Erythropoiesis is a complex, precise, and lifelong process that is essential for maintaining normal body functions. Its strict regulation is necessary to prevent a variety of blood diseases. Normal erythropoiesis is precisely regulated by an intricate network that involves transcription levels, signal transduction, and various epigenetic modifications. In recent years, research on post-transcriptional levels in erythropoiesis has expanded significantly. The dynamic regulation of splicing transitions is responsible for changes in protein isoform expression that add new functions beneficial for erythropoiesis. RNA-binding proteins adapt the translation of transcripts to the protein requirements of the cell, yielding mRNA with dynamic translation efficiency. Noncoding RNAs, such as microRNAs and lncRNAs, are indispensable for changing the translational efficiency and/or stability of targeted mRNAs to maintain the normal expression of genes related to erythropoiesis. N6-methyladenosine-dependent regulation of mRNA translation plays an important role in maintaining the expression programs of erythroid-related genes and promoting erythroid lineage determination. This review aims to describe our current understanding of the role of post-transcriptional regulation in erythropoiesis and erythroid-associated diseases, and to shed light on the physiological and pathological implications of the post-transcriptional regulation machinery in erythropoiesis. These may help to further enrich our understanding of the regulatory network of erythropoiesis and provide new strategies for the diagnosis and treatment of erythroid-related diseases. These reticulocytes are then released into the blood and continue to mature into fully functional biconcave erythrocytes within 1 to 2 days.3Erythropoiesis is the process by which hematopoietic stem cells proliferate, differentiate, and mature into red blood cells within the hematopoietic microenvironment.6 If the process is disrupted, it can lead to a variety of blood-related disorders.6 According to a report by the World Health Organization, an estimated 30% of the population suffers from anemia, and approximately 8.8% of them are unable to care for themselves because of it, significantly affecting their health and quality of life.7 Moreover, an insufficient global blood supply, low erythrocyte expansion in vitro, and low enucleation efficiency are major issues that need to be addressed.10Maintaining a healthy erythropoietic homeostasis is essential for normal body functioning.12 The strict regulation of erythropoiesis is critical for ensuring body homeostasis.6 In recent years, research has shifted focus to transcriptional regulation; however, the correlation between protein levels and transcriptional expression is poor, and post-transcriptional regulation/translation is the main determinant of protein abundance in cells.13 Therefore, an emphasis on post-transcriptional regulation in erythropoiesis has also emerged in recent years. Post-transcriptional regulation refers to the regulation of gene expression at the post-transcriptional level, reflecting a series of modifications and processing of the transcription products of eukaryotic genes.16 Post-transcriptional regulation includes the regulation of RNA alternative splicing, m6A methylation, regulation of mRNA stability by RNA-binding proteins (RBPs) and regulation of noncoding RNAs, such as microRNA.16 It has been demonstrated that these post-transcriptional regulatory pathways are vital for normal erythropoiesis, and any abnormalities can lead to erythroid disorder-related diseases.17Erythropoiesis is precisely regulated by complex networks at transcriptional and post-transcriptional levels, and by signal transduction and epigenetic modifications.18 This program is beneficial for driving differentiation and ensuring the synthesis of the appropriate protein isoforms required for stable red blood cell production.18Erythropoiesis uses a complex and comprehensive alternative splicing program to modify gene expression at the post-transcriptional level, ultimately regulating the structure and function of the proteome in a differentiation stage-specific manner.19 Recently, the activation of the protein 4.1R exon 16 3’ splice site was shown to require coordination between TIA1, PCBP1, and RBM39 during terminal erythropoiesis.20 The mechanism underlying protein 4.1R exon 16 splicing is the binding of TIA1/PCBP1 to the upstream cis elements activating a 3’s through direct interaction with RBM39 and then connecting to SF3b155 for 3’s recognition.20 MBNL-binding motifs are enriched near exons undergoing developmental splicing transitions, and MBNL1 is involved in the regulation of the splicing of transcripts .21 For example, the knockdown of MBNL1 in cultured murine fetal liver erythroid progenitors disrupts the developmentally regulated exon skipping of NDEL1 mRNA, which is bound by MBNL1 and blocks erythroid terminal proliferation and differentiation.21 Similarly, the knockdown of U2AF1 affects the alternative splicing of the gene in erythroblasts. U2AF1 knockdown cells have decreased levels of spliced transcripts encoding MTA1, EIF3B, HNRNPC, and HNRNPD, but increased levels of spliced transcripts encoding BAX and SNHG1.22 Erythrocyte gene expression is also regulated by intron retention, where transcripts are polyadenylated and spliced at most introns but remain unspliced at one or more introns.24 Hundreds of retained introns are dynamically regulated during terminal erythropoiesis, which inversely correlates with expression levels, exhibiting low intron retention in early progenitor cells but higher intron retention in erythroblasts.24Previous reports demonstrated that RBM38 regulated the activation of protein 4.1R (EPB41) exon 16 during terminal erythropoiesis and was a potent activator of exon 16 splicing.25 Alternative RNA splicing can generate a novel short TAL1 transcript variant (TAL1s). Enhancing the interaction between SF3B1 and RBM15 promotes the production of full-length TAL1 (TAL1fl) mRNA, while reducing the RBM15 protein levels through the PRMT1-mediated degradation pathway which alters the TAL1s/TAL1fl ratio in favor of TAL1s expression.25 Because of the structural characteristics of TAL1s, the interaction between TAL1 and ETO2 is blocked, further inhibiting early erythropoiesis.25The mutation of splicing factor SF3B1 disrupts erythroid differentiation via aberrant alternative splicing of transcription factor TAL1. Compared with the SF3B1 wild-type protein, the SF3B1 K700E mutant has stronger binding to the RBP, RBM15, and alters the RNA splicing of the transcription factors TAL1 and GATA1.23 Stage-specific splicing of naturally occurring premature termination codons (PTCs) in SNRNP70 exon 8 is low in proerythroblasts; therefore, most transcripts represent translatable mRNA.23 However, splice transitions convert an increasing proportion of transcripts to NMD-sensitive isoforms in mature cells.23The dynamic regulation of splicing transitions during erythropoiesis can drive changes in protein isoform expression to add novel functions favorable for erythrocytes. The alternative splicing of the SNRNP70 splicing factor coupled with nonsense mutation-mediated mRNA degradation (NMD) has been reported as a post-transcriptional mechanism downregulating gene expression.13RBPs, key players in post-transcriptional regulation, are involved in regulating the mRNA stability of crucial erythroid factors.26 Specifically, RBP-AUF-1 and RBP-YB-1 assemble a messenger ribonucleoprotein β-complex on the β-globin 3′ untranslated region (UTR) to enhance the binding of PABPC1 to the poly(A) tail, thus inhibiting mRNA deadenylation and enhancing β-globin mRNA stability in erythroid progenitors.26 PABPC4 binds and stabilizes GPA mRNA and other erythroid targets, including HBA1/HBA2, HBB, BTG2, and SLC4A1.27 Mechanistically, PABPC4-impacted mRNAs possess a high-value AU-rich motif that enhances the association between PABPC4 and mRNA containing critically shortened poly(A) tails to protect a subset of mRNAs from accelerated decay.27 The m6A-mRNA-binding protein YTHDF2 can mediate mRNA decay; thus, the activity of these RBPs can antagonize the activity of YTHDF2.28 mRNAs with dynamic translation efficiency during erythroid development, whose UTRs are enriched for target sites of specific RNA-binding proteins in hematopoietic cells, regulate the erythrocyte translation program. RBP-RBM38 is specifically induced by GATA1/TAL1 during late erythropoiesis and has been linked to splicing during late erythropoiesis.29 RBP-RBM38 associates with the translation initiation factor EIF4G and promotes the translation of select mRNAs with decreasing mRNA levels in late differentiating or enucleating cells. The inhibition of RBM38 results in a translation defect and blocks reticulocyte generation, suggesting that RBM38 plays a critical role at the post-transcriptional level during erythropoiesis.29 RBP-PCBP1 and PCBP2, which have critical functions in erythropoietic post-transcriptional regulation, modulate the development of erythroid mRNA expression and stability by directly recognizing the cognate poly(C)-rich motif and indirectly mediating alterations in the expression of downstream RNA-binding proteins, such as RBM38.30 In addition, PCBP2 controls the process of erythropoiesis by regulating the functional splicing of Runx1 transcripts.31 The regulatory network of RBPs in erythropoiesis is complex. Gaps exist in our current understanding of the role and mechanism of RBPs in erythropoiesis based on existing reports. Therefore, we obtained the predicted RBP interaction network of erythroid key transcription factors from the POSTAR3 website  to provide unique and helpful insights into the implications of RBP-related regulation in erythropoiesis.The normal expression of beta-globin (HBB) protein in mature erythrocytes critically depends on post-transcriptional events in erythroid progenitors that ensure the high stability of HBB mRNA. RBP-PABPC1 has been shown to bind and stabilize HBB mRNA by inhibiting its deadenylation.32 MiRNAs, such as miR-142, miR-144, miR-451, miR-221/222, and miR-155, play essential roles in erythroid progenitors, erythroid differentiation, and erythrocyte homeostasis54 .MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post-transcriptionally by altering the translational efficiency and/or stability of targeted mRNAs.45 The deletion of miR-142 results in the biconcave shape of red blood cells, impaired structural elasticity, abnormal metabolism of reactive oxygen species (ROS), and shortened overall lifespan.34 MiR-142 also maintains homeostatic erythropoiesis, hematopoietic progenitor proliferation, and normal enucleation by targeting Rac1 mRNA.34 Inhibiting the expression of miR-144 and/or miR-451, which is upregulated in erythropoiesis, impairs erythropoietin (EPO)-driven erythropoiesis by targeting RAB14.38 Recently, miR-144/451 has also been found to block mitochondrial respiration during erythropoiesis by inhibiting the expression of the target gene COX10.39 Additionally, CAP1 is a target of miR-144/451 and negatively regulates erythropoiesis and enucleation.40 The erythroid-specific transcription factor GATA1 can activate miR-144/451 expression, which directly targets MYC in erythroblasts.41 In miR-144/451–depleted erythroblasts, persistently high levels of MYC prevent erythroid differentiation.41 Interestingly, Myc inversely regulates the expression of miR-144/451, forming a miR-144/451-MYC positive feedback to ensure a normal level of MYC during erythropoiesis; thus, the GATA1-miR-144/451-MYC network protects normal erythropoiesis.41 Moreover, miR-144/451 inhibits the LKB1/AMPK/mTOR pathway to promote the survival of erythrocyte precursors during recovery from acute anemia.42MiRNAs target negative regulators of erythropoiesis Table .33–45 Th54 For example, an elevated expression of miR-155 leads to fewer erythroid progenitors and erythrocytes during erythropoiesis, and exerts its function by targeting the key erythroid transcription factors PU-1 and CEBP.50 MiR-669m is not expressed in erythroblasts, and its overexpression targets AKAP7 and XK to inhibit terminal erythroid differentiation.47 The upregulation of miR-152 suppresses GATA1 expression in Antarctic icefish, thereby significantly inhibiting hematopoiesis.48 Moreover, QKI5 can activate the processing of primary miR-124-1 (pri-124-1). When erythropoiesis proceeds, decreased QKI5 expression leads to decreased levels of mature miR-124, which increases the expression of the miR-124 target genes TAL1 and c-MYB to promote erythroid maturation.46MiRNAs target positive regulators of erythropoiesis Table .2,46–54 56 However, XPO7 expression increases as the miR-181a levels decrease during erythrocyte maturation, thereby condensing the nucleus and promoting enucleation.56 Heavy ferritin subunit (FHC) silencing leads to an aberrant GATA switch mediated by miR-150 upregulation, thereby blocking erythroid differentiation of K562 cells.57MiRNAs exert different functions as their expression changes during erythroid development. During the active erythrocyte nuclear transcription stage, a high expression of miR-181a inhibits erythrocyte enucleation by suppressing XPO7 mRNA and protein levels.58 In addition, it has been revealed that differences exist in miRNA expression between adult and fetal erythroblasts via omics analyses. There is a predominant expression of let-7 miRNA family members in adult erythrocytes, whereas the chromosome 14q32 miRNA cluster is significantly upregulated in fetal erythroblasts.59Numerous miRNA analyses based on omics in erythropoiesis have been conducted in recent years. Based on the combined omics analysis of differential miRNA expression, erythroid differentiation gene expression and hemoglobin expression, more downregulated miRNAs than upregulated miRNAs have been found during erythropoiesis, while several lineage-specific miRNAs, such as miR-15b-5p, miR-16-5p, miR-96-5p, and miR-22-3p, were dramatically increased.62 LncRNAs assist RBPs in functioning as negative regulators of miRNAs in erythropoiesis. It has been shown that the lncRNA, Gm15915, is highly expressed in erythroid progenitor cells and red blood cells. Mechanistically, Gm15915 interacts with WDR82 to promote the transcription of KLF1 and globin genes, thereby regulating the early and late stages of erythropoiesis, respectively, and maintaining erythropoiesis.63 The expression of the lncRNA, UCA1, is dynamically regulated during human erythrocyte maturation, with maximum expression in primary erythroid cells, and its deletion primarily impairs heme biosynthesis and prevents erythroid differentiation at the primary erythroid stage.60 In terms of mechanism, UCA1 physically interacts with the RBP, PTBP1, and acts as an RNA scaffold to recruit PTBP1 to ALAS2 mRNA, thereby stabilizing it.60 Taken together, lncRNA-mediated post-transcriptional mechanisms provide a new dimension for the regulation of erythropoiesis.LncRNAs affect multiple steps of gene post-transcriptional expression, including the regulation of alternative splicing of pre-mRNA, the regulation of mRNA stability and abundance, and the function of competing endogenous RNAs.64 However, most duplicated genes accumulate various loss-of-function mutations during evolution, resulting in pseudogenes.65 Since their discovery, pseudogenes have been generally considered nonfunctional. Among various functional mechanisms of pseudogenes, however, antisense transcription-related regulation and competition with miRNA and RBPs appear to be common.66It has long been believed that gene duplication drives evolution by generating new genes.65 Mechanistically, HBBP1 competes with the mRNA of the key transcription factor TAL1 in erythropoiesis to bind the RBP, hnRNPA1, thereby stabilizing TAL1 mRNA, increasing TAL1 protein expression and maintaining normal erythropoiesis65 . HBBP1 deletion leads to the complete loss of the ability of embryonic stem cells to differentiate into erythroid cells and inhibits the differentiation process of hematopoietic stem/progenitor cells in vivo and in vitro.65In erythropoiesis, HBBP1, a duplicated pseudogene, has been identified as a key RBP-competing protein for erythroid transcription factors.6A is the most common modification of mRNA, lncRNA and various other noncoding RNAs in higher organisms.67 The function of m6A modification is mainly determined by “encoder ,” “decoder ,” and “reader .”68 The known methyltransferase complex components are METTL3, METTL14, WTAP, and KIAA1429. The demethylases that reverse methylation include ALKBH5 and FTO, and the m6A-binding proteins include the YTH domain protein and the nuclear heterogeneity protein HNRNP family.69More than 60% of RNA modifications have been shown to involve methylation, and m6A-dependent regulation of mRNA translation in maintaining the erythrocyte gene expression program and promoting erythroid lineage decisions has been clarified. We found that three core components of the m6A MTase complex  are required for the maintenance of GPA expression in HEL cells and erythroid lineage determination in human stem/progenitor cells. Moreover, m6A MTase activity is mediated by the selective translation of approximately 300 m6A-tagged mRNAs to promote the erythrocyte gene expression program.15 The important regulatory role of m6A RNA methylation in erythropoiesis has also been demonstrated by CRISRP-Cas9 comprehensive screening.70 With regard to the physiological role of the m6A-dependent regulation of mRNA translation in maintaining the erythrocyte gene expression program and promoting erythrocyte lineage determination, it was found that the inhibition of the methyltransferase complex-mediated loss of m6A resulted in a disruption of the erythrocyte transcriptional program; this did not directly inhibit previously identified master transcriptional regulators  but resulted in a variety of other gene translation downregulations.15 Furthermore, the maintenance of the erythroid transcriptional program is partially driven by the m6A translational regulation of the SETD1A/B complex, which promotes the recruitment of the transcriptional activation markers H3K4me3 and KLF1 transcription factors to erythroid gene promoters.15Recently, the role of m6 In ineffective erythropoiesis, there is an imbalance due to an increased proliferation of erythroid progenitors accompanied by increased apoptosis and decreased erythrocyte maturation.72 Ineffective erythropoiesis leads to a series of anemia-related blood diseases, including myelodysplastic syndrome (MDS), β-thalassemia (β-TM), and Diamond-Blackfan anemia (DBA).74Maintaining normal erythropoiesis relies on the tight and precise regulation of erythroid development in healthy individuals.75 Therefore, miRNA oligonucleotides or anti-miRNA oligonucleotides may serve as potential therapeutic strategies for the treatment of erythroid anemia-related diseases. We summarize here some potential links to the post-transcriptional regulation of erythropoiesis and related treatment strategies for MDS, β-TM, and DBA.Ineffective erythropoiesis, erythroid hyperplasia, and anemia phenotypes have been reported in the miR-142-/- mouse model. MiR-451/144 deficiency leads to erythroid hyperplasia and mild anemia, whereas miRNAs  are upregulated in individuals with persistent injury, thereby inhibiting erythropoiesis and resulting in the development of persistent injury-related anemia.81 The most common spliceosome mutations observed in MDS include U2AF1, with mutation hotspots in the zinc finger domains at codons S34 and R156/Q157; SF3B1, with hotspots, such as K700 and K666 in the HEAT domains; and SRSF2, with a hotspot at codon Pro95.81 The mutant protein exhibits relatively modest changes in RNA-binding specificity; however, such changes are sufficient to induce aberrant 3′ splice site selection in transcripts in many patients.77 SF3B1 mutations are among the most common events, and in the case of SF3B1 mutants, branch point recognition is altered and can promote aberrant splicing.83 ABCB7 expression is reduced in MDS patients with SF3B1 mutation owing to splicing of the aberrant 3′ splice site that alters the translational reading frame, introduces PTC and activates NMD.84 Studies have demonstrated that the physiologic expression of SF3B1 K700E mutation causes impaired terminal erythropoiesis, erythroid dysplasia, aberrant splicing and sensitivity to pharmacologic spliceosome modulation, which is sufficient to cause the characteristic features of MDS.85 K562 cells with the SF3B1 K700E mutation exhibit accelerated differentiation and increased erythroid-cell death, which is due to mis-spliced MAP3K7 transcripts leading to specifically deregulated p38 MAPK.86 SF3B1 deficiency impairs human erythropoiesis by decreasing the expression of the large MKRN1 isoform to activate the p53 pathway; this improves our understanding of ineffective erythropoiesis in MDS patients with SF3B1 mutations.87 SRSF2 Pro95 hot spot mutations elicit enhanced mRNA decay, which depends on sequence-specific RNA binding and splicing.80 SRSF2 mutants enhance the deposition of exon junction complexes (EJCs) downstream of the PTC through RNA-mediated molecular interactions, whose architecture favors the association of key NMD factors to elicit mRNA decay.80MDS is a clonal blood disorder characterized by abnormal hematopoietic development and cytopenia that is usually caused by mis-sense mutations in genes encoding the splicing factors SF3B1, ZRSR2, U2AF1, U2AF2, or SRSF2.6A mRNA methylation was detected in 70 of 104 known MDS genes, including 8 of the 10 most commonly mutated genes .15 The U2AF1 S34F mutation exhibits lineage specificity in altering the pre-mRNA splicing of downstream target genes, resulting in distinct phenotypes in different myeloid lineages involved in MDS.88 Therefore, the study of post-transcriptional regulation in erythropoiesis is potentially important for understanding MDS and developing corresponding therapeutic strategies.In addition, m89 Based on combined omics analyses of differential miRNA expression, erythroid differentiation gene expression and hemoglobin expression, miRNAs and their associated genes or pathways could be explored as potential targets for new therapies for β-hemoglobinopathies and other erythropoietic disorders.58 During erythroid differentiation, more miRNAs are downregulated, while the expression of several lineage-specific miRNAs  is dramatically increased in the terminal stages.58 It has been reported that in β-TM, 8 miRNAs  were identified as significantly upregulated, while 4 miRNAs  were significantly downregulated.11 Further analysis revealed that these miRNAs are related to a variety of biological processes and molecular functions, including the MAPK and HIF-1 signaling pathways related to the upregulation of HbF.11 Therefore, the high level of HbF in β-TM individuals may be mediated by miRNAs. The upregulation of miR-214 mediates oxidative stress in β-TM by targeting ATF4.90 Significantly increased levels of extracellular vesicle miR-144-3p were observed in β-TM and induced apoptosis, which may lead to organ dysfunction and complications in patients with β-TM.91 MiR-2355p increases the γ-globin levels by targeting KLF6 mRNA to promote normal cell expansion in β-TM patients, providing more information for the clinical management of patients.35 In individuals with severe β-TM, the increased miR-326 expression in reticulocytes was positively correlated with HbF levels.52 Therefore, miR-326 may play an important role in regulating globin turnover during β-TM stress-induced erythropoiesis.52 In plasma, miR-451 acts as a novel hemolytic marker for β-TM/hemoglobin E (HbE) disease.92β-TM is a type of β-hemoglobinopathy known for clinical heterogeneity in which patients exhibit elevated fetal hemoglobin (HbF) levels.65 HBBP1 also has an important protective effect in patients with β-TM. HBBP1 is compensatively increased in β-TM patients and participates in HbF activation and anemia symptom relief in patients by promoting the stable expression of TAL1.65HBBP1 competes with TAL1 mRNA, a transcription factor critical for erythroid development, to bind the RBP, hnRNPA1, thereby improving the stability of TAL1 mRNA and maintaining normal erythropoiesis.14Additionally, point mutations within exons or their flanking introns interfere with the splicing of many erythroid transcripts. Owing to changes in exon skipping or splice site selection that alter the translation reading frame, introduce premature stop codons and activate NMD, splice mutants often manifest as underexpressed alleles, a common cause of β-TM and erythrocyte membrane disorder.93 In DBA patients, mutations in the 3′ end splice site of exon 2 in the GATA1 gene were found to be almost eliminated.93Disturbing the isoform balance between alternatively spliced transcripts may contribute to the development of DBA. Partial skipping of exon 2 in the GATA1 gene occurs naturally to generate transcripts encoding shortened protein isoforms called GATA1s, and the deficiency of full-length GATA1 affects erythropoiesis.94 Similarly, ginsenoside Rb1 upregulates the expression of miR-208, which binds the 3′-UTR of NLK mRNA and targets it for degradation, thereby improving abnormal erythropoiesis in DBA patients.95In addition, miRNA agonists or inducers represent a potential new approach for DBA therapy. It has been reported that metformin induces the expression of miR-26a and recognizes the binding site within the 3′-UTR of Nemo-like kinase (NLK) to promote the degradation of NLK transcripts, thereby promoting erythroid differentiation and improving anemia in DBA patients.96Myeloproliferative neoplasm (MPNs), such as polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are a heterogeneous group of clonal hematopoietic disorders characterized by myeloid progenitor proliferation, which involve an excess of differentiated erythrocytes, platelets, and leukocytes circulating in peripheral blood.97 The short isoform RUNX1a overexpression has been reported in MPN.98 The SRSF2 mutation is found in 3% to 20% of MPN, with lower frequencies in PV and ET compared with PMF.99 In PMF, U2AF1 mutations were associated with inferior survival.100 PMF, PV, and ET patients with U2AF1 mutations have a poorer myelofibrosis-free survival compared with wild-type patients. A total of 65% of U2AF1 mutations affect Q157 and the presence of this mutation is associated with significantly shorter overall survival in MPNs.101RNA splicing factor mutation is one of the most frequent mutations in MPN. The SF3B1 mutation occurs in 5% to 10% of MPN patients. The SRSF2 mutation affected transcriptional regulation through predominant splicing of RUNX1 to form the RUNX1a transcript.The application of RNA splicing factor mutation in the early risk stratification of patients increases the possibility of early intervention to prevent the progression of MPN. However, more research is needed to explain the potential molecular mechanisms and effectively target them.6A-dependent regulation of mRNA translation plays an important role in maintaining erythrocyte gene expression programs and promoting erythroid lineage determination. Noncoding RNAs, such as miRNAs and lncRNAs, are indispensable for maintaining the normal expression of genes related to erythropoiesis and targeted therapy for abnormal erythroid diseases.The importance of erythropoiesis for normal human body function underscores the need for multilevel research. Dynamic regulation of splicing transitions during erythropoiesis leads to changes in protein isoform expression that add new functions beneficial for erythropoiesis. RNA-binding proteins adapt the translation of transcripts to the protein requirements of the cell, resulting in mRNA with dynamic translation efficiency. The mFig. 4). In some instances, splicing factor mutations as exemplified by SF3B1 and U2AF1 have provided diagnostic utility and information on clinical outcomes for erythropoiesis-related diseases, such as myelodysplastic syndrome and MPNs. However, more research is needed to explain the potential molecular mechanisms and effectively target them, so as to achieve the purpose of clinical treatment. Although miRNAs and RNA-binding proteins involved in post-transcriptional regulation are key players in erythropoiesis, their specific roles of these particular pathways in erythroid development remain largely unknown, given the relative infancy of the miRNA/RNA-binding protein field. In addition, the post-transcriptional regulatory network of m6A during erythropoiesis requires further elucidation. Based on our understanding of the existing post-transcriptional regulation mechanism during erythropoiesis, abnormal post-transcriptional regulation during erythropoiesis can lead to a variety of ineffective erythropoiesis-related diseases, such as myelodysplastic syndrome, β-thalassemia, and Diamond-Blackfan anemia. Consequently, further exploration and discovery of new post-transcriptional regulatory mechanisms will enrich our understanding of the regulatory network of erythropoiesis and provide new strategies for the diagnosis and treatment of erythroid-related diseases.In this review, we have summarized recent research in the post-transcriptional regulation of erythropoiesis. These post-transcriptional regulatory patterns can form regulatory networks centered on certain genes that are critical for erythropoiesis, thus controlling the whole process of red blood cell development ; the Fundamental Research Funds of the Central Universities of Central South University (Grant number 2021zzts0562); and the Fundamental Research Funds for the Scientific Research Innovation Project of Hunan Province (Grant number CX20210182).J.L. designed this study. Y.L. drafted the manuscript. H.Z., B.H., and P.W. collected the data. J.L. and W.W. revised the manuscript. All authors have read and approved the final manuscript.


Pseudomonas fluorescens SBW25 strain rendered incapable of flagellar mediated motility in soft-agar plates via deletion of the flagellar master regulator (fleQ). This bacterium can evolve to rescue flagellar motility via gene regulatory network rewiring of an alternative transcription factor to rescue activity of FleQ. Previously, we have identified two members (out of 22) of the RpoN-dependent enhancer binding protein (RpoN-EBP) family of transcription factors (NtrC and PFLU1132) that are capable of innovating in this way. These two transcription factors rescue motility repeatably and reliably in a strict hierarchy – with NtrC the only route in a ∆fleQ background, and PFLU1132 the only route in a ∆fleQ∆ntrC background. However, why other members in the same transcription factor family have not been observed to rescue flagellar activity is unclear. Previous work shows that protein homology cannot explain this pattern within the protein family (RpoN-EBPs), and mutations in strains that rescued motility suggested high levels of transcription factor expression and activation drive innovation. We predict that mutations that increase expression of the transcription factor are vital to unlock evolutionary potential for innovation. Here, we construct titratable expression mutant lines for 11 of the RpoN-EBPs in P. fluorescens. We show that in five additional RpoN-EBPs , high expression levels result in different mutations conferring motility rescue, suggesting alternative rewiring pathways. Our results indicate that expression levels (and not protein homology) of RpoN-EBPs are a key constraining factor in determining evolutionary potential for innovation. This suggests that transcription factors that can achieve high expression through few mutational changes, or transcription factors that are active in the selective environment, are more likely to innovate and contribute to adaptive gene regulatory network evolution.Evolutionary innovation of transcription factors frequently drives phenotypic diversification and adaptation to environmental change. Transcription factors can gain or lose connections to target genes, resulting in novel regulatory responses and phenotypes. However the frequency of functional adaptation varies between different regulators, even when they are closely related. To identify factors influencing propensity for innovation, we utilise a  Propensity for innovation is a key determinant of evolvability – the ability to rapidly generate heritable phenotypic variation – in living organisms . AlteratPseudomonas fluorescens [A key property for evolvability in a transcription factor is its ability to gain novel connections, which often involves gain of promiscuous activity. For a transcription factor, this constitutes gain of illicit or non-canonical regulatory interactions, and is a key factor in revealing a transcription factor to selection and for evolutionary innovation to occur . These iescens . We madeescens  between escens . These pE. coli) [The expression and activation levels of a transcription factor are determined by the architecture and connectivity of the GRN it sits within . Our pre coli) , where m coli)  would be coli) , where l coli) .Similarly, there will be variation in how easily a transcription factor may gain a hyperactivated state. This again depends on the signalling connectivity of the transcription factor within the GRN. Bacterial regulatory networks will sense and transduce internal or environmental stimuli , commonlP. fluorescens SBW25. This was achieved by introduction of the RpoN-EBP coding sequence downstream of a rhaSR-PrhaBADl-rhamnose titratable promoter system [P. fluorescens chromosome using the miniTn7 transposonal insertion system [l-rhamnose to the growth media, simulating an alternative GRN architecture and environment where the transcription factor is highly expressed. Utilising these expression strains, we set out to identify if any other RpoN-EBPs could rescue motility, and if increased expression of an RpoN-EBP would bias evolutionary outcomes in a flagellar motility rescue experiment.This study aims to investigate the combined role of these intrinsic  and extrinsic  factors in determining evolutionary pathways and trajectories followed during phenotypic adaptation, and in particular how variation in transcription factor gene expression can impact adaptive outcomes. To do this we make use of a previously characterised model system of transcription factor evolution. In our model, we have identified two RpoN-EBPs – NtrC and PFLU1132 – capable of rescuing flagellar motility via promiscuous activity. There are 19 other RpoN-EBPs that could also theoretically achieve this  due to sr system , and insn system . This alPseudomonas fluorescens AR2 (SBW25ΔfleQ IS-ΩKm-hah: PFLU2552), or AR2ΔntrBC , rendering it unable to move via flagellar and flagellar-independent spidery-spreading motility respectively, as detailed previously [Escherichia coli strains for cloning were cultured on LB media at 37 °C. For experiments using M9 minimal media, the follow recipe was used: 0.2 % w/v Glucose, 0.1 mM CaCl2, 2 mM MgSO4, 1× M9 salts . In some cases alternatives were used as the sole carbon or nitrogen sources, in which the Glucose or NH4Cl were omitted respectively.All ancestral strains in this study are derived from either eviously . All rourhaBAD promoter and downstream of rhaSR, allowing rhamnose-titratable expression of the RpoN-EBP [E. coli DH5α by chemical-competence heat-shock. The miniTn7 transposon containing the rhaSR genes and the PrhaBAD-stRBS-RpoN-EBP construct was then transferred to the P. fluorescens chromosome by transposonal insertion downstream of the glmS gene via four-parent puddle-mating conjugation [E. coli DH5α pJM220-derived plasmid donor was combined with recipient P. fluorescens AR2 strains, transposition helper E. coli SM10 λpir pTNS2 and conjugation helper E. coli SP50 pRK2073, and Gentamicin resistant Pseudomonas selected for on LB supplemented with Gentamicin sulphate and Kanamycin sulphate. Chromosomal insertion of the correct miniTn7 transposon and RpoN-EBP was confirmed by colony PCR. The rhaSR-PrhaBAD-stRBS-PFLU4895 construct was also transferred to the chromosome by miniTn7 insertion in an AR2ΔntrBC background. Activity of the rhaSR-PrhaBAD and response to l-rhamnose present in growth media tested using a rhaSR-PrhaBAD-stRBS-lacZ construct in a β-galactosidase activity assay  as detailed previously [Inducible expression constructs for a panel of RpoN-EBPs were constructed in the AR2 genetic background. The RpoN-EBP ORF was amplified by PCR and a strong ribosome binding site (stRBS) introduced upstream with a 7 bp short spacer between the stRBS and the start codon. PCR also introduced restriction enzyme cut sites up and down-stream of the gene, which were used to insert the RpoN-EBP into the multiple cloning site of the miniTn7 suicide vector pJM220  by restriction-ligation. This positions the RpoN-EBP gene under control of the PRpoN-EBP  as used jugation . The relEvolutionary rescue of motility was assayed in 0.25 % agar M9 plates as described previously . Pure siP. fluorescens SBW25 reference genome [gcsR_C1 and ΔntrBC_1 have a mutS mutation and subsequently far more additional mutations relative to other samples. These mutations are listed in Table S1 but not included in Fig. 3. For motile isolates where mutations occurred in the RpoN-EBP gene that had been overexpressed, we determined which RpoN-EBP copy the mutation was present in by PCR amplification of the copy present on the rhaSR-PrhaBAD inducible expression construct with primers binding either side of the multiple cloning site (pJM220 300 bp Forward and Reverse primers). PCR products were cleaned up using the Monarch PCR cleanup kit (NEB), and Sanger sequenced using the service provided by Source Bioscience. Sequences of the introduced EBP copy generated by Sanger sequencing were aligned to the coding sequence of their respective EBP from the reference genome using MAFFT v7.511 [To identify motility rescuing mutations, genomic DNA was extracted from motile strains and their ancestral strain using the Thermo Scientific GeneJET Genomic DNA Purification Kit. Genomic DNA was quality checked using BR dsDNA Qubit spectrophotometry to determine concentration and nanodrop spectrophotometry to determine purity. Illumina NextSeq 2000 sequencing was provided by Seqcenter , with a minimum 30× coverage. Returned paired-end reads were further filtered using fastp v0.23.2  with pare genome  and mutae genome . The conT v7.511 . The preP. fluorescens SBW25 proteome was searched for Pfam 35.0 domains [To identify where mutations affect the protein domains of RpoN-EBPs, the  domains  using In domains .Pseudomonas species, complete Pseudomonas genomes were obtained from NCBI RefSeq [Pseudomonadales marker genes were present and ≤2 % were duplicated using BUSCO [To count RpoN-EBPs in I RefSeq . A repreng BUSCO , 140 genrhaSR-PrhaBAD RpoN-EBP expression constructs, l-rhamnose was added to the motility agar with a final concentration of 0.15 % w/v. Activating signals R-hydroxybutyrate (R-HB – for hbcR), or Glycine (Gly – for gcsR) were added to media at concentrations of 30 mM and 10 mM respectively. Six biological replicates of each strain of interest were inoculated into the motility plates by picking single colonies with a sterile toothpick and stabbing into the soft agar. These replicates allow us to discern whether any motility is adaptive, as if all six replicates display the same phenotype it is unlikely that any mutations have occurred.Motility phenotype induction was assayed using soft-agar (0.25 %) plates with varying media compositions and supplements. Soft-agar plates were set up as described previously . For indAll statistical analysis and data handling was performed using R core statistical packages. Differences between the rhamnose absent and present conditions were tested via two-way ANOVA, and differences between the two conditions for individual RpoN-EBPs tested using Wilcoxon tests. For differences in rewiring pathway frequency between rhamnose absent and present conditions, a test was used.P. fluorescens SBW25 RpoN-EBPs genes selected based on structural similarity to the FleQ protein, along with including representatives of orphan regulators (those lacking a cognate kinase) and members of two-component systems [aauR (PFLU1134), algB (PFLU0088), fleR (PFLU4441), PFLU2055, PFLU1132, dctD (PFLU0286), prpR (PFLU2386), hbcR (PFLU2630), gcsR (PFLU4895), mifR (PFLU4954), PFLU2695 and PFLU2209. These expression constructs were introduced into the P. fluorescens SBW25 ΔfleQΔntrBC genetic background. This background lacks both the FleQ flagellar regulator, as well as the highly evolvable NtrBC system studied previously [We constructed rhamnose inducible expression systems for 11  systems  – aauR . This was curious, as there is no known mechanism for FleR to regulate the entire flagellar cascade by itself without the action of FleQ [We began by testing whether increasing the expression of each RpoN-EBP resulted in immediate restoration of motility. Constructs were incubated in M9 motility agar supplemented with 0.15 % w/v  of FleQ , which ml-rhamnose supplement. We incubated plates for up to 6 weeks to allow motility mutants to evolve. In the ΔfleQΔntrBC genetic background, the expected pathway of motility rescue is through the previously studied PFLU1131/2 two-component system [l-rhamnose. The ΔfleQΔntrBC control background evolved with an average time to emergence that was not significantly different with or without l-rhamnose supplement . Broken down by each RpoN-EBP expression system, pairwise comparisons (Wilcox test) indicate no significant difference between presence and absence of l-rhamnose on time to emergence for aauR, algB, PFLU2055, PFLU2695, mifR, and prpR, but significant differences for dctD, hbcR, PFLU1132, PFLU2209, and gcsR , this indicates a strong effect of a negative correlation between TF expression and time for motility mutants to emerge despite gene specific responses to rhamnose.To test if overexpression of each RpoN-EBP altered the mutational pathway for evolutionary rescue of motility, we incubated each expression construct in M9 motility agar with or without 0.15 % and gcsR  . HoweveΔfleQΔntrBC control line  significantly dropping from 26/26 in the no-rhamnose control to 4/36 in the rhamnose condition (P=2.861e-11), with isolates from the hbcR, aauR, gcsR, dctD and PFLU2209 overexpression conditions lacking any mutations in the PFLU1132 pathway. For the ΔfleQΔntrBC genetic background and PFLU1132 expression control lines, all motility rescue mutations in the presence and absence of l-rhamnose occurred in the PFLU1132 pathway . These mutations occurred in inter-domain regions of the RpoN-EBP proteins for most cases, aside from four which all occurred within the response receiver domain (Table S3). The second group do not gain mutations directly in the rhamnose-induced RpoN-EBP. These isolates instead gain mutations in a set of metabolic genes, which likely involve indirect modulation of RpoN-EBP activity through feedback.In some cases, motile isolates evolved in the presence of or genes . MutatiogcsR overexpression, for which 8/10 motile isolates had gained a mutation in gcvP, one in gcvT and one in the intergenic region between PFLU5143 and PFLU5144. Of the eight gcvP mutations, four result in frame shifts, suggesting loss of function of the protein product. These genes encode the GcvP glycine dehydrogenase and the GcvT aminomethyltransferase, both components of the glycine cleavage system catabolic cycle [gcvP mutants in other organisms [aauR shifted the mutational spectrum to include genes involved in acidic amino acid metabolism. Across three motile isolates from the aauR condition there were individual cases of mutations in the genes pyrB (PFLU5758), pyrC (PFLU5759) and pyrF (PFLU1851). These genes encode enzymes aspartate carbamoyltransferase, dihydroorotase, and orotidine 5'-phosphate decarboxylase respectively, which are involved in the biosynthesis of pyrimidine nucleotides by converting aspartate to uridine-monophosphate [aauR overexpression condition gained INDELs in these genes, which likely inactivated these enzymes  – again this likely causes an increase of intracellular aspartate, by which AauR activity is determined through its sensor-kinase AauS [aauR overexpression isolates had mutations in PFLU1583 and PFLU1584. These genes encode a putative anti-sigma factor system, which we have previously shown to facilitate RpoN-EBP promiscuity of PFLU1132 [PFLU2055 and PFLU2209 also resulted in motile isolates with putative feedback mutations. One PFLU2055 motile isolate had a mutation to lptD (an LPS-assembly enzyme) – which may impact cell membrane integrity, which PFLU2055 homolog, PspF, has been shown to respond to [pnp, dcd).The clearest example is that of ic cycle . Loss ofrganisms . As GscRrganisms , these mhosphate . Motile ase AauS . This wopyrB PFLU58, pyrC hbcR and dctD), or the genes associated with its regulatory function, as was the case for gcsR and aauR. These results indicate that many members of the RpoN-EBP family of transcription factors are capable of rewiring to rescue motility in our assay, and that the expression of the RpoN-EBP is a significant factor constraining their potential to evolve novel interactions . Such activating signals could also feasibly be provided by the external environment rather than mutation to internal metabolism. To test this, we selected two RpoN-EBPs for which an activating signal could be provided externally – gcsR, and hbcR. This pair were chosen as contrasting examples – gcsR rescued motility in our experiment through mutations to internal glycine catabolism, whereas hbcR evolved through mutation to one of the two copies of the RpoN-EBP gene itself. However, the HbcR transcription factor can be activated by the presence of (R)−3-hydroxybutyrate (R-HB), a metabolite which cannot be produced by internal metabolism [hbcR itself are the primary mechanism of rescuing motility for this RpoN-EBP, as higher levels of R-HB cannot be produced internally.Our experiments demonstrated that for some cases, an RpoN-EBP that was overexpressed could rescue motility through mutations that likely yielded increased cytosolic concentrations of the signal to which the RpoN-EBP in question responded , and the relevant activating signal resulted in immediate flagellar motility .To test if flagellar motility could be rescued by environmentally providing activating signals for these two transcription factors, we set up M9 soft agar plates supplemented with motility . We addimifR overexpression strain  were run  and were found to remain immotile upon addition of l-rhamnose, R-HB and l-glycine in the relevant combinations. This confirms that rescue of flagellar motility via rewiring of RpoN-EBP transcription factors can also be facilitated by presence of signals that activate the relevant transcription factor in the environment.To confirm that motility depends on overexpression of these RpoN-EBPs and their interaction with their respective signals, control plates of the rhamnose-inducible expression system lacking any RpoN-EBP, and of the P. fluorescens, in addition to those that have been previously capable of innovation to rescue motility without modified expression (NtrC and PFLU1132) [Through manipulation of transcription factor expression, we have identified several novel pathways for evolutionary rescue flagellar mediated motility in FLU1132) . RegulatPseudomonas fluorescens SBW25. For all these regulators, no canonical regulatory link to the flagellum is known, yet flagellar expression is rescued through a small number of mutational steps when these RpoN-EBPs are overexpressed. Why overexpression of the remaining regulators  do not alter the evolutionary pathway for rescue of flagella motility is unknown, however could be due to differences in the precise expression levels of these RpoN-EBPs, or local GRN connections suppressing activity or not providing a suitable regulatory link to the flagellar genes.This set of identified transcription factors capable of rescuing flagellar motility, together with NtrC and PFLU1132, constitute 58 % of the RpoN-EBP transcription factor family tested by our work, and 33 % of the total encoded by P. fluorescens SBW25 can rescue flagellar motility suggests that this transcription factor family may be uniquely suited to regulatory innovation. Why this should be the case is unclear, however the answer may lie in the unique regulatory mechanism of the RpoN-EBP family. The sigma factor, RpoN, plays a pivotal role in localising the EBP so that it may provide ATPase activity to catalyse transcription initiation [P. fluorescens encodes ~20, Escherichia coli ~12, Bacillus subtilis ~5, Chlamydia pneumoniae and Treponema pallidum both encode one each, and Mycoplasma genitalium and Rickettsia prowazekii encode none [Clostridiales species [Pseudomonas genus . It has been suggested previously that RpoN-EBPs provide a biological advantage in regulating genes compared to other regulators, potentially due to providing ‘leaky’ regulation [The fact that so many members of the RpoN-EBP transcription factor complement in itiation . RpoN-EBitiation , and numode none . Even wi species , and betgulation . This cagulation , situatigulation . Our resAlongside showcasing the ability of a set of RpoN-EBPs to rewire when overexpressed, we have also demonstrated that signals that activate transcription factors can also aid innovation. The nature of connections within regulatory networks will not only determine which transcription factors are highly expressed but also which are activated at any given moment via transduction of environmental signals . Both thglnA gene encoding glutamine synthetase was a possible mutational target [Our data also raise the intriguing possibility that transcription factors that are responsive to signals which can be generated via mutation to internal cellular physiology, may more easily evolve novel phenotypes than those than respond to purely external signals. In the case of AauR and GcsR, mutations occurred in metabolic pathways that catabolise the chemical signals to which these regulators respond (Aspartate and Glycine), likely resulting in their activation. Similarly, in our previous studies of NtrBC rewiring, the l target . Loss ofl target . This tyl target . When R-Our results indicate the key role that transcription factor expression can play in constraining avenues of evolutionary innovation and evolvability in their constituent transcription factors. We have demonstrated that by increasing the expression of a transcription factor – representing an altered GRN architecture – that transcription factor can become the preferred candidate for evolutionary rewiring in our model system. We have demonstrated propensity for rewiring in multiple RpoN-EBP family members and highlighted the interplay between GRN architecture and prevailing environmental conditions necessary to facilitate rewiring in certain transcription factors. Our findings help to build a systems-level understanding of how conditions and signalling within gene regulatory networks can influence the evolutionary trajectories of their constituent components and create or constrain opportunities for innovation of transcription factors.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


First-degree relatives of type 2 diabetics (FDR) exhibit a high risk of developing type 2 diabetes (T2D) and feature subcutaneous adipocyte hypertrophy, independent of obesity. In FDR, adipose cell abnormalities contribute to early insulin-resistance and are determined by adipocyte precursor cells (APCs) early senescence and impaired recruitment into the adipogenic pathway. Epigenetic mechanisms signal adipocyte differentiation, leading us to hypothesize that abnormal epigenetic modifications cause adipocyte dysfunction and enhance T2D risk. To test this hypothesis, we examined the genome-wide histone profile in APCs from the subcutaneous adipose tissue of healthy FDR.TFAM, which regulates mitochondrial DNA (mtDNA) content and stability. In FDR APCs, a significant reduction in H3K4me3 abundance at the TFAM promoter was accompanied by a reduction in TFAM mRNA and protein levels. FDR APCs also exhibited reduced mtDNA content and mitochondrial-genome transcription. In parallel, FDR APCs exhibited impaired differentiation and TFAM induction during adipogenesis. In CTRL APCs, TFAM-siRNA reduced mtDNA content, mitochondrial transcription and adipocyte differentiation in parallel with upregulation of the CDKN1A and ZMAT3 senescence genes. Furthermore, TFAM-siRNA significantly expanded hydrogen peroxide (H2O2)-induced senescence, while H2O2 did not affect TFAM expression.Sequencing-data analysis revealed 2644 regions differentially enriched in lysine 4 tri-methylated H3-histone (H3K4me3) in FDR compared to controls (CTRL) with significant enrichment in mitochondrial-related genes. These included TFAM expression and affecting mitochondrial function. Reduced H3K4me3 enrichment at the TFAM promoter renders human APCs senescent and dysfunctional, increasing T2D risk.Histone modifications regulate APCs ability to differentiate in mature cells, at least in part by modulating The online version contains supplementary material available at 10.1186/s13148-023-01556-z. Mitochondrial abnormalities and dysfunctions in metabolically relevant tissues, such as adipose tissue, have long been recognized in obesity and type 2 diabetes , 2; T2D.Dysfunctional adipose tissue is critical to developing chronic low-grade inflammation, insulin resistance and progression toward T2D, as preservation of adipocyte plasticity mitigates the effects of the caloric overflow, preserving peripheral insulin sensitivity and metabolic health , 5. IndeHealthy first-degree relatives of type 2 diabetics (FDR) exhibit subcutaneous adipose tissue (SAT) early senescence, inflammation and hypertrophy, independent of obesity and body fat distribution –12. ThesEpigenetic modifications mediate, to a large extent, the environmentally induced excess risk of T2D , 30. Epimitochondrial transcription factor A (TFAM) gene, crucial for mitochondrial DNA (mtDNA) replication and mitochondrial-encoded gene transcription  × 100%, where [log (X%)/log2)] accounts for the INPUT dilution 1/X.1 × 10https://manticore.niehs.nih.gov/pavis2/). The analysis provides statistics on ChIP enrichment at important genome features. The functional annotation tool Enrichr (https://maayanlab.cloud/Enrichr/#) was adopted to categorize genes exhibiting histone peaks based on cellular component localization.Four study participant pools for the Chromatin Immunoprecipitation Sequencing (ChIP-Seq) assay were prepared accordingly to the aforementioned protocol. Both immunoprecipitated and INPUT samples were sent to IGA Technology Services, and ‘Ovation Ultralow Library System v2’ kit  has been adopted for library preparation following the manufacturer’s instructions. Quantification of both INPUT and immunoprecipitated samples, as well as the final libraries, was performed using the Qubit 2.0 Fluorometer from Invitrogen  and quality-tested by Agilent Bioanalyzer High Sensitivity DNA assay  and Real Time Stratagen Mx3000P . Libraries were then prepared using Illumina cBot for cluster generation on the flowcell, following the guidelines provided by the manufacturer and sequenced on paired-end 125-bp mode on HiSeq2500 . The CASAVA 1.8.2 version of the Illumina pipeline was used to process raw data for both format conversion and de-multiplexing. The ERNE and Cutadapt tools were used to remove low-quality bases and adapters. Trimmed reads were aligned to the human reference genome (human hg19) with Bowtie2 (with default parameters), and uniquely mapping read selection was performed on alignments. Contaminants assessment and examination on how well the signal in the ChIP-Seq sample can be differentiated from the background distribution of reads in the control sample have been done through bamFingerprinting. Peaks were detected with MACS2 (with default parameters), capturing the influence of genome complexity to evaluate the significance of enriched ChIP regions. The qualitative comparison of BED Peak files has been carried out through the BEDTools. The peaks annotation and the identification of the potential association between ChIP regions and functionally important genomic regions were assessed using the online tool PAVIS  or negative control (scramble) using lipofectamine RNAimax reagent . The preparation of the siRNA-lipofectamine mix was carried out in accordance with the guidelines provided by the manufacturer. The cells were then incubated in a humidified incubator at 37 °C and 5% CO2 and harvested after 48 h of culture. CTRL APCs were also differentiated and silenced throughout the entire differentiation process, starting with the confluence stage on day 0 and ending with mature adipocytes on day 15. The siRNA-lipofectamine mixture was replaced together with the differentiation mix every 3 days.APCs were seeded in 6-well plates, and the medium without antibiotics was added, enabling cells to reach 60–80% of confluence. The cells were transfected with 30 pmol/L Total RNA was isolated from APCs of CTRL and FDR subjects using EuroGold Trifast reagent in accordance with the instructions provided by the manufacturer . RNA samples were reverse-transcribed into complementary DNA (cDNA) by SuperScript III Reverse Transcriptase from Life Technologies . The resulting cDNA served as the template for the subsequent qPCR analysis, conducted using the iTAQ Universal SYBR Green Supermix  on QuantStudio 7 Flex system .Ribosomal protein L13a (RPL13A). Relative quantification of gene expression was determined using the comparative 2 − ∆∆Ct method, based on the cycle threshold (Ct) values of the target and housekeeping genes. Absolute quantification  of gene expression was calculated using the comparative 2 − ∆Ct method and shown as individual data points. The primer sequences utilized for the analysis, indicated in Additional file http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The primer sequences were obtained from Sigma-Aldrich .Data normalization was achieved using the housekeeping gene TFAM gene. The bisulphite genomic sequencing was executed according to the protocol described in our previous publication . No. No37]. vs 1167 FDR; Additional file p value = 0.001700; Fisher’ exact test), mitochondrial part (GO:0044429) , and mitochondrial nucleoid (GO:0042645) , suggesting an involvement of this cellular component in the preadipocytes abnormalities observed in those individuals who are FDR. As shown in Fig. ChIP-Seq profiling of the H3K4me3 mark was then performed in APCs isolated from abdominal SAT biopsies in the FDR and CTRL subjects. The H3K4me3 ab-precipitated chromatin isolated from these individuals was analyzed by deep sequencing, and ChIP-Seq bioinformatic analysis provided the entire H3K4me3 map covering the entire genome. The sequencing-reads were regularly distributed across the human genome and detected in all chromosomes (data not shown). As previously published , 41, H3KGlyceraldehyde 3 Phosphate Dehydrogenase—TSS) and one negative genomic control region (Myoglobin exon 2)  and protein . Furthermore, mitochondrial DNA amount in preadipocytes from FDR subjects was significantly decreased compared to the CTRL  and directly correlated with TFAM mRNA expression . Since the C-terminal tail of TFAM is crucial for the activation of promoter-specific transcription of mtDNA  was measured in FDR preadipocytes . In addition to these alterations, we detected a significant reduction in ATP levels .Based on this information, we addressed further efforts on sis Fig. c. The deof mtDNA , we furtase II CO, ATP synase II CO, ATP synTFAM transcription in APCs, we have examined the TFAM upstream transcriptional network, known to be mainly composed of the Peroxisome proliferator–activated receptor γ coactivator 1α (PGC1α), Nuclear respiratory factor 1 (NRF1), and Nuclear respiratory factor 2 (NRF2) transcription factors. We measured their expression by qPCR and detected no significant difference in mRNA levels of NRF1 and NRF2 in the APCs from either FDR or CTRL individuals . Thus, the differential enrichment of H3K4me3 at TFAM promoter was postulated to represent the most proximal change occurring in the FDR subjects and related to the reduced TFAM expression in these individuals.To obtain further insight into the regulation of als Fig. a, b, whiTFAM gene is regulated by DNA methylation of its promoter region, and this methylation has been associated with insulin resistance in adolescents . Fig. S3 Schematic representation of the DNA methylation analysis performed on the TFAM promoter CpG island identified by Emboss Cpgplot. Methylation of all 50 CpGs at the TFAM promoter for ten replica clones. White circles, unmethylated CpGs; black circles, methylated CpGs. A representative experiment with one FDR and one CTRL individual is shown.Additional file 2. Peaks annotation file in CTRL individuals. Genes (1051) annotated near the gene TSS (± 1000 bps), using the PAVIS tool, differentially enriched in CTRL subjects.Additional file 3. Peaks annotation file in FDR individuals. Genes (797) annotated near the gene TSS (± 1000 bps), using the PAVIS tool, differentially enriched in FDR subjects.