that intervention strategies should target all stages of the parasite life cycle with a particular interest in preventing transmission . Plasmodic areas .Plasmodium falciparum is very complex. It includes different hosts, different cell types within the same host, asexual multiplication phases, and a sexual replication phase  and initiate the intraerythrocytic cycle. Within the intraerythrocytic developmental cycle, a small percentage of the parasite commits to sexual differentiation, resulting in the development of the gametocyte forms. The gametocytes will be picked up by the mosquito during their blood meal. Within the mosquito, the sporogonic cycle will take place.Plasmodium development in the mosquito have been the subject of several comprehensive reviews [Plasmodium falciparum developmental process from the pre-sexual stage in the human host to the ookinete stage of the mosquito vector. The review is based on studies conducted with various Plasmodium species, including predominantly P. falciparum and the rodent malaria parasites P. berghei. Although Plasmodium berghei is a rodent malaria parasite, the availability of fast and efficient experimental genetics techniques to access the complete in vivo life cycle made rodent malaria parasites valuable models for understanding some aspects of P. falciparum biology. The review highlights studies assessing the mechanisms involved in the parasite-stage-specific gene expression, sexual stage development in both the vertebrate and mosquito vector, and zygote to oocyst differentiation. This review starts with an outline of genome organization, general gene expression, and regulation mechanisms in the intraerythrocytic asexual stage. Then, we describe the mechanisms driving the development of gametocytes, gametes, fertilization, and zygote to oocyst differentiation. Finally, the review highlights gaps and potential targets for developing new classes of antimalarial drugs or vaccines.Gametocytogenesis and  reviews ,37,38,39Plasmodium parasite genome of ~18–30 Mb is organized into 14 chromosomes containing about 5300 protein-coding genes, with many sub-telomeric multigene families [Plasmodium gene expression [P. falciparum nucleosome organization consists of approximately 155 DNA base pairs (bp) around a histone octamer [Plasmodium histone molecules are characterized by distinct biochemical properties thought to modulate both nucleosome stability and their rapid displacement in a temperature-dependent manner [The families . The pacpression . As in mpression ,41. The  octamer . Site-spt manner .P. falciparum and P. vivax, have a distinct genome organization [A comparative analysis of chromosome organization in different plasmodial species revealed that the two most pathogenic human malaria parasites, nization . Indeed,nization . IdentifPlasmodium parasite differentiation by inducing, preventing, or poising transcriptional activation, DNA replication, damage repair, and chromatin condensation. Recent quantitative chromatin proteomic approaches and studies on the genome-wide localization of various epigenetic features have identified stage-specific histone modifications and their potential implication in the different life cycle stages of the malaria parasite [Histone modifications’ spatial and temporal coordination impacts parasite ,46 , 10 putative histone acetyltransferases (HATs), 10 methyltransferases (HMTs), and 3 demethylases [PfHda2 [PfSir2a and PfSir2b [PfSET2 [PfSET10 [P. falciparum erythrocyte membrane protein 1 (PfEMP1) located in the subtelomeric regions of multiple parasite chromosomes [Reversible gene silencing and activation are mediated by histone-modifying enzymes and histone-associated proteins that will “read, write or erase” histone code. thylases ,56. Thes [PfHda2 , the sir PfSir2b , the H3K [PfSET2 , and the[PfSET10  are invo[PfSET10 , and the[PfSET10 ,57,58,59omosomes . They aromosomes .PfHP1) [PfBDP1 [PfBDP2 [PfBDP7 [PfHP1 is recruited by the H3K9me3 repressive epigenetic mark. The binding of PfHP1 regulates the formation of heterochromatins. On the contrary, the histone post-translational modification histone H3, serine 10 phosphorylation (H3S10ph) impairs the binding of HP1 [Epigenomic and functional genetic studies have also identified several histone-associated proteins, the heterochromatin protein 1 (PfHP1) ,63 and s [PfBDP1 , PfBDP2  [PfBDP2 , and PfB [PfBDP7 . These p [PfBDP7 ,64,65. Pg of HP1 . The autPfMYST  [PfGCN5  [P. falciparum-purified MYST proteins revealed the recruitment of PfMYST to acetylated histone 4 gene activation marks, suggesting PfMYST implication in the expression of var genes and genes regulating asexual intraerythrocytic growth [PfGCN5 highlighted its association with euchromatin marks and the controlled expression of erythrocyte invasion and virulence genes [The activation of gene expression is mediated by the histone acetyltransferase 60-like)  and PfGCsible 5) . An in vc growth . Similarce genes .Pfmc-2tm), the nutrient acquisition multigene family (clag genes and acyl-CoA synthase genes), parasite differentiation genes , and the Plasmodium helical interspersed subtelomeric (PHIST) family.Epigenomic studies have demonstrated that while gene activation marks  are widespread throughout the genome, the repressive marks  appear to be specific for distinct gene families adjacent to subtelomeric var genes . The genPlasmodium appear to be evolutionarily conserved in eukaryotic cells, suggesting a limitation in targeting them for therapeutic intervention. However, an in vitro screening of epigenetic modulator inhibitors against multiple life cycle stages of P. falciparum demonstrates the potential of using HDAC and HMT inhibitors as a new class of antimalarial drugs [Plasmodium histone modification enzymes [Most epigenetic regulators and chromatin proteins implicated in gene silencing and activation in al drugs . Coetzeeal drugs  tested t enzymes .Plasmodium falciparum includes the canonical TATA box binding proteins and the RNA-polymerase-II-dependent RNA expression [P. falciparum genome identified 156 parasite-transcription-associated proteins, including all the 12 subunits of the RNA polymerase II and the general transcription factors (GTFs) for the basal transcription machinery [P. falciparum genome [In addition to genomic organization and epigenetics, the regulatory machinery for gene expression in the pression . A Hiddeachinery . Coulsonm genome . The autPlasmodium [Plasmodium AP2 transcription factors is not restricted to regulating stage-specific gene expression. Indeed, the ApiAp2 proteins PfAP2Tel [PfSIP2 [PfMyb1 protein (a transcription factor belonging to the tryptophan cluster family) [PfPREBP (a protein with four K-homology domains) [PfNF-YB (a protein containing a histone-fold domain) [A low number of transcriptional regulation motifs and transcription factors that could account for the tight regulation of stage-specific gene expression patterns have been identified ,72,73. Tasmodium . These tasmodium . With soasmodium ,78,79,80PfAP2Tel  and PfSI [PfSIP2 , charact [PfSIP2 ,82. Addi family) , PfPREBPdomains) , and PfN domain) ,86. Howe domain) ,84,85,86PfApiAp2 revealed that eight were preferentially associated with heterochromatic regions with differential coverage profiles [PfAP2-HFs) are likely part of the machinery recognizing repressive states in a DNA-motif-independent manner. Indeed, one of these PfAP2-HFs was shown to be strictly recruited by PfHP1 and to be a core component of heterochromatin.Systematic profiling of the genome-wide occupancy of eighteen profiles . Shang eprofiles  suggestePlasmodium gene expression. Since the ApiAP2 protein family originated from the plant lineage and has no homologs in humans, they may be good antimalarial drug targets. An in silico screen to dock thousands of small molecules into the crystal structure of the AP2-EXP AP2 domain identified four compounds that specifically block DNA binding by AP2-EXP [P. falciparum trophozoite stages, characterized by a significant decrease in the abundance of AP2-EXP target genes. Two other DNA-binding inhibitory compounds have multi-stage anti-Plasmodium activity against blood and mosquito-stage parasites. A structure-based drug design combined with in vitro screening, in vivo studies in animal models, and the comprehensive profiling of ApiAp2 genetic polymorphisms in field-isolated parasites from natural human infection are likely to help in the development of a novel class of antimalarial drug targeting the Plasmodium ApiAp2 transcription factor. Furthermore, this compound class can potentially interfere with gene transcription in other Plasmodial species and apicomplexan parasites of medical importance, including Toxoplasma gondii and Cryptosporidium spp. While T. gondii causes many deaths in immunocompromised patients and fetal death when newly acquired during pregnancy [Cryptosporidium spp. Is one of the leading causes of infant diarrhea in developing countries [T. gondii and Cryptosporidium parvum are predicted to encode 24 and 19 ApiAP2 genes, respectively, with some cross-conservation among Apicomplexan parasites [The shortage of transcription and associated proteins strongly suggest an essential role for epigenetics, chromatin structure, genome organization, and posttranscriptional mechanisms in  AP2-EXP . One of regnancy , Cryptosountries . T. gondarasites ,92.Plasmodium falciparum life cycle. These biological stages ensure parasite virulence and transmission. During the erythrocytic cycle, 1–5% of asexual parasites switch from the asexual multiplication pathway to a sexual differentiation pathway. The differentiation of sexually committed parasites into mature gametocytes is divided into five developmental stages (stage I–V) over 14 days. The differentiation process is characterized by dramatic alterations in parasite size, shape, and flexibility. Only early stages (stage I) and mature stage V are detectable in the peripheral blood, as the other stages are sequestered in the bone marrow and possibly other tissues [The development of male and female gametocytes within the vertebrate host and the sexual recombination in the mosquito vector are critical steps in the  tissues . The dif tissues ,95,96. TPfAP2-I [Several environmental and metabolic factors that trigger sexual commitment, including antimalarial drugs ,98, lysoPfAP2-I . The celP. falciparum genome is predominantly acetylated during the asexual developmental cycle, ApiAP2-G appears to be repressed, and its locus was shown to be associated with histone heterochromatin marks, including H3K4ac, H3K9me1, H3K36me2, PfHP1, and the gametocyte essential factor PfAP2-G5 [PfHP1-dependent gene silencing is antagonized by a sexual commitment activator protein called PfGDV1  [PfGDV1 targets heterochromatin and triggers the eviction of PfHP1. Interestingly, PfGDV1 activation is, in turn, controlled by a multi-exon long non-coding gdv1 antisense RNA (Pfgdv1asRNA) that initiates downstream of the gdv1 locus [While the PfAP2-G5 . PfHP1-dpment 1) ,108. PfGv1 locus .PfAP2-G5 binds upstream of the Pfap2-g locus and other exogenic regions, hence suppressing ap2-g expression [PfGDV1 and the activation of PfApiAP2-G remain unknown.The gametocyte essential factor AP2-G5 further prevents sexual commitment . Indeed,pression . AlthougPfAP2-G expression in the schizont of the preceding cycle [PfAP2-G expression in ring stages [P. yoelii, AP2-O3, implicated in a gender-specific transcription program [ap2-g transcription and many heterochromatic genes [Sexual conversion can occur by two possible routes following specific histone modification marks that promote changes in gene expression and sexual differentiation ,109. One program . AP2-O3,ic genes .Gametocyte development from stage I to stage V is characterized by euchromatic post-translational modifications and an abundance of repressive methylation marks on histone 3. Ealy gametocyte stages (I to III) are characterized by the H3K9me3, H3K27me2, H3K27me3, H3K36me2, H3K37me1, H3R17me1, and H3R17me2 modifications ,106. TheP. falciparum genome revealed that the localization and interaction of sexual differentiation genes in a repressive center are critical for regulating sexual conversion [PfHP1 and PfGDV1 will undoubtedly unravel the molecular player involved in parasite conversion.Three-dimensional structure analyses of the nversion . FurtherP. falciparum gametocytes but showed sub-micromolar transmission-blocking activity against the rodent malaria parasite P. berghei. Hence, histone-modifying enzymes are potential targets to prevent malaria infection in the mosquito.Despite their similitude with human enzymes, histone-modifying enzymes could be potential targets for developing antimalarial drugs with transmission-blocking activities. Stenzel et al.  designedAnopheles mosquito and the subsequent spread of competent parasites depend on the coordinated development of the Plasmodium parasite in the mosquito midgut. The differentiation process in the mosquito midgut, lasting almost 20 h, is defined by the rapid conversion of mature stage V female and male gametocytes into female macrogametes and male microgametes, respectively [Plasmodium life cycle as there is a 316-fold loss in parasite abundance from the gametocyte to ookinete stage and a 100-fold loss from the ookinete to oocyst stage [Establishing a successful malaria infection in the ectively ,10,11. Tectively ,11. The st stage . Due to st stage .Gamete formation is morphologically characterized by a rounding-up of the cell, followed by the parasitophorous vacuole membrane (PVM) rupture and parasite egress from the erythrocyte ,114. TheP. falciparum gametocytes and gametes revealed that small transcriptome changes characterize gametogenesis compared to other life-stage differentiation processes [Plasmodium berghei, causing rodent malaria. The rodent parasites are not of direct practical concern to humans. However, the easy access to Plasmodium life stages in the mosquito and the practicability for the experimental study of human malaria made them a good model for human malaria.Transcriptomic analysis of rocesses . Most stPlasmodium berghei, the change in gene expression necessary for female gametogenesis is determined by the translation of a large number of mRNAs maintained in a repressive translational state [Plasmodium berghei model system, Guerreiro et al. revealed that approximately 50% of the transcriptome is maintained in this translationally repressed state by a messenger ribonucleoprotein (mRNP) composed of 16 major factors, including the RNA helicase DOZI (development of zygote inhibited) and the Sm-like factor CITH (homolog of worm CAR-I and fly Trailer Hitch) [P. falciparum DOZI (PfDZ50) and demonstrated that the protein inhibits translation [P. berghei female gametogenesis.In al state ,117. Indr Hitch) . Using anslation , suggestP. berghei gametes revealed that proteins implicated in RNA translation, protein biosynthesis, glycolysis, environmental stress response, and tubulin-associated cytoskeleton dynamics are predominantly regulated during gamete formation [The expression of stage-specific genes that are necessary for microgamete formation is regulated by transient and reversible protein phosphorylation followed by the de novo synthesis of genes involved in DNA replication, axoneme assembly and motility, chromatin condensation, cytokinesis, and exflagellation ,121. Proormation . Gene exormation ,123,124.Plasmodium receptors responsible for sensing temperature drops and binding to XA have not been identified yet. The exposure of P. falciparum gametocytes to XA has been shown to increase cyclic GMP (cGMP) levels in the gametocyte, suggesting parasite guanylyl cyclase (GC) activation [Gametocyte activation is stimulated by environmental stimuli, including a temperature drop by approximately 5 °C, the presence of mosquito-derived xanthurenic acid (XA), and an increase in extracellular pH from 7.2 to about 8 ,126,127.tivation . In the tivation ,130,131.Plasmodium falciparum genome encodes two guanylyl cyclase proteins (GCα and GCβ) that are only expressed in sexual-stage parasites. Functional studies in P. falciparum revealed that these proteins are active guanylyl cyclases and likely important in gametocyte activation [P. falciparum and P. berghei gamete formation have shown that GC activation leads to the formation of a secondary messenger cyclic GMP (cGMP), which in turn activates the protein kinase G (PKG), the unique effector identified in Plasmodium [P. falciparum gametocyte conversion and that premature high cGMP levels are deleterious for gamete formation [Plasmodium endoplasmic reticulum membrane, no ortholog of an IP3 receptor channel has yet been identified. A recent biochemical approach of an IP3 affinity chromatography column combined with bioinformatics has revealed a potential transporter associated with multidrug resistance in P. falciparum [The tivation . Pharmacasmodium ,133. Tayormation . Benninkormation ,136. Altlciparum . AdditioPlasmodium, the intracellular calcium level is sensed by calcium-dependent protein kinases (CDPKs) [The rapid release of calcium in mature gametocytes mediates a stage-specific calcium-dependent effector pathway in all gametogenesis steps, including gamete formation, gamete egress, microgamete mitotic maturation, and exflagellation ,133,135. (CDPKs) . Macro-  (CDPKs) ,139,140.2+ is sensed by CDPK1 [In mature female gametocytes, the increase in cytoplasmic Caby CDPK1 . CDPK1 hby CDPK1 .P. berghei, the lysis of the host cell membrane(s) surrounding the microgametocyte is mediated by CDPK1 [P. berghei, a histone chaperone protein termed FACT-L (facilitates chromatin transcription), which facilitates chromatin transcription, was shown to be involved in male gametocyte DNA replication and the production of fertile microgametes [The differentiation of male gametocytes is regulated by a male-specific calcium-dependent protein kinase CDPK4, which initiates DNA replication, axoneme assembly, and cell motility ,141. In by CDPK1 . In addiby CDPK1 . The celby CDPK1 . Whetherby CDPK1 . In P. bogametes . The forP. berghei have revealed that axoneme assembly and motility require several proteins, including the stage-specific Actin II [Pf16 [Plasmodium falciparum kinome includes four NIMA-related kinases (PfNEK 1 to 4). While PfNEK-1 is expressed in asexual and sexual stages, the mRNA transcripts of PfNEK-2, PfNEk-3, and PfNEK-4 are exclusively expressed in gametocytes. NEK1 and NEK3 have been shown to activate the atypical MAP2 protein through phosphorylation [PfNEK-2 and PfNEK-4 are required for meiosis completion in the ookinete [P. berghei male gametocyte exflagellation [Upon completion of DNA replication, axonemes become motile, facilitating the egress of the microgametes from the host cell. Genetic studies in Actin II , the armII [Pf16 , the spiII [Pf16 , the SR II [Pf16 , and a gII [Pf16 ,150. Therylation ,152. PfNookinete ,154. A mellation .P. falciparum-cGMP-dependent protein kinase identified a thiazole scaffold that kills erythrocytic and sexual-stage parasites [Plasmodium berghei sexual development and transmissibility to the mosquito. Two compounds from the panel effectively inhibited different development stages, from gametogenesis to ookinete maturation. These examples highlight that in silico modeling and screening combined with in vivo and ex vivo approaches using mouse models and human malaria could help identify parasite kinase and phosphatase inhibitors. The kinases regulating male gamete exflagellation  and DNA replication  should be of great interest in the screening.Kinases and phosphatases involved in gamete formation can be considered promising targets for drug development. A high-throughput screening of arasites . Since tarasites  assessedP. falciprum gametogenesis, more studies relying on human malaria parasites are necessary to grasp the complexity of the early stages of malaria transmission. Those studies should consider including field studies to better understand the biology of transmission during natural infection.Although rodent malaria parasites are sound model systems to understand P. falciparum and P. berghei genes have revealed that P48/45 and P230, two members of a protein family defined by a disulfide bonding pattern of six conserved cysteine residues, are essential for male gamete fertility and fusion with macrogametes [P. falciparum, P48/45 and P230 are localized on the gamete surface and have been shown to form a complex necessary for the fusion of the microgamete to the macrogamete. In vitro and in vivo studies in the mouse model have demonstrated the critical implication of P48/45 and P230 in fertilization. These findings led to their use as targets for transmission-blocking vaccines [Pfs47, a paralog of P48/45 was identified [Pfs47 exclusively on the surface of female gametes, but the protein did not appear crucial for fertilization [The zygote results from the fusion between a fertile male microgamete and a female macrogamete. Fertilization of a macrogamete by a microgamete is mediated by stage- and sex-specific proteins synthesized in the respective gamete before the fusion event . The proogametes ,158,159.vaccines ,160. In entified . Functiolization .p48/45 or hap2/gcs1 resulted in sterility due to the inability of the male gamete to either attach or fuse to fertile female gametes [Plasmodium revealed that a histone chaperone protein named FACT (facilitates chromatin transcription) plays an essential role in fertilization. However, the mechanism of action of this nuclear protein is still unclear [The evolutionarily conserved class II gamete fusogen HAP2/GCS1 (hapless 2/Generative-cell-specific 1) has also been implicated in the fusion of the plasma membranes of two haploid gametes . Gene di gametes ,162. Stu unclear .Plasmodium spp., Toxoplasma gondii, the ciliates, and dinoflagellates. This membranous patchwork is anchored to the subpellicular microtubule network. It also interacts with various parasite-specific proteins to coordinate parasite morphological changes, the segmentation of daughter cells during asexual replication, and parasite motility. Interestingly, the localization and interaction of many IMC proteins are regulated by post-translational S-palmitoylation mediated by the palmitoyl-acyl-transferase DHHC2 (the ortholog of PfDHHC1) [P. berghei [The fusion of the two gametes requires the prior disassembly of a peculiar organelle located underneath the parasite plasma membrane and is named the inner membrane complex (IMC) . The IMCPfDHHC1) . Inhibit berghei . Hence, P. falciparum TBVs are undertaken, but the functional activities associated with most antibodies remain modest. Further study must be conducted to determine the players involved in gamete fusion and their association with each other to orchestrate gamete fusion. The disassembly of the IMC is a pre-requisite for gamete fusion, and targeting the enzymes modulating this biological process could be an approach to interfere with the establishment of the infection in the mosquito gut. Plasmodium falciparum is predicted to encode 12 putative palmitoyl acyl-transferases thought to ensure lipid-based palmitoylation of parasite proteins and act as a biological rheostat for protein–protein interactions and subcellular trafficking [The concept of transmission-blocking immunity is mostly antibody-mediated. Therefore, the development of transmission-blocking vaccines (TBVs) focuses on inducing potent antibodies sustained at adequate levels. Extensive efforts toward the clinical development of fficking . Inhibitfficking ,167,168.P. berghei revealed that zygote development and differentiation into ookinetes require the transcriptional activation of several maternal silenced mRNAs in the zygote. A study investigating the parental contribution of transcripts implicated in zygote differentiation revealed that while inherited maternal mRNAs are activated to drive the early stage of zygote differentiation, the paternal alleles are initially silenced and then reactivated [Gene expression studies in ctivated ,169.Plasmodium kinome, combined with genetic studies in P. berghei, revealed that protein kinase 7 (PK7) and cyclin-G-associated protein (GAK) are essential in ookinete formation [Plasmodium phosphatase PPKL (protein phosphatase with kelch-like domains) is essential during ookinete differentiation and is involved in defining ookinete polarity, pellicle morphology and integrity, and ookinete motility [Plasmodium falciparum DHHC1, a palmitoyl-S-acyl-transferase (PAT) containing a conserved DH(H/Y)C motif, was shown to be exclusively localized to the IMC [The derepression of a maternal transcript post fertilization will drive significant morphological changes defined by the formation of the IMC and the secretory organelles, as well as the elongation and apical polarization of the differentiating zygote ,170,171.ormation ,172. GAKormation . PK7 mutmotility . Plasmod the IMC ,173. DHH the IMC ,173.Plasmodium parasite are regulated by NIMA-related kinases (Neks). Gene disruption studies in P. berghei revealed that Nek-4 and Nek-2 are abundantly expressed during the gametocyte stages. Nek-4 and Nek-2 are essential for zygote differentiation into the ookinete stage but not for gamete formation and fertilization [Plasmodium kinases are attractive targets for the next generation of antimalarials. Indeed, ongoing efforts attempt to characterize Plasmodium kinases while evaluating them as antimalarial drug targets [Subsequent regulation of stage-specific gene expression requires a de novo synthesis of ookinete-specific genes by the transcription factor ApiAp2-O . ApiAp2-lization ,154. Giv targets ,179.Ookinete maturation is completed 19 to 36 h post-gametocyte ingestion in the blood meal. The ookinete will quickly exit the midgut lumen via an intracellular or intercellular route . OokinetPlasmodium infection [Plasmodium stages found in the mosquito [The ookinete stage is an attractive target for transmission-blocking strategies as the mosquito immune system naturally kills many ookinetes. Ookinete proteins involved in the attachment and invasion of the midgut epithelial cells are potential vaccine targets. Two ookinetes surface proteins, P25 and P28, that play a role in ookinete adhesion to the midgut and differentiation into oocyst are candidates for transmission-blocking vaccines . Transminfection . The ratnfection . On the mosquito  or mosqumosquito . AntibodPlasmodium multi-stage drugs.As mentioned above, parasite-specific kinases, whose functions are required for parasite motility, are also good targets for Following the traversal of the midgut epithelium, ookinetes settle in the basal lamina surrounding the gut and differentiate into oocysts. Ookinetes can be found in the basal lamina 18–24 h after an infective blood meal . The mecPlasmodium stages has revealed an abundance of antisense transcripts in gametocytes and ookinetes [Compared with other developmental stages, relatively few transcriptomics and proteomics studies assess changes associated with the early stage of oocyst development. Sequencing strand-specific cDNA libraries of seven okinetes . These fokinetes . StudiesPlasmodium sexual differentiation within the vertebrate host and the mosquito vector. During intraerythrocytic development, parasites appear to predominantly regulate the expression of functional gene products in a stage-specific manner via epigenetic and transcriptional regulation. When preparing to change hosts, gametocyte formation necessitates post-translational repression mechanisms and epigenetic and transcriptional regulation. The fast differentiation process within the mosquito gut relies on the post-translational modification of proteins by specific kinases, phosphatases, and lipid modifications.Functional studies have uncovered critical processes associated with Plasmodium sexual differentiation is far from complete. The development of new approaches, including single-cell technologies, will provide greater resolution on transcriptional, epigenomic, and metabolomic variations that regulate parasite differentiation. Current findings suggest that functional genetics data need to be integrated into studies assessing the function of malaria proteins for a better understanding of the developmental processes. Although rodent malaria parasites are good model systems to understand P. falciparum development within the mosquito, more studies relying on human malaria parasites are necessary to grasp the complexity of malaria transmission and infection in the mosquito. Plasmodium functional studies have been carried out using parasites maintained and propagated in vitro. With environmental factors affecting considerable parasite gene expression, additional epigenomic, proteomics, transcriptomic, and metabolomic studies from field isolates are pertinent to better understand the processes that govern parasite differentiation. Finally, these studies will have to target all six malaria species infecting humans to identify conserved and species-specific processes that could be used to discover new vaccine candidates and novel classes of antimalarial drugs. Computer-aided approaches and in silico modeling might be alternatives for developing new classes of antimalarial drugs targeting parasite enzymes.The list of the molecular players involved in


Chromatin is the physical substrate of the genome that carries the DNA sequence and ensures its proper functions and regulation in the cell nucleus. While a lot is known about the dynamics of chromatin during programmed cellular processes such as development, the role of chromatin in experience-dependent functions remains not well defined. Accumulating evidence suggests that in brain cells, environmental stimuli can trigger long-lasting changes in chromatin structure and tri-dimensional (3D) organization that can influence future transcriptional programs. This review describes recent findings suggesting that chromatin plays an important role in cellular memory, particularly in the maintenance of traces of prior activity in the brain. Inspired by findings in immune and epithelial cells, we discuss the underlying mechanisms and the implications for experience-dependent transcriptional regulation in health and disease. We conclude by presenting a holistic view of chromatin as potential molecular substrate for the integration and assimilation of environmental information that may constitute a conceptual basis for future research. The mammalian brain is a complex organ whose functions are strongly influenced by experience. Across life, the brain needs to assimilate and integrate information about the environment, and use this information to adapt and direct actions and behavior. These biological functions are fundamental for animals and human beings and have a social, ecological and evolutionary impact –3. EarlyIn the cell nucleus, genome organization relies on chromosomal interactions and on associations between chromatin and the nuclear lamina at the periphery of the nucleus , 27. ChrRegulatory elements can physically interact via long-range interactions and create chromatin loops . At a hicis-regulatory elements, particularly enhancers and promoters, and modulate gene transcription, for instance in differentiating stem cells, during circadian cycle and even during activation of brain cells [c-Fos and Arc, which leads to gene transcription [The 3D organization of the genome is an essential component of transcriptional regulation in mammals and undergoes major changes during development and cellular activity , 41, 42.in cells –45. Chroin cells , 19, 46.cription , 47. Impcription –50.Mechanistically, the establishment and stabilization of long-range interactions and chromatin loops primarily involve the activity of the architectural proteins cohesin and binding of the CCCTC-binding factor (CTCF) , 52. TheChromatin-based mechanisms that control gene expression contribute to cell differentiation during brain development and to higher-order functions in the adult. Early postnatal life is a time of intense epigenome remodeling in brain cells. In the maturing brain, enhancer–promoter interactions undergo global rewiring, and TADs and chromosomal compartments are reorganized within neuronal and glial cells nuclei , 64. At Chromatin states are also modified by neuronal activity, which involves activation of enhancers . SeveralOverall, changes in chromatin structure and 3D organization have been associated with neurodevelopmental processes and higher-order brain functions in adulthood, but emerging evidence suggests that they can also be implicated in experience-dependent regulatory memory in brain cells. Below we review current evidence supporting a role for chromatin in the maintenance of traces of prior activity in brain cells and we describe potential mechanisms of establishment and functional implications in health and disease.In mammals, neuronal activation induces the expression of genes necessary for neuronal plasticity, a cellular property essential for learning, memory and cognition . At the Unlike transcription, changes in the structure, composition or 3D organization of chromatin that occur after IEGs activation are not transient and chromatin is not necessarily re-established at pre-stimulation states. For instance, neuronal activation by electroconvulsive stimulation increases chromatin accessibility at thousands of genomic sites after one hour in hippocampal slices . But altKainic acid, frequently used to induce seizures in animal models, also persistently affects chromatin in neurons. A single injection is sufficient to strongly activate neurons and increase transcription in excitatory neurons of the adult hippocampus. It extensively changes chromatin accessibility and long-range 3D interactions between regulatory elements . Like wiRemarkably, genomic regions with lasting changes in chromatin accessibility are also engaged in persistent long-range interactions that are independent of CTCF binding. These regions are enriched for the binding motif of the family of TFs AP-1, suggesting a role for AP-1 TFs in the establishment and maintenance of chromatin states (see Mechanisms). Sites with stable changes in occupancy are also located at the promoter or enhancer of genes involved in axonal growth or protein aggregation , suggestEnvironmental exposure and salient experiences, i.e., experiences that stand out, are extreme and emotionally charged, can have long-lasting effects on chromatin in brain cells. Their effects can vary depending on the time window of exposure (whether before or after birth or in adulthood), but also its type  and duration (acute or chronic). While the adult brain is susceptible to changes, the developing brain is probably even more sensitive and may be more profoundly affected since many developmental processes such as cell division and differentiation in prenatal life and cell migration, synaptogenesis and myelination after birth are ongoing . FurtherDot1l and Kdm2b, which are H3K79 methyltransferase and demethylase respectively, in dopaminergic neurons of NAc [Dot1l dysregulation occurs only in early adulthood, the observed changes in H3K79me1/2 are not an immediate consequence of stress at the time of exposure but rather emerge later in development, suggesting a form of molecular memory of prior stress.Early life conditions such as stress before or after birth can have effects on physiology, behavior and cognition that can last until adulthood . These es of NAc . Althougs of NAc  and enhas of NAc . Since DWhile transcriptional and epigenetic changes have been well studied in animal models and humans, changes at the level of chromatin itself remain not well characterized in the brain. Most evidence for changes in chromatin by stress is in immune cells. Perinatal glucocorticoids have been shown to favor chromatin accessibility in cytotoxic T cells at genes carrying glucocorticoid receptor binding sites necessary for interferon production . The effFurther to stress, exposure to chemicals early in life can also affect behavior and cognition in mammals and involve lasting changes in chromatin structure and organization affecting gene expression in the brain. Repeated exposure to midazolam, an anesthetic frequently used in children, can cause neurological abnormalities. In mouse, midazolam exposure from PND10 to PND12 impairs hippocampus-dependent object memory on a novel location recognition task as well as fear memory on a contextual fear conditioning test . At the Memory formation has been associated with long-lasting changes in chromatin accessibility. Neuronal activation by fear conditioning rapidly increases chromatin accessibility at thousands of genomic loci in hippocampal neurons (within 1.5 h). Some of the changes persist for at least five days, suggesting lasting chromatin states . ConditiΔFosB promoter and enhancer elements have increased chromatin accessibility upon acute cocaine administration that persists during prolonged withdrawal and upon relapse [Further to memory, addiction has also been associated with lasting changes in chromatin. Addiction is a pathological dysfunction characterized by a loss of self-control over one’s action that can be harmful. One of the detrimental features associated with addiction is relapse, that can occur even after prolonged abstinence and is caused by “drug” memory. In mice, chronic exposure to cocaine followed by 30-day withdrawal increases chromatin accessibility at thousands of genomic regions including potential enhancer elements, in medium spiny neurons of NAc expressing the D1 dopamine receptor (cocaine target) . Some of relapse . ImportaFurther to neuronal cells, immune cells in the adult brain can also acquire lasting changes in chromatin composition. In adult mice, peripheral inflammation induced by lipopolysaccharides injection causes differential deposition of H3K4me1 and H3K27ac at enhancer elements that persist for at least 6 months in microglia, brain resident macrophages . EnhanceOne of the consequences of the genomic redistribution of DNA binding proteins such as TFs are changes in chromatin accessibility. Because TFs can remain bound to DNA, they have been proposed to regulate the establishment and maintenance of chromatin states –96. ConsAn epigenetic mechanism for dynamic changes in gene expression mediated by lasting modifications of chromatin accessibility has also been proposed in the context of neuronal activation . IncreasWhile still limited in brain cells, there is evidence that TFs play a role in the establishment, maintenance and reactivation of chromatin states in the context of inflammatory memory in adult stem cells , 104. InA hit-and-run mechanism mediated by constitutive and environmentally induced TFs has also been described in adult hematopoietic stem cells to explain long-lasting changes in chromatin accessibility induced by lipopolysaccharides . In thesGenomic regions with chromatin memory in brain cells are also differentially enriched in histone post-translational modifications such as H3K4me1, H3K27ac, H3K79me2/3 among others , 12. ThiAn alternative scenario in which histone modifications may directly influence chromatin memory is via changes in 3D genome organization. Such changes may affect the segregation of genomic regions carrying the modifications into separate compartments (A or B) leading to their isolation and stabilization within the nucleus . Such seH2afz and enhanced association of the H2A.Z chaperone ANP32E to chromatin [H2afz gene itself remains significantly downregulated after chronic cocaine and during withdrawal, H2A.Z incorporation is reduced at promoter regions and in NAc-specific enhancers of genes such as FosB [1165,97. This suggests that the reduced genomic binding of H2A.Z during withdrawal might result from lower H2afz transcription and increased H2A.Z removal, potentially affecting chromatin accessibility genome-wide.Another mechanism that contributes to the maintenance of chromatin states is the replacement of histone variants. H2A.Z is one of the variants known to be reduced by chronic cocaine administration in the rodent brain and that remains low for up to 30 days after drug withdrawal . H2A.Z ihromatin . Interes as FosB . However as FosB , it may The establishment and maintenance of chromatin states induced by exposure are largely influenced by the developmental time window and duration of exposure. While long-lasting changes in chromatin structure can be potentially established any time during development, the molecular mechanisms allowing their stabilization and their cellular and functional consequences on the brain largely depend on the time of exposure. In particular, postnatal stages of brain development are periods of increased susceptibility because NSCs proliferate and differentiate into post-mitotic neurons that no longer divide nor undergo molecular “renewal” by replication. This means that epigenetic modifications acquired at that time may remain associated to the genome and constitute a form of chromatin memory in newborn neurons , 102. FuIn brain cells, changes in chromatin accessibility have been associated with differential gene expression. Exposure of NSCs to midazolam results in transcriptional upregulation of quiescence genes which correlates with increased chromatin accessibility at the promoter of these genes that persists in adult cells . Howeverc-Fos expression associated with a lasting gain in chromatin accessibility at Jdp2, a gene located downstream of c-Fos that encodes an AP-1 repressor [Pathological neuronal activation such as during epileptic seizure (or status epilepticus) can also result in long-lasting changes in chromatin accessibility at key regulatory elements and potentially affect transcriptional programs. Mice subjected to seizure by kainic acid have memory deficits and blunted epressor . Changesepressor . OverallExperiences are major determinants of brain functions that shape developmental and molecular trajectories of brain cells and have lasting effects influencing neural responses and behavior in adulthood. While synaptic processes are classically thought to be the main site for neural stability and plasticity, new findings indicate that chromatin also has these properties. Its composition, structure and 3D organization within the cell nucleus can contribute to transcriptional programs activated by experience and act as mediator of information assimilation in the genome in the form of chromatin memory.Mechanistically, the establishment and maintenance of chromatin memory rely on the activity of TFs that bind to genomic sites upon stimulation, and on the stabilization of activated chromatin states. Such mechanisms have been implicated in inflammatory memory in immune cells and epithelial stem cells, and we postulate that they are also at play in brain cells. We propose that experiences can lead to persistent changes in chromatin structure and organization via differential activity of AP-1 TFs. Since these TFs have been implicated in signal-induced long-range interactions between regulatory elements , they maWhile important progress has been made in the understanding of chromatin regulation in the brain, several major questions about the mechanisms of chromatin memory and their implication in brain functions and diseases remain open and need experimental work to be addressed. In particular, studies on the formation of chromatin memory in brain cells with a better temporal resolution (from minutes/hours to days or months after exposure) coupled with genome-wide characterization of binding profiles of AP-1 TFs, histone modifications and cell-type specific and constitutive candidate TFs are needed. Such experiments should ideally be conducted in vivo but also in vitro using primary cultures, differentiated ESCs or even brain organoids, and analysis may focus on activated cells to maximize the detection of effects. Experiments designed to identify and characterize the temporal dynamics of chromatin memory formation and maintenance should also help clarify the stability of changes. It is also still unclear how long after exposure changes in chromatin can be considered stable or still a remnant of regulatory dynamics operating after stimulation. Further, studies characterizing the acquisition of chromatin memory in brain cells in response to the same stimulus but at different period of life  should inform on the effect that time of exposure can have on the vulnerability or resilience of the genome to lasting changes in chromatin and whether this correlates with cognitive and behavioral alterations.The available evidence at the moment suggests that chromatin memory is a more widespread phenomenon than previously anticipated. Therefore, its study in the context of brain functions should not be limited to neurons, unlike done so far, but should also include glial cells such as microglia, and even epithelial cells, since environmental exposures are likely to affect all cell types in the brain. Further, multiple brain regions in addition to classical hippocampus, such as the medial pre-frontal cortex should be examined. Critically, the functional consequences of chromatin memory in the brain remain to be clarified but likely involve transcriptional programs that affect brain cells physiology and therefore, behavior and cognition. The manipulation of regulatory sequences with chromatin memory in vitro or in vivo could inform on their contribution to transcriptional programs after experience and to changes in behavior and cognition.Finally, while accumulating evidence strongly supports the existence of chromatin memory in brain cells, this evidence is mostly derived from animal models, rodents in particular. Therefore, there is also a need to address the existence of chromatin memory, its mechanisms and potential functional implications in human brain cells. In this regard, the use of neurons and glia derived from induced pluripotent stem cells (iPSCs) in culture or in the form or organoids coupled with different types of exposure should provide valuable models to characterize chromatin memory in human brain cells. We anticipate that mechanisms of chromatin memory identified in rodents also operate in human cells, however, direct experimental evidence is needed to support such claim. We postulate that lasting changes in chromatin structure and organization in human cells, acquired during our life time, might not just influence cognition and behavior but also the likelihood for the development of psychiatric and mood disorders as well as the origin and evolution of neurodegenerative diseases.


Protein lipidation, the attachment of lipids to proteins, influences how proteins fold, their stability, trafficking, interaction with other proteins, and association with specific domains on cellular membranes. Various types of lipids, such as phospholipids, isoprenoids, fatty acids, cholesterol, GPI (glycosylphosphatidylinositol)-anchors, and LDEs (lipid-derived electrophiles), modify proteins covalently. S-palmitoylation or S-acylation is a reversible type of lipidation where a fatty acid called palmitate attaches to specific cysteine residues through a labile thioester bond. N-palmitoylation (via a stable amine bond) and O-palmitoylation (via a stable ester bond) are less common and irreversible kinds of palmitoylation . S-palmiThe objective of this Research Topic is to compile the most recent insights on the impact of protein lipidation on protein function and explore how alterations in these modifications are linked to the pathophysiology of human diseases, paving the way for the development of novel therapeutic strategies.Wirth and Ponimaskin review the literature describing that the small GTPase Cdc42 splice variant which is palmitoylated  is mainly expressed in the brain, in contrast to its prenylated ubiquitous form (Cdc42-prenyl). It would be interesting to further explore in the future whether these are anecdotal cases or whether the regulation of splice variants and protein isoforms are more widespread modulators of protein palmitoylation.One important feature of many post-translational modifications (PTMs) is how they can be regulated by gene-splicing variants. For example, the splice variant of the AMPA scaffolding protein GRIP1, GRIP1b, can be palmitoylated, whereas GRIP1a cannot, affecting its subcellular distribution . In thisWirth and Ponimaskin discuss the possible association between aberrant palmitoylation of Cdc42 and activation of inflammatory responses and autoinflammatory diseases. Furthermore, West et al. introduce PATs as novel regulators of T-cell function in their literature review. For example, zDHHC7 and zDHHC21 have been shown to be critical for T-helper cells differentiation, and key proteins in T-cell signalling such as CD4 and CD8 are palmitoylated. Despite these advances, the role of lipidation in the immune system remains mostly unknown.Recent studies have revealed that numerous immune-associated proteins undergo S-palmitoylation. These findings have shed light on the vital roles played by palmitoylation in immune function, particularly in directing immune signalling proteins to the membrane and lipid rafts. Furthermore, alterations in PAT activity and variations in palmitoylation have been linked to human immunological disorders . To streRamzan et al.)]. In the future it will be pivotal to further investigate the crosstalk between lipidation and other PTMs to understand the consequences for protein localization and function, as well as the impact on organelle function.Diverse findings point to a complex interrelationship of palmitoylation and other PTMs such as phosphorylation, S-nitrosylation, acetylation and ubiquitination. Thus, for example, palmitoylation may inhibit, compete with, or facilitate another PTM, and palmitoylation/depalmitoylation can also regulate subsequent phosphorylation of certain substrates [reviewed by    and humaRamzan et al.) and palmitoylation-independent functions of PATs in future studies.Dysregulation of lipid metabolism and protein lipidation is associated with different pathologies, but understanding the underlying molecular mechanisms remains a challenge. This is partly due to limited knowledge regarding how lipidation and its regulating enzymes are controlled by cellular signalling pathways. While lipidation-targeted therapies are being investigated for cancer treatment, for example, their application to other diseases is still largely unexplored. Furthermore, while certain forms of protein lipidation, like S-palmitoylation, have garnered substantial attention in recent years, others, such as N-palmitoylation, remain relatively unexplored. Therefore, it is crucial to advance the knowledge of the regulatory mechanisms that control lipidation, the interplay between lipidation and other post-translational modifications, methodologies to detect, measure, and manipulate protein lipidation, the interplay between lipid metabolism and protein lipidation, and less common or studied varieties of lipidation. Besides, it is worth to highlight the importance of prioritising studies investigating putative sex difference in palmitoylation . Main et al. used publicly-available RNA-seq datasets from patients with heart failure to evaluate how palmitoylation and depalmitoylation pathways remodel in these patients as well as in animal models and clinical biopsies. A complex picture emerges in which the surface-membrane acyl transferase zDHHC5 expression increases significantly during early remodeling but declines once heart failure is established. These expression changes correlate very poorly with substrate palmitoylation, highlighting the continuing paucity in our understanding of how zDHHC-PAT activity is controlled.The contribution of aberrant palmitoylation to development of human pathologies is an important consideration for this field. This Research Topic gives a broad overview of the importance of these PTMs in neurodegeneration, X-linked intellectual disability, metabolic disease, cancer, and cardiac pathologies  can be stabilized by GCP16 to support enhanced enzymatic activity. This suggests an additional layer of control for activity of some of these enzymes than has hitherto been under-appreciated. Clearly the expression level of the accessory protein, as well as the expression level of the enzyme itself, will be an important determinant of substrate palmitoylation.With this in mind, the findings of Congreve et al. focused on the cardiac voltage sensitive ion channel HCN4, which is expressed in the sino atrial node and cardiac conduction system, and has the unique property of being activated by hyperpolarization. HCN4 controls heart rate by controlling the rate of depolarization in nodal tissue, and its activity is significantly enhanced by palmitoylation of two cysteines in the channel’s amino terminus. This raises the intriguing possibility that a fundamental feature of multicellular organisms, the heartbeat, is controlled by the coordinated activities of palmitoylating and depalmitoylating enzymes on a single ion channel.Numerous ion transporters and channels in excitable tissues are regulated by palmitoylation. Gupta and Jenkins identified numerous palmitoylation sites in ankyrin B which are palmitoylated by zDHHC17. When ankyrin B is not palmitoylated it loses its ability to correctly localize voltage sensitive sodium channels to dendritic membranes, highlighting the importance of palmitoylation not only of ion channels but also of their targeting partners for the control of tissue excitability.The scaffolding protein ankyrin B, the small cousin of the giant ankyrin G, controls the localization of ion channels and transporters in numerous tissues. In the last 20 years, progress has been made in the study of regulatory mechanisms of palmitoylation, the role of this posttranslational modification in physiology and diseases, and the methodology for its study. However, there is still much research to be performed in these aspects and the interplay of the findings with the overall regulation of lipid metabolism in the organism.


How molecules interact governs how they move. Single-molecule tracking (SMT) thus provides a unique window into the dynamic interactions of biomolecules within live cells. Using transcription regulation as a case study, we describe how SMT works, what it can tell us about molecular biology, and how it has changed our perspective on the inner workings of the nucleus. We also describe what SMT cannot yet tell us and how new technical advances seek to overcome its limitations. This ongoing progress will be imperative to address outstanding questions about how dynamic molecular machines function in live cells. An increasing number of labs around the world deploy various methods to track single endogenous protein molecules in cells, tissues, and even whole organisms to probe their biochemical behavior in their native environment. Here we will discuss how single-molecule tracking (SMT) is performed, what we can learn from it, and how ongoing technical advances are making SMT more informative. SMT has diverse applications in cell and molecular biology, from studying the translocation of molecular motors along the cytoskeleton to monitoring interactions of membrane receptors. Each biological problem poses its own distinct challenges and requires different methodologies . In thisSwitching a gene on or off involves multitudinous interactions between hundreds of proteins. Protein-encoding genes are transcribed by RNA polymerase II (Pol II), which initiates transcription following assembly of general transcription factors (GTFs) into a pre-initiation complex (PIC) at the gene promoter ,5. Transin vitro mixtures [SMT involves localizing and tracking individual fluorescently labeled molecules over time. More than a decade of advances in microscopy ,14, genomixtures  to cell mixtures ,25, to tmixtures . Severalmixtures ,42. Secomixtures . Third, mixtures , passes mixtures . A schemSMT experiments fall into two broad categories: ‘Slow' SMT observes molecules bound to slow-moving scaffolds, such as chromatin ,51–53. AThe first step in SMT data analysis is to localize and track single molecules, which can be done using various algorithms . In slowFor fast SMT, fitting the mean squared displacement (MSD) as a function of time provides a simple way to estimate diffusion coefficients. However, this approach suffers from systematic bias when applied to three-dimensional samples, because faster-moving molecules are more rapidly lost to defocalization. This bias can be corrected mathematically ,39,56. Swww.mir-lab.com/dynamics-database). It should be noted that the accuracy of residence time measurements depends sensitively on correction for photobleaching and defocalization of fluorophores, and it will be important to validate the consistency of these measurements across multiple labs.SMT has enabled a new form of live-cell biochemistry, illuminating previously unseen behaviors of transcription regulators. For instance, it is tempting when gazing at chromatin immunoprecipitation sequencing (ChIP-seq) peaks to imagine that proteins form stable complexes on chromatin. However, SMT has shown that this is generally not the case. Residence time measurements by slow SMT have revealed that different transcription regulators interact with chromatin over a wide range of timescales, from milliseconds to minutes ,55,64,71in vivo order of assembly of GTFs and Pol II into the PIC [Fast SMT measurements can reveal how the chromatin bound fraction of a protein changes in response to genetic or chemical perturbations, providing a powerful way to dissect the biochemistry of protein–DNA interactions in live cells. In the case of Type I nuclear receptors, fast SMT revealed an increase in chromatin binding upon ligand treatment ,69,75,76 the PIC .in vitro have directly observed 1D sliding by TFs, this would be difficult to resolve in live-cell SMT experiments [Fast SMT has also allowed us to directly visualize how TFs locate their binding sites. The classic ‘facilitated search' model of von Hippel and Berg proposes that DNA-binding proteins find specific sites by alternating between three-dimensional diffusion and one-dimensional sliding along DNA . While seriments ,78,79. Heriments ,39,80,81Another intriguing feature of target search has emerged from analyzing angles between successive displacements. The motion of the transcriptional activator c-Myc is well described by regular Brownian motion, in which these displacement angles have a uniform (isotropic) distribution . In contSMT also provides a way to detect weak interactions between proteins that are otherwise difficult to study, such as those mediated by intrinsically disordered regions (IDRs). Particularly enriched in TF activation domains, IDRs mediate weak, flexible, and multivalent interactions between many types of proteins . IDRs caAlthough SMT experiments are data-rich, there is much more that we would like to discern. We can determine what fraction of a given transcription regulator binds chromatin and measure its residence time, but we don't know where on the genome it binds. We can resolve populations of TFs with different diffusion coefficients, but this does not reveal what those complexes are biochemically. We would like to monitor transitions between protein complexes, yet photobleaching and camera frame rates limit how fast and how long we can track individual molecules. Molecules move three-dimensionally in the nucleus, but we typically track them in two dimensions. Fortunately, technical advances and new approaches provide opportunities to overcome many of these limitations .lacO and tetO arrays, ParB-parS , and dCas9 [To understand nuclear processes like transcription, it is crucial to measure the binding of proteins to the specific genomic loci where these processes occur. While various tools exist for locus labeling — nd dCas9 , monitorDrosophila polytene chromosomes, naturally occurring gene arrays consisting of bundles of aligned, endo-replicated sister chromatids [lacO sites [One solution is to image arrays of multiple gene copies. This increases the number of localizations detectable at the specific target while spatially separating those localizations from molecules bound to the rest of the genome. Locus-specific binding of single proteins has been visualized on romatids . Naturalromatids . Slow SMromatids ,51. SlowcO sites . A drawbIn some cases, PPIs can be inferred from SMT data by analyzing the effect of chemical or genetic perturbations. However, these perturbations may have unanticipated effects on the conformational and binding equilibria of proteins, possibly resulting in inaccurate interpretation of results. It would therefore be advantageous to detect PPIs directly.For some proteins, such as cell surface receptors imaged by total internal reflection fluorescence (TIRF) , PPIs caPPIs can also be detected by fusing the two interacting partners to two halves of a split fluorescent protein or HaloTag, which are reconstituted into a complete tag when the fusion proteins come together ,103. WhiWe recently reported a new method to detect PPIs by SMT . While iMany fluorescent dyes can undergo chemical reactions when excited, which render them non-fluorescent or ‘dark'. Dark fluorophores can reactivate either spontaneously or through direct reactivation (DR) by near-UV light. Additionally, excitation of one cyanine (Cy) dye (e.g. Cy3) can reactivate a second nearby Cy dye (e.g. Cy5) . Cy dye Although the mechanism of PAPA between JF dyes is unclear, it offers distinct advantages as a proximity sensor. Unlike Cy dye photoswitching, it occurs in live cells with membrane-permeable dyes, and it is more permissive of large inter-dye separations than either Cy dye photoswitching or FRET . Proof-oTracking a single protein molecule throughout its lifetime, from synthesis to degradation, would reveal how fast it enters and exits the nucleus, how its diffusion changes over time due to association with binding partners, and how often it binds and dissociates from chromatin. Unfortunately, tracking a single molecule for an extended period of time is difficult, particularly in fast SMT, where trajectories of diffusing molecules last no more than a few camera frames . While aTwo technical limitations stand in the way: First, fluorophores photobleach rapidly at the laser powers required for SMT. Approaches to overcome photobleaching include (1) using more stable fluorophores, (2) labeling each protein with multiple fluorophores, and (3) using a structured illumination approach called MINFLUX. Second, while molecules move three-dimensionally in the cell, SMT typically images a single two-dimensional focal plane, and molecules that exit this plane can no longer be tracked ,56.in vitro experiments. The development of genetically encoded, self-labeling tags like Halo and SNAP, together with cell-permeable fluorophores like JF dyes, has combined the best of both worlds, making it possible to label target proteins with bright, photostable synthetic fluorophores in live cells [Fluorescent proteins (FPs) transformed microscopy by making it possible to label target proteins using simple molecular cloning. Synthetic organic fluorophores, although much brighter and more photostable than FPs, have historically required chemical conjugation to target proteins, mostly restricting their use to ve cells . These tve cells ,116.Labeling a protein with multiple fluorophores increases the total fluorescence signal and allows it to be tracked longer before it is lost to photobleaching. A simple option is to fuse multiple tandem copies of HaloTag to the target protein . The SunMINFLUX localizes one fluorophore at a time by continuously re-centering a ‘doughnut' beam to find the point of minimum excitation . Fewer pz-planes simultaneously on a camera [Loss of trajectories due to defocalization could be prevented by imaging and tracking in 3D. While light-sheet microscopy can be used for 3D slow SMT , its vola camera ,112,123.a camera ,125.Angular anisotropy: Deviation of the angles of successive single-molecule displacements from the uniform distribution characteristic of regular Brownian motion.Brownian motion: An idealized random walk in which the subsequent position of an object is drawn from a Gaussian distribution centered on its current position.Defocalization: Movement of objects out of the focal plane.nt, where t is time and n is the number of dimensions. E.g. in two dimensions, <r2> = 4Dt.Diffusion coefficient: A measure of how fast a molecule diffuses. More precisely, the constant of proportionality between its MSD and 2Direct reactivation (DR): Reactivation of a fluorophore from a dark state in response to absorption of UV or near-UV light.Highly inclined and laminated optical sheet (HILO): A type of illumination similar to TIRF in which a laser beam is allowed to propagate obliquely through a sample, illuminating the region closest to the coverslip. Unlike TIRF, the beam propagates through the sample rather than undergoing total internal reflection, permitting excitation of fluorophores slightly farther from the coverslip (e.g. in the cell nucleus).Intrinsically disordered regions (IDRs): protein sequences that do not adopt a fixed three-dimensional fold. IDRs have been dubbed the ‘dark matter' of the proteome—extremely common, yet difficult to characterize using the standard tools of biochemistry and structural biology.Localization error: Uncertainty in the spatial localization of a molecule, arising from a finite number of emitted photons, molecular motion, camera noise, etc.Mean-squared displacement (MSD): the mean of the squared displacement from the starting position of a random walk. For regular Brownian motion, MSD increases linearly with time.minimal flux of emitted photons.MINFLUX: A method in which fluorophores are localized and tracked by actively re-centering a ‘doughnut' beam on the fluorophore. This is accomplished by fast electronic feedback, which locates the beam position that gives a Photobleaching: destruction of fluorophores by chemical reactions that occur in the excited state.Proximity-assisted photoactivation (PAPA): Reactivation of one fluorophore (e.g. JFX650) from a dark state upon excitation of a second nearby fluorophore (e.g. JF549).Residence time: How long a molecule remains bound to its target (e.g. chromatin).o Slow SMT: Tracking molecules at a relatively slow frame rate (hundreds of milliseconds to seconds) to monitor how long molecules remain bound to a slow-moving scaffold (e.g. chromatin) and how they move while bound.o Fast SMT: Tracking molecules with a relatively fast frame rate  to measure their diffusion. Short 1–2 ms stroboscopic excitation pulses can also be used to reduce motion blur.Single-molecule tracking (SMT): a method that monitors the motions of individual fluorescently labeled molecules under a microscope. SMT is a type of single-particle tracking (SPT), which includes the tracking of objects larger than a single molecule, although in practice the terms SMT and SPT are often used interchangeably.Total internal reflection fluorescence (TIRF): A type of illumination in which light is reflected off the interface between the coverslip and the sample at an angle greater than the critical angle. The resulting total internal reflection produces an exponentially decaying evanescent field that excites fluorophores within a few hundred nanometers of the interface.Single-molecule tracking (SMT) probes interactions of biomolecules in live cells.Key observables of SMT include diffusion rate and anisotropy, chromatin-bound fraction and residence time, subcellular localization, and proximity to other labeled molecules. SMT can distinguish subpopulations of a biomolecule based on differences in these parameters.New technologies are enhancing our ability to track molecules for extended periods of time and study specific protein–protein and protein–nucleic acid interactions.


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.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.The online version contains supplementary material available at 10.1007/s00109-023-02352-1. Regulation of gene expression based on mutual epigenetic mechanisms, mainly DNA methylation levels, differential transcription of ncRNA species, and recruitment of histone modifying and chromatin remodeling systems, is attracting research attention in recent years. Of particular interest is the regulation of ncRNA expression mediated by TFs imitating the classical protein coding genes’ transcriptional regulation mechanisms. Correct tissue-specific and time-defined production of miRNAs and lncRNAs, of the most studied ncRNA varieties, constitutes the endogenous chromatin signaling towards a synchronized gene expression regulation. Among other ncRNA-dependent cellular processes, human erythropoiesis and hemoglobin switch from embryonic to HbF and to HbA during gestation are orchestrated by specific sets of miRNAs, lncRNAs, and co-operation of TFs –4. Gene DANCR-lncRNA (Differentiation Antagonizing Non-Protein Coding) promotes erythroid differentiation [MONC- (Megakaryocytic Oncogenic Non-Coding RNA) and MIR100HG-lncRNAs reside within cell nucleus and are involved in the regulation of erythro-megakaryocytic development. Their abnormally high expression has been evaluated in acute megakaryoblastic leukemia (AMKL) [2γ2 chains) to HbA (α2β2 chains), which under normal conditions occurs at the latest stage of gestation before birth, has been combined with BGLT3-lncRNA (Beta Globin Locus Transcript 3) expression [lncRNAs have long been implicated in human hematopoiesis by contributing to the conformation of an accessible chromatin landscape in a tissue and stage specific manner . Cross-antiation , whereasa (AMKL) . Along eUCA1- and ZEB1-AS1-lnRNAs with miR-548j, miR-3646, miR-937, and miR-19b-3p is confirmed to regulate HbF [NR_001589- and uc002fcj.1-lncRNAs in a high-HbF patient group were significantly correlated by bioinformatics methodology with certain sets of miRNAs and mRNAs [MiRNAs have been also reported to affect hemoglobin switch from HbF to HbA and in certain circumstances to alleviate anemia symptoms in β-hemoglobinopathies patients. Research efforts to reactivate HbF expression in adult β-hemoglobinopathies patients are ongoing with the ultimate goal of benefiting patients from anemia symptoms and support their de-addiction from constant blood transfusions. Repressing effects towards BCL11A (B-Cell CLL/Lymphoma 11A), the most fully studied γ-globin repressor, are exerted by miR-15a/16–1, miR-486-3p, and miR-210; therefore, HbF levels are increased with valuable results for the patients . MiRNAs A yet unsolved issue is decoding the pathway directing the epigenetic elements to a favorable repertoire of action during healthy state or disorder. Genome architecture and epigenetic landscape support positively or negatively ncRNA transcription, depending on the conditions prevailing across genetic loci coding for miRNA clusters, lncRNAs, and other ncRNA species, not sufficiently studied. lncRNAs are considered mainly products of RNA polymerase II transcription , and sevBGLT3, encoding for a lncRNA spanning the intergenic sequence between γ- and β-globin gene and simultaneously at hypersensitive sites of β-globin Locus Control Region (βLCR), supporting hemoglobin transversion from HbF to HbA [So far, CxxC zinc finger protein 1 (or CFP1) has been shown to bind CpGs associated with active transcription start sites and enhance gene expression . Also, oF to HbA .cis and trans regulatory elements, mainly modifying TFs acting to the β-type globin locus [To assess the expression levels of miRNAs and lncRNAs influenced by LRF and implicated in hemoglobin switch from HbF to HbA, we have developed an in vitro system of transgenic human erythroleukemia cell line K562, over-expressing LRF transcription factor. Natural process of hemoglobin switch in humans is under control of the βLCR and other in locus , 25, witZBTB7A gene encoding for LRF, as previously described. Reporter gene eGFP has been included within the same transcriptional cassette, and long-term expression of the exogenous gene has been confirmed by fluorescence microscopy . LRF/ZBTB7A expression levels were evaluated and were up to 11-fold increased. In parallel, transfections were carried out with the vector backbone. Results obtained by qPCR analyses displayed insignificant differences versus untransfected K562 cells. Treatment of transfected and untransfected K562 cells with 30 μΜ hemin  and 5 ng/mL erythropoietin (EPO)  for 3 h resulted to erythroid differentiation, hemoglobinization, and induction of β-globin expression [Human K562 erythroleukemia cells were cultured and transfected with episomal vector encompassing the coding sequence (exons 1 and 2) of pression  Genomic https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) to assess quality. Minion [https://github.com/FelixKrueger/TrimGalore) to remove contaminants and low quality reads with parameters “-q 20–length 20.” The remaining reads were aligned to the human reference genome (GRCh38) with STAR v2.7.10a [Picard Tools. Broad Institute. http://broadinstitute.github.io/picard/) was subsequently applied to minimize the enrichment of PCR duplicates. Reads that were aligned to the mitochondrial genome were excluded from downstream analyses. LRF peaks were identified with MACS2 [LRF-binding sites were identified by analyzing existing K562 UT, UT+hemin/EPO, LRF-OE, and LRF-OE+hemin/EPO cell samples from chromatin immunoprecipitation assays with antibodies against LRF (Gene Expression Omnibus accession ID GSE200135), combined with next-generation sequencing (NGS) of precipitated DNA fragments (ChIP-Seq), previously conducted by Chondrou et al. . Reads w. Minion  was alsov2.7.10a  using path MACS2  with par® technology , according to manufacturer’s instructions and as previously described [Pyrosequencing CpG assay was utilized to assess DNA methylation status across CpG islands located at 5′ prime ends of lncRNAs’ genetic loci. Genomic DNA was extracted from K562 cultivated cells  with phenol:chloroform:isoamyl alcohol in 25:24:1 ratio (Sigma-Aldrich), according to standard protocol . 1.5 μg escribed . PCR andGAPDH control gene. Undetectable expression levels were assigned to lncRNAs with Cq values above acceptable limits (above 32–33 PCR cycles). The 2^- ΔΔCt method or Pfaffl equation with PCR efficiency correction was used for data analysis [Total RNA was extracted from K562 cultivated cells  using Trizol Reagent  according to the manufacturer’s instructions. One microgram of total RNA was converted to cDNA by Quantitect Reverse transcription kit . lncRNA expression was assessed by qPCRs with sequence specific primer sets , and a library consisted of all four RNA samples was created with QIAseq miRNA library kit . As an initial step, adapters were ligated to the 3′ and 5′ ends of miRNAs, and subsequently, universal cDNA synthesis with UMI (Unique Molecular Indices) assignment, cDNA cleanup, library amplification, and library cleanup were performed. By this method, the presence of adapter dimers, the major contaminant in sequencing libraries, is eliminated. Downstream RNA sequencing reactions were performed in Illumina ISEQ  instrument in single-ended mode with 75 bps and read length ranging from 1–1.5 million per sample, which is considered adequate, due to the short length of mature miRNAs (21–25 bp). The in-house generated miRNA-Seq samples from K562 UT, UT+hemin/EPO, LRF-OE, and LRF-OE+hemin/EPO cell clones were initially processed with custom Python scripts to remove reads that did not include the complete UMI sequence. Due to the distinctive size distribution of miRNA sequences and the FastQC quality assessment results, Trimgalore was used with parameters “-q 30–length 18.” Three alignment rounds were applied to account for the uniqueness of miRNA Biology related to the fact that some miRNA sequences originate from multiple genomic loci. Reads were initially aligned on mature miRNA sequences derived from miRBase v22.1  using STp value cutoff of 0.05 (Supplementary Table DEGseq  was applThe transcription start site (TSS) of miRNA genes were downloaded from DIANA-miRGen v4  repositot-test was applied to compare expression levels between the different groups. p values < 0.05 was considered statistically significant.Statistical analysis was performed with SPSS software version 20.0. One-way ANOVA with Tukey’s multiple comparisons post hoc tests or lncRNAs selected for the present study met specific criteria; preserved LRF occupancy at promoter regions either co-occurring with CpG islands or not and/or functions related to erythropoiesis were already assigned to these molecules. To define LRF:lncRNA interactions upon hemin/EPO treatment, LRF peaks were divided into three groups using BEDTools intersect : peaks uHOTAIRM1), Deleted In Lymphocytic Leukemia 1/2 (DLEU1/2), Maternally Expressed 3 (MEG3), Nuclear Paraspeckle Assembly Transcript 1 (NEAT1), Differentiation Antagonizing Non-Protein Coding RNA (DANCR), Plasmacytoma Variant Translocation 1 (PVT1), Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1), and Imprinted Maternally Expressed Transcript H19 are already associated with hematological disorders [ENSG00000225806, ENSG00000236140, ENSG00000236617 (CHFR-DT), ENSG00000248925 (PDCD6-DT), ENSG00000249494 (DMXL1-DT), ENSG00000254821, ENSG00000263923, ENSG00000267338, and ENSG00000273391 genetic loci, which are found in Homo sapiens and annotated by several databases , were selected for investigation as well. Publications related to these transcripts (when exist) refer to genome-wide surveys or analysis of RNA-seq data; however, any affiliated molecular processes are not yet evident. All above-mentioned lncRNA transcripts were flanked by CpG islands. Sequences and chromosomal locations of CpG islands were tracked from UCSC Genome browser (https://genome-euro.ucsc.edu/cgi-bin/hgGateway)  , whereas erythropoiesis induction with hemin/EPO treatment raised LRF affinity to DNA and peaks up to 5.7 × 10ENSG00000225806-lncRNA, both CpGs 45 and 78 of MEG3-lncRNA, and CpG 94 of PVT1-lncRNA were hypermethylated (methylation levels above 70% and up to 92%) in every single cytosine followed by guanosine deoxynucleotide with methylation potential  across CpG island’s sequence, whereas downstream sequence of CpG 116 as well as CpGs 175 and 53 of ENSG00000267338- and ENSG00000254821-lncRNAs, respectively, were methylated in low rate, between 16 and 26%. The rest examined lncRNA loci were hypomethylated with methylation mean values below 10% , and analogous results were obtained from DLEU2-lncRNA and ENSC00000236617-, ENSC00000248925-, ENSC00000249494-, and ENSC00000263923-lncRNAs, comprising the group with hypomethylated CpG islands (bellow 10%)  was found exceptionally active. Notably, DLEU2-lncRNA shares the same CpG island with DLEU1-lncRNA, an alternative transcript; however, they were differentially expressed in all experimental conditions of the present study.Transcription efficacy of studied lncRNA genetic loci demonstrated three differentially expressed groups: those with undetectable expression, those that exhibited equal levels of expression regardless of different cell conditions, and those with decreased expression levels, apparent during LFR-OE and LRF-OE post-erythropoiesis induction. Correlations between DNA methylation status of CpG islands and expression levels led to the following results: transcription was undetectable in DANCR-, BGLT3-, DLEU1-, MALAT1-, NEAT1-, H19-, ENSG00000273391-, ENSG00000236140-, ENSG00000254821-, and ENSG00000287483-lncRNAs, which were downregulated. Transcription levels of DANCR-, BGLT3-, DLEU1-, H19-, ENSG00000273391-, ENSG00000236140-, and ENSG00000287483-lncRNAs were decreased with significant values (p ≤ 0.001–0.05), compared to UT as reference . LRF overexpression (LRF-OE) underscored its effect in DLEU1-lncRNA) were more thoroughly repressed, and further, reduction of expression displayed by DANCR-, BGLT3-, MALAT1-, NEAT1-, ENSG00000273391-, ENSG000002236140-, and ENSG00000287483-lncRNAs was statistically significant compared to UT+hemin/EPO as reference (p ≤ 0.001–0.05)  and 50% with invariant expression levels (ENSG00000249249- and ENSG00000271833-lncRNAs).In LRF-OE+hemin/EPO cell clones, all mentioned lncRNAs  differentially expressed between the four cell subgroups  were depicted by NGS and DEGseq  data anap value < 0.05 and log2FC > │0.5│) and total reads above 100. In consistency with results obtained from lncRNA expression profiles, miRs were systematically suppressed during high levels of LRF expression within transfected cells and some particular miR species prevailed upon erythropoiesis induction (miR-99b-5p and miR-125a-5p), whereas miR29a-3p, miR10a-5p, and miR29c-3p were apparent only in LRF-OE+hemin/EPO. The depicted miRNA sets from NGS followed by bioinformatics analysis are implicated in essential cell pathways presented in the “LRF-OE cell clones exhibited only decreased expression levels for several miRNA sets. The same cluster of miR-125a-5p and miR99b-5p (upregulated during hemin/EPO treatment) was downregulated in LRF overexpressing cells. Also, miR-29a-3p, miR-29c-3p, and miR-10a-5p  were exclusively downregulated in LRF-OE treated with hemin/EPO. Between LRF-OE versus UT and LRF-OE+hemin/EPO versus UT+hemin/EPO, only downregulated miRNAs with significant values were displayed and are illustrated as Venn diagram  embedded in ENSG00000225806, ENSG00000273391, and DLEU1/2 genetic loci respectively. Although these miRs show insignificant values of expression, the corresponding lncRNA hosts have demonstrated variable (detectable) levels of expression   [DLEU1/2 gene, are expressed with a distinct mode [MEG3-lncRNA expression has been suggested as a potential biomarker for therapy response and survival profile in childhood acute lymphoblastic leukemia (cALL) [NEAT1-lncRNA is upregulated in multiple myeloma and it is involved in mechanisms of cellular stress response [DANCR-lncRNA, apart from its role to erythroid differentiation [PVT1 maps at 8q24 encodes linear and circular transcript isoforms and encompasses miRNAs  inhibits the erythroid myeloid lymphoid cell proliferation [MALAT1-lncRNA is recognized as a biomarker for atherosclerosis [H19 Imprinted Maternally Expressed Transcript has been observed in chronic myeloid leukemia (CML), and myelomonocytic leukemia (CMML), as well as in AML [LRF transcription factor, encoded from the human gene ecisions , influenecisions  and alsopression . The prentiation . DLEU1-lia (CLL) . Notablynct mode  and alsoa (cALL) , whereasresponse . DANCR-lntiation . PVT1 maesponses . Malat1-feration . In humaclerosis . Reduceds in AML , and is s in AML . In the PVT1-lncRNA)  self-renewal and also have a prominent attribute to discrete hematopoiesis stages , 59. ThePVT1- and MALAT1-lncRNAs among them [In cell subgroups of LRF-OE and LRF-OE+hemin/EPO, only significantly downregulated miRNA species were obtained Table . MiR-15bong them . In the ong them , 24, 61.ong them . MiR-26bong them . Pathwayong them . MiR30e-ong them , 66. MiRong them , 68. MiRong them . Conclusong them , TGF-β, ong them  expressiong them , 66 and ong them –69 with This study provided clear evidence for the repressing capability of LRF in ncRNA molecules, mainly lncRNAs and sets of miRNAs. lncRNAs exhibited differential methylation and expression patterns during LRF overexpression status and further, during erythropoiesis terminal differentiation with concurrent hemoglobin switch form γ- to β-globin. Also, specific miRNA species were significantly downregulated, as yet, remains to be elucidated the actual role of this network of non-coding RNAs towards the last stage of hemopoiesis and simultaneous transition from fetal to adult stage of hemoglobin production, in human non-malignant tissue-specific cell lines. Gene silencing is not necessarily dependent on the continuous residence of a sequence‐specific repressor at a control element. Assays that detect genome‐wide TF‐binding activity, such as ChIP‐seq typically, provide a static snapshot of occupancy precisely at the time point of the assay; however, LRF potentially exerts transcriptional repression based in DNA tethering by a fleeting way.Supplementary file1 (DOCX 42 KB)Supplementary file2 (DOCX 21 KB)Supplementary file3 (DOCX 710 KB)Supplementary file4 (DOCX 16 KB)Below is the link to the electronic supplementary material.